participant booklet of the 2015 RQR Symposium

Transcription

Bienvenue à tous les membres du RQR ainsi qu’aux autres participants,
Le Symposium annuel du RQR est l’évènement de réseautage le plus important du RQR, donnant l’opportunité
aux chercheurs, étudiants, personnel de recherche et utilisateurs finaux de se rencontrer et de discuter des
projets de recherche, de leurs collaborations et des futures activités du Réseau.
Nous avons une excellente liste de conférenciers invités fournissant les informations les plus récentes sur trois
sujets. Lors de la première journée, la Dre Catherine Racowsky, du Brigham & Women’s Hospital à Boston,
Massachusetts, présentera une conférence intitulée ‘Embryo selection in clinical IVF: The relevance and current
status’. La deuxième journée, le Dr Christopher Geyer, de la East Carolina University, présentera une conférence
intitulée ‘Defining the essential role for retinoic acid (RA) in spermatogonial differentiation’. La Dre Rebecca Krisher,
de la National Foundation for Fertility Research, présentera aussi la seconde journée. Le titre de sa présentation
sera ‘The Mammalian Oocyte: Metabolic Challenges and Opportunities’.
Le programme inclut également 15 présentations orales, ainsi que 66 présentations par affiche. Il y aura
également une session de courtes présentations de 5 minutes mettant en évidence certaines affiches.
En espérant une rencontre excitante et interactive ici à Montréal, nous vous encourageons à profiter de toutes les
opportunités offertes par cet événement.
Meilleures salutations,
Bruce Murphy
Directeur du RQR
Welcome to all RQR members and other participants,
The annual RQR Symposium is the most important networking event of the RQR, providing the opportunity to
researchers, students, research professionals and end users to interact and to discuss research projects,
collaborations, and future activities of the network.
We have an exciting program with an excellent slate of speakers providing the newest information on three
topics. On the first day, Dr. Catherine Racowsky, from the Brigham & Women’s Hospital in Boston,
Massachusetts, will present a talk entitled ‘Embryo selection in clinical IVF: The relevance and current status’. On the
second day, Dr. Christopher Geyer, from East Carolina University, will present a talk entitled ‘Defining the essential
role for retinoic acid (RA) in spermatogonial differentiation’. Dr. Rebecca Krisher, from the National Foundation for
Fertility Research, will also present on the second day. The title of her presentation will be ‘The Mammalian
Oocyte: Metabolic Challenges and Opportunities’.
The program also includes 15 oral presentations, as well as 66 poster presentations. A session of short 5 minute
presentations highlighting posters, will also be held.
We look forward to an exciting and interactive colloquium here in Montréal. Please take every opportunity to
profit from this event.
Best wishes,
Bruce Murphy
RQR Director
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8 Symposium du RQR • 8 RQR Symposium
1
Table des matières
Mot du directeur
1
Table des matières
2
Programme
3
Résumés session I (présentations orales)
5
Conférence Catherine Racowsky
9
Résumés session II (présentations orales)
10
Présentations d’affiches sélectionnées
15
Résumés session d’affiches I
16
Résumés session d’affiches II
52
Conférence Christopher Geyer
88
Résumés session III (présentations orales)
89
Conférence Rebecca Krisher
93
Résumés session IV (présentations orales)
94
Partenaires financiers
101
Liste des participants
102
Table of contents
Message from the Director
1
Table of content
2
Agenda
3
Session I abstracts (oral presentations)
5
Conference Catherine Racowsky
9
Session II abstracts (oral presentations)
10
Presentation of selected posters
15
Poster session I abstracts
16
Poster session II abstracts
52
Conference Christopher Geyer
88
Session III abstracts (oral presentations)
89
Conference Rebecca Krisher
93
Session IV abstracts (oral presentations)
94
Financial Partners
101
List of participants
102
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Programme préliminaire du 8e Symposium du Réseau Québécois en reproduction
Tentative Agenda of the 8th Symposium of the Réseau Québécois en reproduction
Mardi 17 novembre – Tuesday November 17th
9h00 – 10h00
Inscription
Inscription
Foyer
10h00 – 10h15
Mot de bienvenue
Welcome
Salon des
Saisons A & B
10h15 – 11h00
Présentations : Session I
Presentations: Session I
Salon des
Saisons A & B
Conférencière invitée : Catherine Racowsky
Invited Speaker: Catherine Racowsky
11h00 – 12h00
Embryo selection in clinical IVF: The relevance
and current status
Salon des
Saisons A & B
12h00 – 13h30
Dîner
Lunch
Pierre de
Coubertin
13h30 – 14h30
Présentations : Session II
Presentations: Session II
Salon des
Saisons A & B
14h30 – 15h00
Présentations de 5 minutes : Affiches
5 Minute Presentations: Posters
Salon des
Saisons A & B
15h00 – 16h30
Pause-café et session d’affiches I
Coffee Break and Poster Session I
Foyer
16h30 – 16h45
Remise du Prix MdC du RQR
RQR KT Award Presentation
Salon des
Saisons A & B
16h45 – 19h30
Cocktail
Cocktail
Pierre de
Coubertin
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Mercredi 18 novembre - Wednesday November 18th
9h00 – 10h30
Session d’affiches II
Poster Session II
Conférencier invité : Christopher Geyer
Invited Speaker: Christopher Geyer
10h30 – 11h30
Defining the essential role for retinoic acid (RA)
in spermatogonial differentiation
Foyer
Salon des
Saisons A & B
11h30 – 12h15
Présentations : Session III
Presentations: Session III
Salon des
Saisons A & B
12h15 – 13h30
Dîner
Lunch
Pierre de
Coubertin
Conférencière invitée : Rebecca Krisher
Invited Speaker: Rebecca Krisher
13h30 – 14h30
The Mammalian Oocyte: Metabolic Challenges
and Opportunities
Salon des
Saisons A & B
14h30 – 14h45
Pause-café
Coffee Break
Foyer
14h45 – 16h00
Présentations : Session IV
Presentations: Session IV
Salon des
Saisons A & B
16h00
Rapports des comités – Fermeture de la session
Committee reports – Closing of session
Salon des
Saisons A & B
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Présentations – Presentations : Session I
17 novembre – November 17th
Président – Chair : Daniel Dufort, McGill University
Co-président – Co-Chair : Omar Farah, McGill University
I. Ca² dynamics in oocytes from naturally-aged mice.
Jenna Haverfield, Université de Montréal (Page 6)
10h15 – 10h30
II. Activation du système de réparation de l’ADN des spermatogonies de rat en
réponse à la doxorubicine.
Hermance Beaud, INRS – Institut Armand-Frappier (Page 7)
10h30 – 10h45
III. Early embryonic response to in vitro cultured environment enriched with
global methyl donor: phenotype, transcriptome and epigenome analysis.
Habib A. Shojaei Saadi, Université Laval (Page 8)
10h45 – 11h00
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Ca² dynamics in oocytes from naturally-aged mice
Jenna Haverfield¹ ², Shoma Nakagawa¹, Daniel Love³, Elina Tsichlaki4, Michail
Nomikos³, F. Anthony Lai³, Karl Swann³, Greg FitzHarris¹ ² 4
¹Centre Recherche CHUM, Montreal, Canada, H2X 0A9.
²Department of Obstetrics and Gynaecology, University of Montreal, Montreal, Canada,
H3T 1J4.
³Institute of Molecular and Experimental Medicine, Cardiff University School of
Medicine, Heath Park, UK, CF14 4XN.
4
Department of Cell and Developmental Biology, University College London, London,
UK, WC1E 6BT.
Oocyte aging is a complex, multifactorial process resulting in deterioration of oocyte
viability with advancing maternal age and is a leading cause of age-related female
infertility. Clinical reports reveal that the ability of the oocyte to resume meiosis and
initiate embryogenesis, termed egg activation, following assisted reproductive
technologies declines with maternal age. Egg activation is triggered by repetitive
increases in intracellular Ca² concentration ([Ca² ]i) in the ooplasm as a result of
sperm-egg fusion. We therefore hypothesised that eggs from older females feature a
reduced ability to mount appropriate Ca² responses at fertilisation. To test this
hypothesis we performed the first examination of Ca² dynamics in eggs from young
and naturally-aged mice using live epifluorescence microscopy. Strikingly, we find that
Ca² stores and resting [Ca² ]i are unchanged with maternal age. Although eggs from
naturally-aged mice feature a reduced ability to replenish intracellular Ca² stores
following depletion, this difference had no effect on the duration, number, or amplitude
of Ca² oscillations following intracytoplasmic sperm injection or expression of
phospholipase C zeta, the sperm-borne trigger for Ca² release. In contrast, we
describe a substantial reduction in the frequency and duration of oscillations in
naturally-aged eggs upon parthenogenetic activation with SrCl . We conclude that the
ability to mount and respond to an appropriate Ca² response at fertilisation is largely
unchanged by advancing maternal age, but subtle changes in Ca² handling occur that
may have more substantial impacts upon commonly used means of parthenogenetic
activation.
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Activation du système de réparation de l’ADN des spermatogonies de rat
en réponse à la doxorubicine
Hermance Beaud, Géraldine Delbès
INRS-Institut Armand Frappier, Laval, Québec
Les effets secondaires des chimiothérapies peuvent atteindre la fertilité masculine en
ciblant les spermatogonies en division. Nous avons récemment montré que certains
composés de chimiothérapie, la doxorubicine seule et en combinaison avec la
vincristine (MIX), causent des cassures de l’ADN en fonction du temps et de la dose
sur une lignée de spermatogonies de rat (GC-6spg). Nous soutenons l’hypothèse que
les spermatogonies stimulent leur système de réparation de l’ADN en réponse à des
doses non cytotoxiques de chimiothérapies.
Nous avions montré que le MIX augmentait significativement le taux de cassures de
l’ADN dans les GC-6spg à une dose non cytotoxique de 0,1uM après 24h de
traitement. Dans ces mêmes conditions, nous avons criblé par puce à ADN ciblée, 84
gènes impliqués dans la réparation de l’ADN, le cycle cellulaire, et l’apoptose. Nos
résultats révèlent que le MIX mais aussi la doxorubicine seule affectent
ssion de 13 et 14 gènes respectivement. La
similarité des gènes dérégulés supporte l’idée du rôle majeur de la doxorubicine dans
l’action du MIX. Cdkn1a, impliqué dans le cycle cellulaire, est le gène le plus stimulé
suggérant l’arrêt du cycle cellulaire. Huit gènes de réparation de l’ADN sont affectés
par les traitements. Mgmt code une alkyltransférase formant à elle seule une voie de
réparation. De façon surprenante, cette voie de réparation est la plus activée dans les
spermatogonies. Notre étude démontre pour la première fois, la stimulation des
systèmes de réparation de l’ADN dans les spermatogonies suite à un traitement de
chimiothérapie.
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Early embryonic response to in vitro cultured environment enriched with
global methyl donor: phenotype, transcriptome and epigenome analysis
Shojaei, H., Gagné, D., Fournier, E., Baldoceda, L.M., Sirard, M.A., Robert, C.
Centre de recherche en biologie de la reproduction, Université Laval, Quebec City, QC,
G1V 0A6, Canada
Mammalian early embryos react to their immediate microenvironment and it is believed
that conditions provided by maternal nutrition or stress can induce short (transcriptome)
and long term (epigenome) adaptation. It was hypothesized that early bovine embryos
would react both at the transcriptomic and epigenomic levels when placed in a
microenvironment rich in methyl donor.
The impacts of supraphysiological S-Adenosyl methionine (SAM) supplementation, as
a global methyl donor and final substrate of folic acid, on bovine early embryonic
development was tested in vitro. The culture medium was supplemented with SAM
starting at the 8-cell stage and cultured with the treatment up to the blastocyst stage.
Day-7 blastocysts were collected and analyzed in parallel for genome-wide DNA
methylation and transcription using our dedicated arrays.
The SAM treatment induced a phenotypic response by significantly increasing
embryonic hatching and skew in the sex ratio in favor of male embryos.
Transcriptomics analysis identified 247 transcripts including lncRNAs. DNA methylome
analyses identified 4056 differentially methylated regions (DMRs) mainly in SAMtreated embryos. The distribution of DMRs showed enrichment for exonic regions and
CpG islands. DNA methylome pathways analysis revealed that he most affected
differentially methylated genes involved in ESCs pluripotency and BER pathways
suggesting an epigenetic compensatory mechanisms in early embryos. No association
between the DNA methylation and transcription were detected. In conclusion, this study
shows that methyl donor (SAM) supplementation during in vitro bovine early embryo
development profoundly influences embryonic gene expression and DNA methylation
as well as developmental phenotypes.
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Conférencière invitée - Invited Speaker
Dr. Catherine Racowsky
Dr. Catherine Racowsky is Director of the ART
Laboratory at Brigham & Women’s Hospital in
Boston, Massachusetts and Professor of Obstetrics
and Gynecology at Harvard Medical School. Having
graduated with a Bachelor of Arts in Zoology at the
University of Oxford, she received her Ph.D. in
Reproductive Physiology from the University of
Cambridge in England and then undertook her postdoctoral fellowship at Harvard Medical School.
Her research interests include investigating the effects of maternal and
environmental factors on egg quality, studying the molecular and cellular
mechanisms underlying egg maturation in vivo and in vitro, and expanding
methodologies for assessing human embryo developmental competency. She
supervises Harvard medical students, residents, fellows and junior faculty
members. She has authored over 100 peer-reviewed papers on various aspects
of mammalian egg and embryo physiology, has published numerous news
releases and book chapters, and has co-edited five books. She is also a peerreviewer for numerous journals in reproductive medicine and has served on a
number of editorial boards.
Her main clinical focus concerns quality control and quality improvement in
clinical embryology, with specific attention being given to ensuring validation of
procedures before routine implementation. She is particularly known for her
contributions in establishing single-step medium for growing human blastocysts
in vitro.
On Tuesday, Dr. Racowsky will give a talk entitled: “Embryo selection in
clinical IVF: The relevance and current status”.
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Présentations – Presentations : Session II
Présidente – Chair : Géraldine Delbès, INRS – Institut Armand-Frappier
Co-président – Co-Chair : Fabien Joao, INRS – Institut Armand-Frappier
I. Differential effects of the mycotoxin deoxynivalenol (DON) and its major
metabolite de-epoxy DON on bovine theca cells
Hilda Guerrero-Netro, Université de Montréal (Page 11)
13h30 – 13h45
II. MEF2 and COUP-TFII cooperate to regulate Akr1c14 gene expression in
mouse MA-10 Leydig cells
Mickaël Di-Luoffo, Université Laval (Page 12)
13h45 – 14h00
III. The role of p53 in Hydroxyurea Embryotoxicity
Nazem El Husseini, McGill University (Page 13)
14h00 – 14h15
IV. Les acides gras non estérifiés induisent une augmentation la production
d’androgènes en présence d’insuline dans des cellules thécales bovines
Samuel Leblanc, Université de Sherbrooke (Page 14)
14h15 – 14h30
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Differential effects of the mycotoxin deoxynivalenol (DON) and its major
metabolite de-epoxy DON on bovine theca cells
Hilda M. Guerrero Netro, Hugues Duret *, Younes Chorfi, Christopher A. Price
Centre de Recherche en Reproduction Animale (CRRA), Faculté de médecine
vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada
* École nationale vétérinaire de Toulouse
Deoxynivalenol (DON) is a major mycotoxin found in animal feed, and its mechanism of
action is through the ribotoxic stress response (RSR) in different cell types. DON has
been shown to inhibit estradiol and progesterone (P4) secretion from bovine granulosa
cells, but its effect on theca cells is unknown. The objective of this study was to
determine the effects of DON and its major metabolite, de-epoxy DON (DOM), on
bovine theca cells in vitro.
Bovine theca cells from follicles 3-5 mm diameter were placed in serum-free culture.
Dose and time-course studies with DON and DOM were performed with a maximum
dose of 1 ng/ml. P4 and testosterone secretion was measured by RIA, apoptosis was
measured by Annexin V flow cytometry, activation of major RSR pathways was
assessed by western blot.
Treatment with DOM resulted in a significant inhibition of P4 (P<0.005), and
testosterone (P<0.05) secretion, and an increase in the proportion of apoptotic cells
(P<0.05), while DON only inhibited P4 (P<0.005) and did not alter testosterone
secretion or percentage of dead cells. Western blot demonstrated that DON and DOM
significant increased phosphorylation of ERK1/2 (P<0.005) after 5 min, of doublestranded RNA-activated protein kinase (PKR) after 5-15 min (P<0.005) and JNK at 30
min (P<0.05). Interestingly, phosphorylation of p38 was significantly (P=0.001) upregulated by DOM but decreased (P<0.05) by DON. Taken together, these results
suggest that DON and DOM activate different signaling pathways and differentially
impact theca cell health.
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MEF2 and COUP-TFII cooperate to regulate Akr1c14 gene expression in
mouse MA-10 Leydig cells
Mickaël Di-Luoffo1, Catherine Brousseau1, Raifish E. Mendoza Villarroel1, and
Jacques J. Tremblay1,2
1
Reproduction, Mother and Child Health, Centre de recherche du CHU de QuébecUniversité Laval, Québec Canada
2
Centre de recherche en biologie de la reproduction, Department of Obstetrics,
Gynecology, and Reproduction, Faculty of Medicine, Université Laval, Québec, Canada
Dihydrotestosterone (DHT) is a potent androgen and its bioavailability must be tightly
regulated. In mouse Leydig cells, the Akr1c14 gene codes for the 3aHSD enzyme
which catalyzes the interconversion of DHT into 3a-diol, allowing a balance between
DHT synthesis and elimination. Nothing is currently known regarding the regulation of
Akr1c14 expression in Leydig cells. Recently, the transcription factors MEF2 and
COUP-TFII were identified in the mouse testis, including in Leydig cells. MEF2 and
COUP-TFII are present in Leydig cells throughout fetal and adult life. Analysis of the
transcriptome of MEF2- or COUP-TFII-deficient MA-10 Leydig cells revealed a
significant decrease in Akr1c14 expression. The aim of this study was to determine the
role and mechanism of MEF2/COUP-TFII action in Akr1c14 expression in Leydig cells.
By qPCR, we confirmed that Akr1c14 mRNA levels were decreased by ~55% in MEF2and in COUP-TFII-deficient MA-10 Leydig cells. Conversely, overexpression of MEF2,
COUP-TFII or both in MA-10 cells increased endogenous Akr1c14 mRNA levels. In
silico analysis of the Akr1c14 promoter revealed the presence of a MEF2 and a COUPTFII binding site. Recruitment of MEF2 and COUP-TFII to this Akr1c14 promoter region
was confirmed by ChIP and DNA precipitation assays. In functional promoter studies,
MEF2 and COUP-TFII were found to cooperate in Akr1c14 promoter activation.
Deletion or mutation of the MEF2/COUP-TFII sites abolished this cooperation. In
conclusion, our results identify a novel cooperation between MEF2 and COUP-TFII in
the expression of the Akr1c14 gene involved in regulating DHT levels. Supported by
CIHR.
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The role of p53 in Hydroxyurea Embryotoxicity
Nazem El Husseini, Barbara F. Hales
Department of Pharmacology & Therapeutics, McGill University, Montreal, Canada
Hydroxyurea (HU), an anticancer agent and potent teratogen, is used as a model drug
to study the embryonic stress response during organogenesis. Previously, we
demonstrated that HU activates a DNA damage response (DDR) pathway in the
gestation day (GD) 9 mouse embryo (Toxicol Sci. 2013 133(2):298-308). The p53
tumor suppressor gene is a possible downstream effector of this pathway. P53 plays an
important role in embryonic development, however its response to teratogens is
debatable. We hypothesize that HU exposure at the organogenesis stage activates p53
which then mediates cell cycle arrest and cell death that result in the observed
malformations. To test this hypothesis, CD-1 embryos at GD9 were exposed in vivo to
saline (Control-CO) or two doses of HU (HU400= 400 mg/kg; HU600= 600 mg/kg);
embryos were extracted after 3 hours and samples prepared to examine gene and
protein expression. Microarray analysis showed that the expression of 1346 genes
significantly changed in embryos exposed to HU vs. control and that they were
significantly associated with the p53 signaling pathway. The active form of p53
(phospho-p53) was significantly upregulated in HU exposed embryos; western blot and
confocal
microscopy
showed
increasing
protein
concentrations
and
nuclear
translocation in major embryonic tissues. qRT-PCR showed that p53-regulated genes
(Cdkn1A, Fas, p53inp1) were significantly upregulated and p53inp1 protein increased
in a dose-dependent fashion with HU. Together, these data show that p53 signaling is
the main pathway activated in response to HU during organogenesis, and leads to the
upregulation of cell cycle arrest and cell death promoting factors.
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Les acides gras non estérifiés induisent une augmentation la production
d’androgènes en présence d’insuline dans des cellules thécales bovines
Leblanc S, Battista MC, Baillargeon JP
Département de Médecine, Service d’endocrinologie, Université de Sherbrooke,
Sherbrooke, Québec, Canada.
Le syndrome des ovaires polykystiques (SOPK), caractérisé par une hyperandrogénie
(HA) et une anovulation chronique, est souvent associé à une résistance à l’insuline
(RI). Une surexposition in vivo à des acides gras non estérifiés (AGNE) entraîne une RI
et une HA chez l’humain. Notre objectif est de déterminer si les AGNE et l’insuline
provoquent une hypersécrétion d’androgènes ovarienne. Des cellules thécales bovines
(CTB; n=5) ont été exposées 48h en absence ou en présence de palmitate (Pa, 25 µM2x/j), oléate (Ol, 50µ M-2x/j), insuline (0-200 nM-1x/j) et de forskoline (Fsk; 10 µM1x/2j). Les androgènes androstènedione (A4) et DHEA, ainsi qu’un marqueur de stress
oxydatif, le 8-isoprostane (ELISA), ont été mesurés dans le milieu de culture.
L’activation diminuée d’ERK1/2 ayant été associée à l’HA, sa phosphorylation a été
évaluée (Western blot). Sous Fsk, le mélange Pa/Ol augmente la production d’A4 en
présence d’insuline (maximum : 100 nM: +72±22% vs Fsk seul; P=0,04), alors que le
palmitate ou l’oléate seuls n’ont pas d’effet significatif. Sous Fsk, le Pa augmente le 8isoprostane (+123±58% vs Fsk seul; P=0,03), qui demeure inchangé par le mélange
Pa/Ol. Sous 100 nM d’insuline, le mélange Pa/Ol tend à augmenter la réponse en
DHEA sous Fsk (+85±50%; P=0,06) et à diminuer la phosphorylation de ERK1/2
(24±12%, P=0,12). En présence d’insuline, une surexposition des CTB aux AGNE
augmente leur production en androgènes suite à l’activation de la voie de la LH,
supportant un rôle de l’insuline et de la lipotoxicité dans la surproduction ovarienne en
androgènes, la caractéristique principale du SOPK.
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Présentations d’affiches – Poster Presentations
14h30 – 15h00
I. Binucleation in the Preimplantation Mouse Embryo Cause Higher Frequency
of Chromosome Segregation Errors.
Candice Taguibao, Université de Montréal (Page 33)
14h30 – 14h35
II. Paternal Lifetime Folate Deficiency and Supplementation Effects
Reproductive Health and Induces Aberrant Sperm DNA Methylation.
Lundi Ly, McGill University (Page 34)
14h35 – 14h40
III. Rôle des microARNs miR-34b/c-miR-449 dans la régulation de gènes
épididymaires.
Olivia Jerczynski, Université Laval (Page 44)
14h40 – 14h45
IV. Ubiquitin ligase Huwe1 modulates male germ cell development by regulating
spermatogonial differentiation and meiotic entry.
Rohini Bose, McGill University (Page 72)
14h45 – 14h50
V. Regulation of gene expression in murine granulosa cells.
Milena Taibi, McGill University (Page 80)
14h50 – 14h55
VI. Random unilateral chromosome inheritance is a novel explanation for
mosaic embryo aneuploidy.
Cayetana Vázquez de Castro Diez, Université de Montréal (Page 85)
14h55 – 15h00
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Session d’affiches I – Poster Session I
15h00 – 16h30
1. Importance du facteur de transcription PAX8 dans les cellules endométriales
stromales. Carol-Ann Joly, UQTR (Page 19)
2. Meiotic
Chromosome
Synapsis
In
Murine
Heterozygous
Robertsonian
Translocation Carrier Oocytes. Yuri Chung, McGill University (Page 20)
3. The crucial role of myo-inositol in the protection of developing embryos from
ethanol exposure. Florence Pagé-Larivière, Université Laval (Page 21)
4. Regulation of Ankyrin-repeat and SOCS-box protein 9 (ASB9) in ovarian follicles
and identification of binding partners. Kalidou Ndiaye, Université de Montréal
(Page 22)
5. Characterization of a new genetically-induced mouse model for PCOS syndrome.
Mostafa Esmael, UQAM (Page 23)
6. Changes in the maternal immune system in late onset and postpartum
preeclampsia. Adriana Carbonaro, Université de Montréal (Page 24)
7. Uric acid-induced placental inflammation and dysfunction is dependent on IL-1.
Marie-Eve Brien, Université de Montréal (Page 25)
8. Transcriptome analysis of granulosa cells from poor follicle quality in heifer and
adult cows. David Landry, Université Laval (Page 26)
9. Effects of brominated flame retardants on KGN cells, a human granulosa cell
line. Pavine Lefevre, McGill University (Page 27)
10. Analyse épigénétique de semence et d'embryons provenant de boeufs
péripubères. Simon Lambert, Université Laval (Page 28)
11. Disturbance of spindle function in aged mice oocytes is cytoplasm dependent.
Shoma Nakagawa, Université de Montréal (Page 29)
12. Addition of oocyte can differentialy regulate cumulus expansion in the presence
of FSH or AREG. Paula Fernanda de Lima, Université de Montréal (Page 30)
13. Identification and localization of Phosphodiesterase 10A (PDE10A) in bull
spermatozoa. Loïze Maréchal, Université Laval (Page 31)
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14. In utero exposure to an environmentally relevant dose of DEHP targets the adult
adrenal gland for endocrine disruption. Sunghoon Lee, McGill University
(Page 32)
15. Binucleation in the Preimplantation Mouse Embryo Cause Higher Frequency of
Chromosome Segregation Errors. Candice Taguibao, Université de Montréal
(Page 33)
16. Paternal Lifetime Folate Deficiency and Supplementation Effects Reproductive
Health and Induces Aberrant Sperm DNA Methylation. Lundi Ly, McGill
University (Page 34)
17. Binder of sperm gene deletion by CRISPR genome editing and the effects on
fertility. Marzieh Eskandari Shahraki, Université de Montréal (Page 35)
18. Early growth response 1 (EGR1) activates fibroblast growth factor 8 (FGF8)
signaling in bovine granulosa cells. Peng Han, Université de Montréal (Page 36)
19. Activin induction of follicle-stimulating hormone expression requires protein deacetylation in immortalized gonadotrope-like cells. Gauthier Schang, McGill
20. Specific alterations in the histone modification landscape as a consequence of
transient Dnmt1 deficiency in mouse ES cells. Lisa-Marie Legault, Université de
Montréal (Page 38)
21. Characterization of a novel and differentially expressed long non-coding RNA
(lncRNA) in granulosa cells of bovine dominant follicle. Jacques Lussier,
Université de Montréal (Page 39)
22. Effect of Diet-Induced Magnesium Deficiency on Bone Health and Glucose
Metabolism in Obese-Prone and Obese-Resistant Rats. Sophia Rahimi, Health
Canada (Page 40)
23. Nr5a2 regulates granulosa cells proliferation in vitro and in vivo. Marie-Charlotte
Meinsohn, Université de Montréal (Page 41)
24. Exercise training improves the fertility of female mice. Asaftei Olga, Université de
Montréal (Page 42)
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17
25. The alarmin uric acid-induced fetal growth restriction: new animal model of noninfectious inflammation during pregnancy. Ines Boufaied, Université de Montréal
(Page 43)
26. Rôle des microARNs miR-34b/c-miR-449 dans la régulation de gènes
épididymaires. Olivia Jerczynski, Université Laval (Page 44)
27. Effet d’une exposition in-utero à l’éthinyl-œstradiol sur le développement des
gonocytes de rats mâles. Bintou Gaye, INRS - Institut Armand-Frappier
(Page 45)
28. Caractérisation des voies de signalisation PKA et PKC dans les cellules de la
granulosa humaine en culture. Patricia Tremblay, Université Laval (Page 46)
29. La culture organotypique : un bon modèle d’étude de la reprogrammation
épigénétique dans les cellules germinales fœtales du rat mâle. Arlette
Rwigemera, INRS, Institut Armand-Frappier (Page 47)
30. Régulation de l'activité du promoteur distal 1b du gène Gata4. Vanessa
Théberge, Université Laval (Page 48)
31. Impact de la génistéine sur la régulation de p53 au cours de la décidualisation.
Roch Tremblay, UQTR (Page 49)
32. Contrôle de la stimulation ovarienne par diagnostique de la qualité folliculaire.
Chloé Fortin, Université Laval (Page 50)
33. Single patient with recurrence of the same mechanism in three spontaneous
abortions: digynic triploidy due to failure of meiosis II. Yassemine Khawajkie,
McGill University (Page 51)
18
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Importance
du
facteur
de
transcription
PAX8
dans
les
cellules
endométriales stromales
Carol-Ann Joly, Roch Tremblay, Valérie Leblanc, Céline Van Themsche
Université du Québec à Trois-Rivières (UQTR), Groupe de Recherche en Signalisation
Cellulaire (GRSC)
À chaque cycle menstruel, les cellules endométriales stromales (CES) utérines se
différencient en cellules déciduales, capables d’interagir adéquatement avec un
éventuel embryon. La protéine p53, un facteur de transcription important pour la
complétion de la zone déciduale autour de l’embryon, s’accumule dans les CES en
décidualisation de manière PKA-dépendante. Toutefois, l’identité des facteurs
intracellulaires impliqués demeure inconnue. Puisque l’expression de PAX8 dans
certains types cellulaires est PKA-dépendante, et que PAX8 peut lier le promoteur du
gène p53 et en réguler l’expression, nous posons l’hypothèse que la quantité de PAX8
pourrait être augmentée de manière PKA-dépendante dans les CES pendant la
décidualisation et contribuer à l’accumulation de p53 par la suite. OBJECTIF
GÉNÉRAL DES TRAVAUX : Déterminer la régulation de PAX8 dans les CES au fil de
la décidualisation et le lien fonctionnel entre PAX8 et p53 dans ce contexte. MODÈLE
EXPÉRIMENTAL : Les cellules endométriales stromales humaines immortalisées
(HIESC) sont utilisées comme modèle cellulaire pour la décidualisation in vitro, induite
à l’aide d’AMPc et d’acétate de médroxyprogestérone (MPA). RÉSULTATS
PRÉLIMINAIRES: Augmentation transcriptionnelle et protéique de Pax8 aux jours 3 et
6 de la décidualisation. PERSPECTIVES : Ces travaux pourraient permettre de mettre
en lumière une expression finement régulée de PAX8 dans les CES pendant la
décidualisation, et révéler pour la première fois un rôle pour PAX8 dans la régulation de
p53 et par conséquent, dans le bon déroulement de ce processus essentiel à la
fonction reproductrice féminine.
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Meiotic Chromosome Synapsis In Murine Heterozygous Robertsonian
Translocation Carrier Oocytes
Yuri Chung and Teruko Taketo
Department of Biology, McGill University; Research Institute of McGill University Health
Centre
A major oocyte loss occurs during fetal/neonatal ovarian development, limiting the
ovarian reserve and female reproductive life span in mammals. This oocyte loss
coincides with Meiotic Prophase I, during which homologous chromosomes pair,
synapse, and recombine. Failure in these processes will result in aberrant chromosome
segregation and aneuploidy. Accordingly, it has been hypothesized that a surveillance
mechanism operates to eliminate oocytes with synaptic errors to improve the average
quality of oocytes in reserve. However, the evidence is limited to XO and XY female
mice, which show synaptic failure of sex chromosomes and a greater loss of oocytes
compared to XX females. In the present study, we examined meiotic synapses and
progression in heterozygous Robertsonian Translocation (RbT) carrier females,
involving
chromosomes
8
and
12,
as
a model for
autosomal
asynapsis.
Immunofluorescence detection of synaptonemal complexes in microspread ovarian
cells showed that the RbT region transiently unsynapsed with their counterpart wildtype chromosomes, but eventually fully synapsed by the end of pachytene stage. The
higher percentage of pachytene oocytes showing
H2AX-domains, a DNA damage
response element, was higher in the heterozygous RbT carrier ovaries than the control
ovaries. However, many H2AX domains were observed over unsynapsed non-RbT
chromosomes rather than the RbT region. The total number of oocytes was significantly
higher in heterozygous RbT carrier ovaries than the control ovaries, contrary to our
expectation. These results may suggest that the presence of heterozygous RbT impairs
the surveillance mechanism of oocyte elimination.
20
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The crucial role of myo-inositol in the protection of developing embryos
from ethanol exposure
Florence Pagé-Larivière1, Céline Campagna2, Marc-André Sirard1
1
Centre de recherche en reproduction, développement et santé intergénérationnelle,
Université Laval, Québec, Québec
2
Institut national de santé publique du Québec, Québec, Québec
Alcohol consumption during pregnancy is known to impair foetuses physical and
neurological development that further impacts their health after birth and all lifelong.
Consequently, a majority of women with confirmed pregnancy are recommended to
stop drinking alcohol. Unfortunately, since the pregnancy began several weeks before a
positive pregnancy test, the early embryo might have already been exposed to alcohol
during one of the most critical windows of its development. Little is known about the
impact of alcohol on preimplantation embryos and the molecular pathways it affects.
Here we show that pig embryos exposed to 0.2% of ethanol during their development
had significantly decreased viability and speed of embryonic development. It was also
confirmed that the surviving embryos had a strongly reduced expression of myo-inositol
oxygenase (MIOX), an enzyme that degrades myo-inositol, suggesting that the
capability of increasing the level of myo-inositol is a key element for survival after
alcohol exposure. Interestingly, the addition of myo-inositol to the culture media of
treated embryos resulted in an increased blastocyst rate. Thus, our results indicate that
myo-inositol is a crucial molecule that can protects developing embryos from ethanol
exposure.
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21
Regulation of Ankyrin-repeat and SOCS-box protein 9 (ASB9) in ovarian
follicles and identification of binding partners
Gabriel Benoit, Jacques Lussier et Kalidou Ndiaye
Centre de recherche en reproduction animale (CRRA), Département de biomédecine
vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, P.O. Box 5000,
St-Hyacinthe, Québec, Canada, J2S 7C6, Canada.
Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the large SOCS-box
containing family and acts as a specific substrate recognition component of E3 ubiquitin
ligases in the process of ubiquitination and proteasomal degradation. ASB9 is known to
interact with creatine kinase B (CKB) and ubiquitous mitochondrial creatine kinase
(uMtCK) to negatively regulate cell growth. We previously identified ASB9 as a
differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles and
aimed to further investigate ASB9 regulation and to identify its binding partners in
bovine GC. To this end, GC were obtained from small follicles (SF), dominant follicles
at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG
injection (OF). Steady-state levels of ASB9 mRNA expression was greater in GC of OF
than in DF when analyzed by RT-qPCR (P < 0.0001). In follicular walls, analyses
showed a significant induction of ASB9 expression at 12 and 18 h (P<0.001), reaching
a maximum induction at 24 h post-hCG injection (P<0.0001) as compared to 0 h. These
results were confirmed in western blot analysis showing highest ASB9 protein amounts
at 24h post-hCG. Yeast two-hybrid screening of OF-cDNAs library with ASB9 showed
activation of the GAL4-responsive reporters suggesting physical interactions between
ASB9 and specific proteins in GC, which were confirmed by co-IP analyses. These
results support a physiologically relevant role of ASB9 in the ovulation process, and
provide, for the first time, insights into the mode of action and function of ASB9 in GC.
22
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Characterization of a new genetically-induced mouse model for PCOS
syndrome
Mostafa Esmael, Karl-F. Bergeron, Robert Viger, Catherine Mounier and Nicolas
Pilon
Université du Québec à Montréal (UQAM)
Polycystic ovarian syndrome (PCOS) is the 2nd most common cause of female
infertility. It has particular phenotypic characteristics including obesity, hormonal
imbalance, subfertility and multiple ovarian cysts. There is currently no cure for this
disease mainly because the underlying pathogenic mechanism is very poorly
understood.
Here, we report the generation of a new genetic mouse model of PCOS by random
genomic insertion of a Gata4p-RFP transgene. Importantly, we located the transgene
insertion site to a gene of unknown function on chromosome 2, called Gm10800.
Histological sections of mutant mouse ovaries revealed multiple follicular cysts, few
growing follicles and absence of mature oocytes compared to control tissues. A
hyperplasia of blood vessels consistent with PCOS was also observed in mutant
ovaries. In addition, we observed that about 50% of transgenic female mice gain weight
in an accelerated way compared to controls starting at 4 months of age. This weight
gain is mainly visceral fat and concomitant with subfertility. Taken together, these data
suggest that we might have generated the first mouse model that recapitulates the full
spectrum of PCOS phenotypes. In the upcoming years, we will use this unique genetic
model to better understand the pathogenic mechanism of PCOS and the role played by
the uncharacterized gene Gm10800. We hope this work will help finding a cure for
PCOS, a devastating condition for many women.
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23
Changes in the maternal immune system in late onset and postpartum
preeclampsia
Agnes Ditisheim, Adriana Carbonaro, Kevin Ippersiel, Ines Boufaied, Sylvie
Girard
Ste-Justine Hospital Research Center, Department of Obstetrics and Gynecology,
University of Montreal, Qc, Canada
Background: Preeclampsia (PE) is a syndrome specific to pregnancy and one of the
leading causes of maternal-fetal mortality and morbidity worldwide. Classically, PE
occurs during pregnancy but can also develop in the postpartum period. Inflammation is
involved in PE but the actual changes in the maternal immune system and association
with placental pathology are incompletely understood.
Methods: Women were recruited prospectively at the Sainte-Justine Hospital (N=49)
with either uncomplicated pregnancies with term delivery (Ctrl, N=20), PE after 34
weeks of gestation (PrePE, N=14) or normal pregnancies with postpartum occurrence
of PE (PostPE, N=15). Blood samples were collected prior to delivery and 24h after
birth.
Results: Delivery was associated with changes in the maternal circulating immune
system with elevated leukocytes and neutrophils numbers and decreased proportion of
lymphocytes and monocytes in Ctrl pregnancies. In PrePE, leukocytes and neutrophils
count were elevated before birth vs Ctrl and remained high after delivery, whereas
lymphocytes were significantly elevated after delivery (vs Ctrl). In PostPE, neutrophils
counts were similar to Ctrl before delivery but did not increased after and were lower
when PostPE occurred. The proportion of lymphocytes was elevated at diagnosis in
PostPE.
Conclusion: Altogether, this suggests that changes in the maternal immune system
occur around delivery in normal pregnancies and that these are abrogated in PrePE.
Differences are seen in women with seemingly normal pregnancies that will later
develop PostPE. Detailed understanding of these changes is important to address the
mechanisms of PE and for early identification of women at high-risk of PostPE.
24
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Uric acid-induced placental inflammation and dysfunction is dependent on
IL-1
Marie-Eve Brien, Adriana Carbonaro, Ines Boufaied, Sylvie Girard
Background: The alarmin uric acid is elevated in multiple pregnancy pathologies
including preeclampsia and fetal growth restriction. Uric acid is a known activator of the
inflammasome leading to interleukin (IL)-1 production in immune cells. The role of uric
acid in term placenta is, however, not fully understood. We addressed how uric acid
impacts
the
placenta
in
order
to
identify
new
therapeutic
targets.
Methods: Placental explants were made from human term placentas, from
uncomplicated pregnancies, obtained after caesarean section. Explants were treated
with either uric acid (monosodium urate, 100 g/ml), or IL-1 (10ng/ml) with or without
caspase-1 inhibitor (10 M) or IL-1 receptor antagonist (IL-1Ra). Supernatants and
explants were harvested at 24 or 48h and either frozen for protein analysis (ELISAs), or
fixed for histology.
Results: Uric acid induced the expression and secretion of IL-1 , IL-6 and MCP-1. The
secretion of IL-1
was caspase-1-dependent and IL-6 and MCP-1 levels are also
decreased following caspase-1 inhibition, which strongly suggests that they are
produced in an IL-1-dependent manner. The importance of IL-1 was confirmed using
IL-1Ra, which also abrogated IL-6 and MCP-1 secretion. Uric acid also affected
placental cell function, and induced cytotrophoblast apoptosis, which was protected by
IL-1 blockage (either caspase-1 inhibition or IL-1Ra).
Conclusion: In summary, this work suggests that uric acid induced inflammation in term
placental explants in an IL-1-dependent manner. Due to the known deleterious effects
of IL-1 elevation in pregnancy it offer an attractive therapeutic targets to protect the
placenta and subsequently the newborn.
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25
Transcriptome analysis of granulosa cells from poor follicle quality in
heifer and adult cows
David Landry, Christian Vigneault, Patrick Blondin, Marc-André Sirard
Université Laval, Alliance Boviteq, Alliance Boviteq, Université Laval
Bovine IVF has made substantial progress over the last few years but success is not
always 100%. It is known that the follicle differentiation status is associated to oocyte’s
competences. The recent ability to investigate how genes are expressed by
transcriptomics leads to new perspectives in exploring the link between follicle
environment and oocyte.
The major goal of this project is to define the best follicular scenario to improve oocyte
quality for a better success rate in the assisted reproductive technique. We
hypothesizes that a specific molecular signature is associated with the optimal
response for hormonal stimulation and indicative of corrective measure. Therefore
granulosa cells were obtained from mature and immature animals (n =100) and divided
according to blastocyst rates ( 0-25, 25-75 and over 75 %) of one cycle. Using this
information, a bioinformatic analysis (IPA) was done to identity the major reasons to
explain outcome and several gene pathways were identified particularly in relation to
the metabolism. Then a list of 21 biomarkers have been chosen to assess the timeless
of coasting and the individual response of each animal.
The main outcome of this project is to create a diagnostic tool to custom-design ovarian
treatment for individual cows or heifer’s when failure occurs.
26
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Effects of brominated flame retardants on KGN cells, a human granulosa
cell line
Pavine L.C. Lefevre 1, Mike Wade 4, Bernard Robaire 1,2, Cindy Goodyer 3 and
Barbara F. Hales 1
1
Depts of Pharmacology and Therapeutics 2 Obstetrics and Gynecology, and 3
Pediatrics, McGill University and the McGill University Health Centre, Montreal, QC,
H3G 1Y6, Canada. 4 Health Canada, Environmental Health Science & Research
Bureau, Ottawa, ON, K1A 0K9, Canada.
Brominated flame retardants (BFRs) are incorporated into consumer products to
prevent flame propagation. They leach out into the environment and are absorbed by
inhalation and ingestion. The detection of BFRs in human follicular fluid is associated
with pregnancy failure, raising serious questions regarding the effects of BFRs on
reproductive health, specifically on ovarian function. Here, we evaluated the effects of
BFRs on a human immortalized ovarian granulosa cell line (KGN cells). KGN cells were
exposed for 48 hours to 0-130 µM of a mixture of polybrominated diphenyl ethers
(PBDEs) reflecting the relative composition of the major class of BFR congeners in
North American breast milk and follicular fluid. Cell viability as demonstrated by
decreased cell density and increased propidium iodide staining was significantly
affected at 100 µM. Mitochondrial activity was evaluated by MTT and Mitotracker
assays and was significantly decreased at 100 µM and 130 µM, respectively. The
MitoSox assay and calcein staining revealed signs of oxidative stress in cells exposed
to 100 µM. Concentrations of progesterone and estradiol secreted in the medium were
significantly reduced by PBDE concentrations as low as 5 µM. Microarray analysis
showed that PBDEs decreased expression of INHA, and increased CYP1A1, FZD8 and
IL6 expression at 5 and/or 20 µM. Analysis of the expression of miRNAs revealed
significant changes in five miRNAs after exposure to PBDEs at 1, 5 and 20 µM; two of
these miRNAs, hsa-miR328 and hsa-miR1973, are reported to regulate CYP1A1 and
FZD8 expression. Our results indicate that BFRs are cytotoxic, induce oxidative stress,
disrupt steroidogenic activities and modulate specific gene expression in KGN cells.
Supported by grant RHF100625 from the IHDCYH/CIHR. PLCL has a postdoctoral
fellowship from the FRQS.
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27
Analyse épigénétique de semence et d'embryons provenant de boeufs
péripubères
Lambert Simon1, Blondin Patrick2, Vigneault Christian2, Sirard Marc-André1
1
Université Laval, département des sciences animales
Alliance boviteq
2
Chez le bovin mâle, il y a très peu d’études évaluant l’impact de la maturité sur la
qualité du sperme. Les informations sur l’effet que peut avoir un jeune géniteur sur
l’embryon sont rares. Jusqu’à présent, les seules observations portent sur des critères
morphologiques ou sur le taux de fécondation. La différence entre une défaillance
embryonnaire et une fécondation échouée est difficile à étudier in vivo. Par
conséquent, l’information sur l’impact de l’âge des taureaux sur la qualité de l’embryon
est manquante.
Ce projet vise à évaluer l’impact de l’âge sur la conservation de l’information
épigénétique porté par le spermatozoïde bovin. Il est connu qu’entre 10 et 16 mois, la
motilité, la viabilité ainsi que la morphologie des spermatozoïdes ne cesse d’évoluer. Il
est également documenté que l’utilisation de sperme d’animaux immatures pour
l’insémination génère un taux de succès moindre que la semence issu de mâles de 16
mois. Puisque le génome ne varie pas avec l’âge il est possible que le phénotype
observé soit dû à une fonctionnalité déficiente des spermatozoïdes (fécondation) ou à
une programmation épigénétique différente (embryon). L’analyse des profils de
méthylation en fonction de l’âge a permis de visualiser que des modifications sont
présentes sur l’ADN et que plusieurs sont conservées (fold change= 1.5, p-value=0.05)
chez l’ensemble des individus évalués (n=4). Ces sites représentent des biomarqueurs
qui permettront de suivre ces modifications dans l’embryon et ainsi de voir si elles
contribuent au plus faible taux de survie des embryons issus de ces taureaux.
28
e
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Disturbance of spindle function in aged mice oocytes is cytoplasm
dependent
Shoma Nakagawa, Greg FitzHarris
Centre Recherche CHUM, Université de Montréal
Chromosome segregation errors in mammalian oocytes increase with maternal age,
and are a major cause of infertility. Accurate chromosome segregation depends upon
organizing microtubules (MT) into a spindle. Here we use live 4D imaging and
micromanipulation approaches to investigate differences in spindle function between
oocytes from young mice (6weeks old) and aged mice (15 months). To directly
compare the MT generation dynamics, we expressed the MT plus-end marker
EB1:EGFP in young and aged mice oocytes to perform live 4D imaging. In young mice
oocyte, spindle assembly proceeded by MT assembly from spindle pole to spindle
midzone, as expected. Interestingly however, aged mice oocytes exhibited a multipolar
spindle formation. Multipolar spindle formation was confirmed by immunostaining with
pericentrin antibody, and by tracking MT plus-end movement using high-speed videomicroscopy. To discern whether the multipolar spindle formation in aged oocytes is
caused by defects in the chromosomes or MTs, we exchanged the young and aged
mouse oocyte cytoplasm and chromosomes using micromanipulation technique.
Multipolar spindle formation was observed only in reconstructed oocytes with aged
mouse oocyte’s cytoplasm, regardless of the source of the chromosomes. Consistent
with this, we show differences in MT dynamics in enucleated (chromosome-free)
oocytes between aged and young mice. We conclude that spindle function is disrupted
in aged oocytes, and that this defect is caused by altered microtubule generation rather
than altered chromosome structure. We are now investigating whether targeted
manipulation of protein levels within oocytes from aged mice can prevent age-related
spindle defects.
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29
Addition of oocyte can differentialy regulate cumulus expansion in the
presence of FSH or AREG
Paula Fernanda de Lima1, Christopher Alan Price2, José Buratini1
1
Sao Paulo State University - UNESP
Université de Montréal - UdeM
2
Current dogma is that, unlike in the mouse, in cattle the presence of the oocyte is not
required for expansion of cumulus-oocyte complex (COC). This accepted wisdom is
derived from studies with FSH-induction of expansion, which is not a physiological
event. We hypothesized that expansion in response to the physiological trigger
amphiregulin (AREG) is impacted by the presence of the oocyte in cattle.
COCs were aspirated from follicles 3-8 mm diameter, and COCs, oocytectomized
COCs ('OOX') or OOX plus denuded oocytes (DO) were matured in groups of 20 (n=4).
IVM was performed in TCM199 with either FSH (1µg/mL) or AREG (100 µg/mL).
Degree of expansion was visually assessed, and abundance of mRNA encoding HAS2,
COX2 and AREG in cumulus cells was measured by real-time RT-PCR.
Oocytectomy significantly reduced FSH-induced expansion (OOX 66.25%; COC
81.6%), and coculture of OOX with DO restored expansion to levels not different from
COC controls. Oocytectomy had no effect on AREG-induced expansion. Levels of
mRNA encoding AREG and COX2 were significantly higher in OOX compared with
COC with both FSH and AREG, and coculture with DO restored mRNA levels to those
of COC controls under AREG not FSH stimulation. Abundance of mRNA encoding
HAS2 was decreased in the presence of either FSH or AREG by the absence of the
oocyte, and restored to control levels by addition of DO only under stimulation by
AREG. These data suggest that the stimulus used to provoke COC expansion alters
the ability of the oocyte to regulate cumulus cell function.
30
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Identification and localization of Phosphodiesterase 10A (PDE10A) in bull
spermatozoa
MARECHAL Loïze 1 2, GOUPIL Serge 1 2, FLEYS Charlotte 1 2, RICHARD François J.
1
, LECLERC Pierre 1 2.
1
2
Centre de recherche en biologie de la reproduction, Université Laval,
Centre de recherche du CHU de Québec-CHUL, Québec, QC, Canada G1V 4G2
Cyclic AMP (cAMP) is essential in sperm intracellular signalling. It is implicated in
motility and in sperm capacitation and acrosome reaction, two events essential for
fertilization. Cellular cAMP concentration is principally regulated by the adenylyl
cyclases that synthesize cAMP from ATP, and the phosphodiesterases (PDE), which
degrade cAMP into 5’AMP. PDE10A is one of the most recently discovered PDE.
Although it can degrade cAMP and cGMP it is more efficient on cAMP. PDE10 mRNA
is expressed principally in the brain where it is well studied, due to its implication in
neurodegenerative diseases, but also in the testis. We demonstrated the presence of
Pde10A transcript variants X3 and X5 in the bovine testis and the isoform X4 in
spermatozoa. Our goal is therefore to determine whether this enzyme plays a role in
bovine spermatozoa and to understand how it is regulated. With immunoblots
procedures, PDE10 was detected principally in sperm cytosol and, to a lesser extent,
membranes.
PDE10
was
localized
in
sperm
acrosomal
region by
indirect
immunofluorescence procedure. After separation of cryopreserved sperm by Percoll
density gradient (healthier, highly motile cells are denser), the abundance of PDE10
was related to sperm quality. However, PDE10 inhibitors did not significantly affect
sperm motility, when assessed with Computer Assisted Sperm Analyzer (CASA),
suggesting that this enzyme is not involved in this sperm function. Our next step is to
determine if PDE10 translocates between the cytosol and membranes during
capacitation and mechanisms involved. PDE10 activity will also be assayed in
spermatozoa incubated under capacitating conditions.
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31
In utero exposure to an environmentally relevant dose of DEHP targets the
adult adrenal gland for endocrine disruption
Sunghoon Lee1, Daniel B. Martinez-Arguelles2, Enrico Campioli2, Vassilios
Papadopoulos2
1
Department of Biochemistry and the Research Institute of the MUHC
Department of Medicine and the Research Institute of the MUHC
2
Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in the production of
polyvinyl chloride (PVC) products. Because DEHP is not covalently bound to the
material, it is able to leach out and contaminate the environment. In humans, DEHP
has been detected in umbilical cord blood and amniotic fluid suggesting exposure
begins at the fetal stage.
Previous work in the rat model has revealed that an fetal exposure to >100 mg
DEHP/kg/day results in a reduction of serum testosterone and aldosterone and affects
various adrenal pathways including the potassium, angiotensin, and de novo
cholesterol biosynthesis pathways. Furthermore, transcriptomic studies in the adrenal
gland showed fetal DEHP exposure deregulates the adrenal PPAR pathways at doses
as low at 1 mg DEHP/kg/day. Similar changes in DNA methylation patterns were found
at the 1 mg dose suggesting that a low and environmentally relevant dose of DEHP
may constitute the first hit to the adrenal epigenome.
We hypothesize this first hit of DEHP may leave the adrenal gland vulnerable to
adverse effects from a secondary stressor later in life. To this end, male offspring
exposed in utero to 1 mg DEHP/kg/day received an acute treatment of various stressor
compounds from PND54-59. Specific treatment with the PPAR antagonist, T0070907,
resulted in a significant decrease of serum aldosterone in males exposed to 1 mg
DEHP. Additionally, gene expression studies of the adrenal reveal altered expression of
Kcnk5 and the nuclear receptors, Rxr
and Rxr . Together, these results suggest in
utero exposure to environmentally-relevant doses of DEHP predisposes the adult zona
glomerulosa to endocrine disruption.
32
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Binucleation in the Preimplantation Mouse Embryo Cause Higher
Frequency of Chromosome Segregation Errors
Candice Taguibao, Greg Fitzharris
University of Montreal
Embryos from routine fertility treatments frequently contain binucleated cells, which are
presumed to be tetraploid. Whilst binucleated cells are considered to be indicative of a
poor quality embryo, no mechanistic studies have addressed the impact of binucleation.
Here, to address this, we developed a method to induce binucleated blastomeres in 4cell-stage embryos by treating embryos with the cytokinesis inhibitor, cytochalasin B,
and used live-cell fluorescence and fixed imaging approaches to observe the impact of
binucleation. In contrast to somatic cells, which possess a tetraploidy cell-division
checkpoint, we found that embryos with binucleated cells continue to divide and
develop apparently normally. Thus early mouse embryos do not have an effective
binucleation/tetraploidy checkpoint. However, we observed that previously binucleated
embryos exhibited a large number of micronuclei (17%, compared to 5 %, in controls),
which is suggestive of increased chromosome segregation error. Therefore, to directly
assess the fidelity of chromosome segregation, embryos were labeled with H2B-RFP
and observed using low-damage long-term 4D confocal analysis. Strikingly, binucleated
cells displayed a variety of chromosome segregation defects, that were generally not
seen in mononucleated controls, including lagging anaphase chromosomes leading to
micronuclei (36%), chromosome misalignment (23%), and more severe missegregation
phenotypes (23%). Thus, chromosome segregation errors are more common in
binucleated cells than in mononucleated cells (P=0.012). We therefore propose that
embryo binucleation may be an unforeseen stepping stone in the generation of embryo
aneuploidy.
Candice Taguibao was an RQR-CREATE scholarship recipient.
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33
Paternal
Lifetime
Folate
Deficiency
and
Supplementation
Effects
Reproductive Health and Induces Aberrant Sperm DNA Methylation
Lundi Ly1,2, Donovan Chan2, Mylene Landry2, Nathalie Behan3, Amanda
MacFarlane3, and Jacquetta Trasler1,2,4
1
Department of Human Genetics, McGill University, Montreal QC, Canada,
Research Institute of the McGill University Health Centre at the Glen Hospital, McGill
University, Montreal QC, Canada
3
Nutrition Research Division, Health Canada, Ottawa, ON
4
Departments of Pediatrics & Pharmacology and Therapeutics, McGill University,
Montreal QC, Canada
2
Epigenetic modifications such as DNA methylation (DNAme) have essential roles in
developmental programs. Disruptions in gamete epigenetic reprogramming are
associated with adult disease and transgenerational effects. The fetal period is the key
to DNAme pattern acquisition in developing male germ cells and adequate supply of
methyl donors is required. Previous studies showed that postnatal folate deficiency
(FD) or supplementation (FS) could alter the sperm epigenome. This study aimed to
determine if lifetime FS/FD induces aberrant epigenetics in germ cells detrimental to
offspring. Female mice (n=15) were placed on one of four amino acid controlled diets:
-supplemented diet (20FS),
-
-deficient diet (7FD). Females were
mated to produce F1 litters whose germ cells were exposed to the FA diets throughout
development. F1 males were weaned onto their respective prenatal diets. F2 and F3
litters, unexposed to the folate treatments, were subsequently generated. Tissues of
epresentation
bisulfite sequencing (RRBS) was performed. Offspring death was observed in F2 litters
from 7FD and 20FS exposed fathers and resulting in significantly smaller litters at
weaning, in comparison to F2 litters from FCD exposed fathers. Preliminary DNAme
results from F1 sperm (n=5) demonstrate that perinatal exposure to 7FD, 10FS, and
20FS diets resulted in 153, 132 and 114 differentially methylated loci, respectively,
affecting intergenic and genic regions. These results suggest that lifetime FD/FS can
impact sperm development and offspring health.
(Supported by CIHR and CEEHRC).
34
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Binder of sperm gene deletion by CRISPR genome editing and the effects
on fertility
Marzieh Eskandari-Shahraki1,2, Bruno Prud’homme1 and Puttaswamy
Manjunath1,2
1
Maisonneuve-Rosemont Hospital Research Centre, Montréal, Québec, Canada
Department of Medicine and of Physiology, Faculty of Medicine, University of
Montréal, Montréal, Québec, Canada.
2
Fertilization occurs when receptor proteins on sperm and egg surface recognize each
other and interact. Various molecules are implicated in epididymal sperm maturation
and capacitation which are the two most important processes gained inside the male
and female reproductive tracts. A family of protein secreted in epididymis has been
identified in our laboratory, named Binder of Sperm (BSP) proteins, which induce
important changes in the lipid composition of the sperm membrane leading to
capacitation. One form of this family is expressed in the human epididymis (BSPH1),
and two forms are expressed in the murine epididymis (BSPH1 and BSPH2). In order
to elucidate the functions of these BSP proteins on fertilization in vivo, mice carrying
single and double mutations in Bsph1 and Bsph2 genes were generated using a
revolutionary new technology, the Clustered Regularly Interspaced Short Palindromic
Repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) systems. The progeny
carrying the mutated Bsph1 and Bsph2 alleles were screened by PCR and the targeted
deletion and/or recombination by guided RNA were determined. We have begun mating
Bsph2 null mice with wild-type mice. Bsph1 null mice are currently being generated. In
due course, we will attempt to generate mice lacking both Bsph1 and Bsph2 genes. We
will evaluate fertility and sperm functions (motility, viability, capacitation, acrosome
reaction) of those single and double knockout mice, as well as morphology of the
reproductive organs. This model should help elucidate the in vivo functions of mouse
BSP proteins, which are the counterparts of the human (BSPH1). (Supported by CIHR)
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35
Early growth response 1 (EGR1) activates fibroblast growth factor 8
(FGF8) signaling in bovine granulosa cells
Peng Han1,2, Christopher A. Price2
1
Northwest A&F University
Université de Montréal
2
Fibroblast growth factor 8 (FGF8) alters ovarian function by increasing proliferation,
reducing apoptosis and decreasing steroidogenesis in granulosa cells. Early growth
response 1 (EGR1) is a transcription factor that mediates protein-protein interactions
and it is rapidly up regulated in an acute manner after granulosa cells have been
exposed to FGF8. We hypothesis that EGR1 has a key role in granulosa cells response
to FGF8.
Bovine granulosa cells from follicles between 3-5mm were placed in a serum-free
culture. According to previous experiments, granulosa cells were challenged with EGR1
adenovirus at a MOI of 200 during 18 hours, relative mRNA abundance was measured
by real-time PCR and major pathways assessed by western blot. Overexpression of
EGR1 resulted in an increased in phosphorilation of ERK1/2 in the same patter like
FGF8 treatment. It also increased mRNA abundance for FGF8 downstream genes such
as EGR3, SPRY 1, SPRY2, SPRY4, NR4A1 NR4A2, NR4A3 and GADD45B. Moreover
EGR1 had the ability to dreacreased the steroidogenic enzyme CYP19. It also
increased phosphorilation of p38. We conclude that EGR1 has a main role in FGF8
function in granulosa cells.
36
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Activin induction of follicle-stimulating hormone expression requires
protein de-acetylation in immortalized gonadotrope-like cells
Gauthier Schang, Daniel J. Bernard
Department of Pharmacology and Therapeutics, McGill University, Montreal QC,
Canada
Follicle-stimulating hormone (FSH), a product of pituitary gonadotrope cells, regulates
ovarian follicle development in females and spermatogenesis in males. FSH is a
dimeric hormone, with its biological activity determined by its
subunit. Hypothalamic
gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are considered the
primary drivers of FSH subunit synthesis. Comparatively, mechanisms of GnRH action
on FSH synthesis are poorly understood. Recently, however, a model was proposed in
which GnRH induces the nuclear export of histone deacetylases (HDACs), relieving
their tonic repression of the FSH subunit gene (Fshb) in the immature gonadotropelike cell line, T3-1. Treatment with the Class I/II HDAC inhibitor, trichostatin A (TSA),
similarly stimulated Fshb mRNA levels in these cells, which do not express this gene
under basal conditions. Our attempts to reproduce these findings have proven
unsuccessful. However, in the course of our investigations, we made the serendipitous
discovery that TSA inhibited activin-induction of Fshb mRNA expression in the more
mature gonadotrope-like cell line, L T2. We observed similar results when treating cells
with entinostat, a class I HDAC inhibitor. Activins signal via SMAD proteins and the
forkhead transcription factor FOXL2 to regulate Fshb expression. TSA failed to block
activin stimulated SMAD2 phosphorylation, suggesting that HDAC inhibition has its
effects at the post-receptor level. We are currently testing the hypothesis that following
HDAC inhibition, FOXL2 becomes hyper-acetylated in its forkhead domain, blocking its
DNA binding activity. In summary, our data show that class I HDACs play a necessary
role in activin induction of the Fshb gene in immortalized gonadotrope-like cells.
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37
Specific
alterations
in
the
histone
modification
landscape
as
a
consequence of transient Dnmt1 deficiency in mouse ES cells
Lisa-Marie Legault (1), Maxime Caron (2), Perrine Gaub (1), Rich Chaillet (3), Nicolas
Gévry (4), Serge McGraw (1).
1
Centre de recherche du CHU Ste-Justine, Département Obstétrique-Gynécologie,
Université de Montréal.
2
Centre de recherche du CHU Ste-Justine
3
Department of Microbiology and Molecular Genetics, University of Pittsburgh
4
Département de Biologie, Université de Sherbrooke.
Genome-wide demethylation and remethylation of DNA during early embryogenesis is
essential for mammalian development and genome integrity. Through mostly unknown
mechanisms, imprinted germline differentially methylated domains (gDMDs) and
gDMDs-like regions are able to retain their methylation profiles during the
reprogramming period. However, a temporary lack of DNMT1 activity triggers the
inherited loss of gDMDs and gDMD-like DNA methylation profiles, whereas other
regions of the genome are able to recover original DNA methylation levels. We believed
that following the transient inactivation of DNMT1, rearrangements in the histone mark
landscape occur and prevent the proper recruitment of DNMT1 in regions with inherited
DNA methylation loss. Here, we used an embryonic stem (ES) cell line, in which Dnmt1
is regulated by a tet-off system (Dnmt1tet/tet ES cells), to emulate lack of DNMT1
activity in embryonic cells and trigger inherited DNA methylation loss on the genome.
We then generated DNA methylation profiles using reduced representation bisulfite
sequencing (RRBS) and histone modifications (H3K4me3, H3K27me3, H3K27ac)
profiles using chromatin immunoprecipitation-sequencing (ChIP-Seq) to investigate the
specific interactions between histone marks and aberrant DNA methylation. We found
that the inherited loss of DNA methylation is linked with changes in histone marks.
Especially with the occurrence of H3K4me3, an active mark that can impede the
recruitment of DNMTs; suggesting that alterations in the histone modification landscape
may prevent the proper recruitment of DNMT1. The present study presents new
perspectives on how alterations in normal DNA methylation and histone modification
cross-talk could explain epigenetic inherited dysregulation events during embryo
development.
38
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Characterization of a novel and differentially expressed long non-coding
RNA (lncRNA) in granulosa cells of bovine dominant follicle
Jacques Lussier and Kalidou Ndiaye
Centre de recherche en reproduction animale (CRRA), Département de biomédecine
vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, StHyacinthe, Québec, Canada, J2S 2M2, Canada
In a previous study, we identified differentially expressed genes in granulosa cells (GC)
establishment of a subtracted cDNA library. The current study aimed at characterizing
the full-length cDNA of a fragment that did not match expressed sequence tag (EST) in
GenBank (CF752000), and analyzing its mRNA regulation. The EST fragment (377 bp)
was used as probe to screen a GC cDNA library. The 2151 bp cDNA clone
characterized has a polyadenylation signal followed by a polyA tail but no open reading
frame, which matched the features of a long non-coding RNA (lncRNA). BLAST
analysis showed localization to an intergenic region of chromosome 7 between JAK3
and RPL18A genes. Analysis of mRNA expression was compared among different
follicular stages and in various tissues. Granulosa cells were obtained from small
follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), ovulatory follicles
24h following hCG injection (OF) and CL at day 5. Levels of mRNA were greater in GC
of DF than in SF, OF or CL when analyzed by semi-quantitative RT-PCR (P<0.0001). A
rapid decline at 6h post-hCG injection was observed as compared to 0h in follicular wall
samples (P<0.0001). Comparison of mRNA expression pattern among various bovine
tissues showed expression only in GC of DF. We report for the first time the
characterization of a differentially expressed lncRNA in GC of dominant follicle. Further
investigation will be required to address its physiological role.
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39
Effect of Diet-Induced Magnesium Deficiency on Bone Health and Glucose
Metabolism in Obese-Prone and Obese-Resistant Rats
Sophia Rahimi (1, 2), Christopher Lavergne (1, 3), Hiba Rachid (1, 4), Nina A. Vu (1, 2),
Louise J. Plouffe (1), Eleonora Swist (1) and Jesse Bertinato (1, 2)
1
Nutrition Research Division, Health Products and Food Branch, Health Canada
Department of Biochemistry, Microbiology & Immunology, University of Ottawa
3
Department of Biology, University of Ottawa
4
Food Science and Nutrition Program, Carleton University
2
Inadequate intake of magnesium (Mg) is common amongst Canadians and may have
negative effects on bone health and glucose metabolism. This study investigated the
effect of low dietary Mg intake on indicators of bone health and glucose metabolism;
and the possible modifying effect of obesity. Obese-prone (OP; n=25/diet group) and
obese resistant (OR; n=25/diet group) rats were fed a high-fat diet containing low (LMg;
0.116±0.001 mg/g diet) or normal (NMg; 0.516±0.003 mg/g diet) Mg for 14 weeks. At
week 13, an oral glucose tolerance test (OGTT) was performed on a subgroup of the
rats (n=10/diet group). Rat strain differences were observed for indicators related to
bone health and glucose metabolism. Rats fed the LMg diet exhibited lower (P<0.05)
urine and femur Mg concentrations than rats fed the NMg diet indicating depressed Mg
status. OP and OR rats fed the LMg diet demonstrated lower body weight and lean
body mass. Urine deoxypyridinoline (DPD):creatinine ratio, serum osteocalcin and
femur density were unaffected by diet. OR rats fed the LMg diet had lower femur width,
weight and volume measurements. Serum parathyroid hormone (PTH) concentration
was lower in OR rats fed the LMg diet and was positively correlated with percent body
fat in these rats. Glucose and insulin area under the curve in the OGTT were similar
deficient in Mg does not affect glucose homeostasis but induces changes related to
bone in rats.
40
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Nr5a2 regulates granulosa cells proliferation in vitro and in vivo
Marie-Charlotte Meinsohn, Kalyne Bertolin, Fanny Morin, Vickie Roussel and
Bruce D. Murphy
Centre de recherche en reproduction animale (CRRA), Faculté de médecine
vétérinaire, Université de Montréal
The orphan nuclear receptor Nr5a2 is essential for fertility as mice with Nr5a2 depleted
in granulosa cells are infertile due to multiple causes. We hypothesized that Nr5a2 is
important for granulosa cell proliferation. We generated granulosa-specific knockout
mice (Nr5a2f/fAmhr2Cre/+; cKO) with Nr5a2 depletion from preantral follicles forward.
Immature cKO and control mice were treated with eCG, and ovaries or granulosa cells
were isolated 44h later. We studied the proliferative competence of cKO granulosa cells
by injecting mice with bromodeoxyuridine 20h after eCG. The replicating cells were
detected by immunofluorescence and quantified. Results showed that the number of
proliferating cells was dramatically reduced (p<0.001) in the cKO ovary. To establish
how Nr5a2 depletion in granulosa cells affects genes involved in proliferation we
determined transcript abundance by qPCR. There was a greatly reduced (p<0.05)
abundance of transcript of Ccnd1, Ccnd2, Ccne1, Ccne2, E2f1, E2f2 in cKO compared
to control. To further explore this phenomenon, we reproduced this Nr5a2 cKO model
in vitro. We collected granulosa cells from Nr5a2f/f mice at 44 h after eCG treatment.
These cells were infected with adenovirus Cre and harvested after 24h. We showed the
same reduction of the cyclins and E2f transcripts (p<0,05), with the exception of cyclin
E, and, further, a downregulation in Rb1 (p<0,05) confirming the trend observed in vivo.
We conclude that Nr5a2 is essential for proper proliferation of granulosa cells and its
depletion has an impact on downstream targets such as cyclins and transcription
factors involved in the G1/S phase transition of cell cycle in vivo and in vitro.
Funded by a grant to BDM from CIHR
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Exercise training improves the fertility of female mice
Olga Asaftei1,2, Aida Kasaei Roodsari1,2, Sonia Kajla1, Bruce Murphy4, Julie
Lavoie1,2,3
1
CRCHUM
Département de Kinésiologie, Université de Montréal,
3
CRDM, Montréal;
4
CRRA, Université de Montréal, Saint-Hyacinthe, Québec
2
Introduction: Exercise training has been shown to reduce the prevalence of pregnancy
disorders such as gestational diabetes and preeclampsia. Dr Lavoie’s laboratory has
developed and characterised a mouse model of preeclampsia superimposed on chronic
hypertension,
the
R+A+
mice,
which
overexpress
both
human
renin
and
angiotensinogen. Sedentary R+A+ mice have smaller litter size when compared to
exercise-trained mice. Therefore, exercise training could potentially improve fertility in
this
model
as
well
as
ameliorate
infertility
due
to
other
problems.
Hypothesis: We hypothesise that exercise training restores fertility by modifying the
process of embryo implantation in the R+A+ mice.
Methods: R+A+ mice and their non-transgenic littermates were given access to a freerunning wheel four weeks prior to mating and throughout pregnancy. Mice were
sacrificed on the 6th day of gestation and embryo implantation sites were counted and
measured.
Results: Interestingly, the R+A+ mice tend to have a smaller number of implantation
sites compared to their non-transgenic littermates. Moreover, exercise training
increases
the
number
of
implantation
site
in
both
phenotypes.
Conclusion: Our preliminary results demonstrate that exercise can improve the fertility
of female mice. However, its effect on the fertility on R+A+ females is still to be
determined.
42
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The alarmin uric acid-induced fetal growth restriction: new animal model
of non-infectious inflammation during pregnancy
Marie-Eve Brien, Ines Boufaied, Sylvie Girard
Introduction: Inflammation occurring during pregnancy has deleterious effects on fetal
development. Infection is a cause of inflammation but is not detectable in most cases of
pathological pregnancies whereas inflammation is still detectable. Alarmins are
endogenous inducers of inflammation elevated in pathological pregnancies and another
plausible cause of inflammation. Currently, preclinical models of prenatal inflammation
use infectious stimuli. Our objective was to develop a model of non-infectious
inflammation during pregnancy.
Methods: We used pregnants Sprague-Dawley rats, injected intraperitoneally with the
alarmin uric acid (250, 500 or 1000 g/kg/12h) or vehicle from gestational day (G)18 to
21. Potassium oxonate (uricase inhibitor) was also injected (125mg/kg/day).
Lipopolysaccharide (LPS) a pathogenic stimulus, was administered for comparison. On
G22, ceasarean section was performed and placentas collected and either frozen for
protein analysis (ELISAs) or fixed for histology.
Results: Administration of uric acid at the end of gestation in rats led to FGR, with over
45% of the pups with fetal weight below the 5th centile of pups from vehicle-injected
dams. Significantly elevated levels of IL-1 , IL-6 and TNF- were found in placentas
from uric acid exposed dams. Histological analysis revealed clusters of immune cells
on the fetal side of the placenta within the labyrinth, even though placental morphology
remained intact.
Conclusion: In summary, we found that elevated uric acid is a strong inducer of
placental inflammation in rats leading to FGR. This new model of non-infectious
inflammation during gestation will be used to address the placental mechanisms
involved and test new therapeutic strategies.
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43
Rôle des microARNs miR-34b/c-miR-449 dans la régulation de gènes
épididymaires
Olivia Jerczynski1, Lore-Anne Carré1, Wei Yan2, Clémence Belleannée1.
1
2
CHU de Québec, Université Laval, Québec, Canada.
University of Nevada, Reno, USA.
L’épididyme est régulé par les microARNs (miARNs) qui sont d’importants régulateurs
de l’expression génique et de la fertilité mâle. Le but de notre étude est d’évaluer le rôle
de miR-34b/c et miR-449 dans la régulation de fonctions épididymaires à partir de
souris infertiles doublement invalidées pour ces miARNs (dKO). La caractérisation de
l’expression de miR-34c/miR-449 a été réalisée dans le testicule et dans les trois
grandes régions anatomiques de l’épididyme à partir de souris contrôles. La queue de
l’épididyme a été aussi microperfusée et le fluide collecté et incubée sous différentes
conditions pour déterminer la localisation sub-extracellulaire de ces miARNs. Après
extraction et purification, les niveaux d’expression des miARNs ainsi que ceux de leurs
gènes cibles ont été évalués par PCR en temps réel. Alors que miR-449/miR-34c sont
enrichis dans les extraits de testicule, ils sont aussi retrouvés dans la lumière de
l’épididyme. L’expression de certains gènes cibles, incluant le gène codant pour la VATPase, est réduite dans la région proximale de l’épididyme de souris dKO vs Ctrl. Les
changements protéiques et fonctionnels sont en cours d’évaluation. En conclusion,
miR-449 et miR-34c sont des facteurs testiculaires transportés par la fluide dans
l’épididyme et dont l’invalidation modifie l’expression de gènes dans cet organe.
Puisque 30% des cas d’infertilité masculine sont idiopathiques et susceptibles de
résulter de dysfonction de l’épididyme, les miARNs sont des cibles potentielles pour le
diagnostic et traitement de problèmes d’infertilité.
44
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Effet d’une exposition in-utero à l’éthinyl-œstradiol sur le développement
des gonocytes de rats mâles
Bintou Gaye (1, 2, 3), Géraldine Delbès (1, 2, 3)
1
INRS-Institut Armand Frappier (1), Laval, Qc, Canada ;
Centre de recherche BioMed (2), Montréal, Qc, Canada;
3
Réseau Québécois en Reproduction (3), Québec, Qc, Canada.
2
L’infertilité masculine représente un problème de santé publique. Certains cas
pourraient être dus à une exposition aux xénoestrogènes lors du développement fœtal
du testicule. Il a été montré que les gonocytes, précurseurs fœtaux des cellules
souches spermatogoniales, sont sensibles aux œstrogènes exogènes spécifiquement
pendant la phase de prolifération fœtale. Notons que cette phase coïncide avec la
reprogrammation épigénétique caractérisée par la reméthylation de l’ADN et des
variations de méthylation de lysines sur l’histone H3.
Notre hypothèse est que les xénoestrogènes altèrent l’expression des gènes, en
affectant
la
méthylation
de
l’ADN
et
les
modifications
des
histones.
Pour cette étude, nous utilisons un modèle d’exposition in-vivo de rats transgéniques
exprimant la GFP uniquement dans les cellules germinales permettant de purifier
spécifiquement les gonocytes. Des rattes gestantes (n=4) sont traitées par gavage à
2µg/kg/jour d’éthinyl-œstradiol (EE2), du jour 13 au jour 19 de gestation, incluant donc
la phase de prolifération et de reprogrammation des gonocytes. Les rattes sont
sacrifiées à 20 jours de gestation.
Nos résultats sur le nombre de fœtus par portée, le poids des fœtus et des placentas et
les distances ano-génitales montrent que l’EE2 n’a pas d’effet sur le développement
des fœtus mâles. Nos analyses histologiques montrent aussi que l’EE2 n’a pas d’effet
sur le développement testiculaire fœtal. Finalement, nous étudierons l’effet de l’EE2 sur
l’expression des gènes par puce à ADN à partir des ARN extraits de gonocytes triés.
Cette étude permettra ainsi la compréhension des effets génotoxiques des
xénoestrogènes sur la mise en place des cellules germinales mâles.
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Caractérisation des voies de signalisation PKA et PKC dans les cellules
de la granulosa humaine en culture
Patricia Tremblay, Isabelle Dufort, Marc-André Sirard
Centre de Recherche en Biologie de la Reproduction, Département des Sciences
Animales, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval,
Sainte-Foy, Québec, Canada, G1V 0A6
La compétence ovocytaire peut se définir comme étant la capacité de l’ovocyte à
compléter la maturation, à réussir l’étape de fécondation et à atteindre le stade de
blastocyste. L’acquisition de la compétence se fait en étroite collaboration avec les
cellules somatiques du follicule. Les cellules du cumulus et de la granulosa supportent
la construction de l’ovocyte alors que l’ovocyte en retour influence le patron
d’expression de gènes biomarqueurs dans les cellules de la granulosa. Des études
antérieures laissent croire que l’hormone folliculo-stimulante (FSH) et l’hormone
lutéinisante (LH) joueraient un rôle primordial dans l’acquisition de la compétence
ovocytaire via les voies de signalisation PKA et PKC dans la granulosa. Le projet
consiste en une analyse transcriptomique des gènes différentiellement exprimés entre
deux traitements dans les cellules KGN, une lignée cellulaire secondaire de la
granulosa humaine, avec comme objectif de faire la caractérisation des voies de
signa
-myristate 13-acetate (PMA), des activateurs
connus des voies PKA et PKC respectivement. Les résultats de l’analyse du
transcriptome par biopuces montrent plus de 2000 gènes différentiellement exprimés
(FC de 1,5 avec P de 0.05) entre les traitements ce qui permettra possiblement
l’établissement d’un profil génique spécifique, d’une signature unique à chacune de ces
deux voies de signalisation importantes dans la granulosa.
46
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La culture organotypique : un bon modèle d’étude de la reprogrammation
épigénétique dans les cellules germinales fœtales du rat mâle
Arlette Rwigemera, Géraldine Delbès
INRS-Institut Armand-Frappier, Laval (Québec), Canada
La reprogrammation épigénétique est une étape critique du développement fœtal des
cellules germinales du testicule. Elle est caractérisée par le remodelage de différents
marqueurs épigénétiques dont la reméthylation de l’ADN (5mC) et les variations de
modifications post-traductionnelles de l’histone H3. Ce processus est important pour
effacer d’éventuelles épimutations et les marqueurs épigénétiques acquis durant cette
phase peuvent avoir un rôle déterminant sur le devenir des cellules subséquentes et
éventuellement sur la qualité des spermatozoïdes. La culture organotypique est un bon
modèle de développement ex-vivo du testicule fœtal, car elle permet de conserver la
cinétique de développement du tissu et de préserver l’interaction entre les cellules.
Notre hypothèse est que ce modèle permet de reproduire la dynamique in vivo de la
reprogrammation épigénétique dans les cellules germinales. Ainsi, des testicules
prélevés à 16 jours post-coïtum ont été mis en culture pendant 5 jours dans différentes
conditions : deux supports (filtre ou insert) et avec/sans FBS. Sur insert et sans FBS,
les résultats obtenus par immunofluorescence montrent une augmentation dans le
temps de la 5mC et du marqueur d’histone H3K4me3 tandis que le marqueur
H3K4me2 augmente puis diminue comme in vivo. Nous montrons, ainsi, que ce
modèle permet de rétablir la dynamique de changement de trois marqueurs de la
reprogrammation épigénétique. Ces résultats suggèrent que la culture organotypique
peut reproduire le processus de reprogrammation épigénétique. Elle serait donc un bon
modèle pour étudier les effets de polluants environnementaux sur la mise en place des
marques épigénétiques dans les cellules germinales fœtales.
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Régulation de l'activité du promoteur distal 1b du gène Gata4
Vanessa Théberge1,2, Francis Bergeron1,2, Robert Viger1,2,3
1
Reproduction, santé de la mère et de l’enfant, Centre de recherche du CHU de
Québec (CHUL), Québec, Canada;
2
Centre de recherche en biologie de la reproduction (CRBR)
3
Faculté de médecine, Département d’obstétrique, gynécologie et reproduction,
Université Laval, Québec, Qc, Canada
Le facteur de transcription GATA4 joue un rôle clé dans la morphogenèse et le
fonctionnement de plusieurs organes, notamment les gonades. Le gène Gata4 est
régulé selon deux transcrits majeurs et indépendants qui diffèrent selon la séquence de
leur premier exon, soit l'exon proximal 1a (promoteur 1a) et l'exon distal 1b (promoteur
1b). L’objectif du projet de recherche est d'étudier la régulation du promoteur 1b du
gène Gata4 in vivo. Pour ce faire, nous avons généré un modèle de souris
transgénique exprimant le marqueur fluorescent GFP sous le contrôle de 2 kb du
promoteur Gata4 distal 1b murin. Notre hypothèse est que ce promoteur contribue au
profil d'expression endogène de la protéine GATA4 dans certains tissus. Nous avons
analysé la présence de la protéine GFP dans différents organes par épifluorescence,
par immunohistochimie et par Western Blot aux stades e13,5, e16,5, P35 et P90. Selon
nos résultats, cette séquence de 2 kb n’est pas suffisante pour diriger l’expression du
transgène chez la souris. D’autres éléments de régulation nécessaires à la transcription
endogène du gène Gata4 sous le promoteur 1b sont probablement manquants. Afin
d’identifier ces éléments, nous avons utilisé des outils bio-informatiques tels que la
base de données Fantom5 ainsi qu’un algorithme IM-PET. Quatre enhancers
potentiellement impliqués dans la régulation du promoteur Gata4 1b ont ainsi été
identifiés. Chacun de ces enhancers a été cloné devant un gène rapporteur luciférase
afin de mesurer leur activité en lignées cellulaires. Les résultats obtenus nous
permettront ainsi d’ouvrir une voie dans la compréhension des mécanismes entourant
la régulation transcriptionelle du gène Gata4.
48
e
th
Impact de la génistéine sur la régulation de p53 au cours de la
décidualisation
Roch Tremblay, Carol-Ann Joly, Jessica Dion, Valérie Leblanc, Céline Van
Themsche
Université du Québec à Trois-Rivières (UQTR), Groupe de Recherche en Signalisation
Cellulaire (GRSC)
Le soya est de plus en plus présent dans la diète occidentale. Il contient une grande
quantité de phytoestrogènes, principalement la génistéine. Ce composé est susceptible
d’interférer avec tous les processus impliquant des estrogènes par sa capacité de lier
et d’activer les récepteurs à l’estradiol des cellules humaines. À chaque cycle
menstruel, sous l’action des œstrogènes entre autres, les cellules endométriales
stromales (CES) sont le site d’une décidualisation, un processus de différenciation
cellulaire essentiel à l’implantation de l’embryon puis au maintien de la grossesse.
Notre hypothèse est que l’exposition à la génistéine pourrait interférer avec certains
mécanismes moléculaires activés pendant la décidualisation. Dans un modèle in vitro
de CES humaines immortalisées, nous avons caractérisé une accumulation au fil de la
décidualisation du facteur de transcription p53, ainsi qu’une hausse de l’expression de
cette protéine sous l’action de la génistéine. Notre objectif est maintenant d’investiguer
si l’exposition à la génistéine peut modifier la quantité et l’activité de p53 dans les CES
au cours de la décidualisation. Nous avons obtenu des résultats préliminaires qui
suggèrent que pendant la décidualisation des CES induite par l’AMPc et l’acétate de
medroxyprogestérone, l’activité de p53 serait modifiée par l’ajout de génistéine. Nos
travaux constituent une première étape très importante permettant d’évaluer l’impact
d’un facteur alimentaire sur un processus reproductif et nous planifions réaliser des
expérimentations dans un modèle de décidualisation chez le rat pour déterminer si
l’exposition à la génistéine interfère avec la décidualisation et la fonction reproductrice
in vivo.
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49
Contrôle de la stimulation ovarienne par diagnostique de la qualité
folliculaire
Chloé Fortin, Marc-André Sirard
Université Laval, Centre de recherche en biologie de la reproduction
Les cycles de fécondation in vitro échouent plus souvent qu’ils ne réussissent, mais
malheureusement, il n’y a pas d’outils permettant de comprendre les raisons de l’échec
et d’adapter conséquemment le traitement lors des cycles subséquents. Lors d’études
précédentes, un panel de biomarqueurs fut développé pour évaluer les conditions
menant à des ovocytes de moindre qualité. L’objectif du projet est de déterminer si ces
marqueurs peuvent être utilisés pour modifier le résultat lors d’un deuxième cycle
d’IVF. Jusqu’à maintenant, les 24 biomarqueurs ont été confirmés après réalisation de
deux micropuces. Des échantillons (pools de granulosa) ayant mené à un résultat
négatif après implantation à J3 ou J5 ont été comparés à des échantillons positifs
(grossesse). 164 et 101 gènes ont été exprimés de façon significativement différente
(ratio > 1.5, P-value <0.05). Les fonctions de ces gènes indiquent des niveaux de
différentiation variables entre les pools de follicule. Les 200 échantillons provenant de 4
cliniques seront ensuite testés pour identifier les 12 meilleurs biomarqueurs du panel
(présence de biomarqueurs liés aux problèmes folliculaires vs résultats). Les résultats
obtenus seront fournis aux cliniques qui pourront modifier le traitement prévu pour le
deuxième cycle en fonction des indicateurs de mauvaise réponse au premier cycle. Les
résultats seront comparés à ceux obtenus lorsqu’aucun diagnostic n’a été établi au
premier cycle et donc qu’aucune modification de traitement n’a été faite pour le
deuxième cycle.
50
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Single patient with recurrence of the same mechanism in three
spontaneous abortions: digynic triploidy due to failure of meiosis II
Y. Khawajkie1, W. Buckett2, A. Ao2, SL. Tan2, M. Lemoine3, R. Slim1,2,3
1
Departments of Experimental Medicine, 2Human Genetics, and 3Obstetrics and
Gynecology, McGill University Health Centre, Montreal H3G 1A4, Canada
Spontaneous abortions (SAs) affect 15% of clinically recognized pregnancies.
Recurrent spontaneous abortions (RSAs) are defined by the occurrence of at least two
pregnancy losses and affect 1% to 5% of couples trying to conceive. RSAs are clinically
and genetically highly heterogeneous. One of the many factors that has hampered the
identification of causative or susceptibility genes for RSAs lies in the high genetic
heterogeneity of this clinical entity.
In an attempt to categorize the entity of RSAs and divide the patients into more
categories according to the mechanisms leading to their recurrent fetal losses, we first
used flow cytometry to assess the ploidy of 97 POCs from 60 patients. We identified 6
triploid POCs (6%), of which three are from unrelated patients and three are from the
same patient. We then used fluorescent in situ hybridization to confirm the triploidies
and fluorescent microsatellite genotyping with distal and pericentromeric markers to
determine their parental origin and the mechanisms leading to their formation. We
found that all six triploidies are digynic. Among the three triploidies from unrelated
patients, one was due to an error in meiosis I and two due to an error in meiosis II. The
three triploidies from the same patient were found to be due to an error in meiosis II.
Identifying such patients with the same recurrent mechanism will pave the way for the
identification of causative genes for RSAs and will allow for the provision of better
genetic counselling and appropriate ART services for the patients.
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Session d’affiches II – Poster Session II
9h00 – 10h30
1. Établissement d’une lignée de gonocytes fœtaux chez le rat. Fabien Joao, INRS
- Institut Armand-Frappier (Page 55)
2. Analyses épigénétiques et transcriptomiques sur embryons bovins obtenus à
partir d'ovocytes de donneuses en période péripubertaire. Léonie Morin-Doré,
Université Laval (Page 56)
3. Inflammation in human placental trophoblastic cells: a melatonin target? Lucas
Sagrillo-Fagundes, INRS - Institut Armand-Frappier (Page 57)
4. Comment protéger le testicule pré-pubère de la cytotoxicité des chimiothérapies?
Amélie Tremblay, INRS - Institut Armand-Frappier (Page 58)
5. Melatonin activates NRF2 and autophagy in human placental cells. Josianne
Bienvenue-Pariseault, INRS - Institut Armand-Frappier (Page 59)
6. Myoid cells of the rodent epididymis: implications of functions linked to sperm
motility. Regiana Oliveira, McGill University (Page 60)
7. Analysis of the association between maternal NODAL polymorphisms and
preterm birth. Lisa M. Starr, McGill University (Page 61)
8. In vitro cell reprogramming using porcine oocyte extract. Werner Glanzner,
9. La concentration lipidique du liquide folliculaire des femmes suivant une
fécondation in vitro est associée à une augmentation de la production intraovarienne d’androgènes. Joanie Faubert, Université de Sherbrooke (Page 63)
10. Angiogenic activity of PGF2a in women with endometriosis. Halima Rakhila,
Université Laval (Page 64)
11. In Vitro Embryo Production from Holstein Calf Oocytes Recovered by
Laparoscopic Oocyte Pick-up. Luke Currin, McGill University (Page 65)
12. Integrative meta-analysis of the transcriptome of three follicular compartments
reveals shared pathways associated with oocyte competence in bovine ovaries.
Daulat Raheem Khan, Université Laval (Page 66)
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13. Maternal Casein-Based Diet Sensitizes Male Offspring to in-Utero Endocrine
Disruptor (ED) Induced Reproductive Toxicity. Annie Boisvert, McGill University
(Page 67)
14. Deciphering the role of the Nodal signaling pathway in preterm birth. Taghreed
Heba, McGill University (Page 68)
15. Wnt4 and Wnt5a play redundant roles in the development and function of the
uterus, but not of the ovaries. Guillaume St-Jean, Université de Montréal (Page
69)
16. Nuclear YAP does not regulate oocyte development in mammals. Laleh Abbassi,
17. Expression of two Sperm Adhesion Molecule 1 (SPAM1) isoforms in bull testis
and epididymis. Andrée-Anne Saindon, Université Laval (Page 71)
18. Ubiquitin ligase Huwe1 modulates male germ cell development by regulating
spermatogonial differentiation and meiotic entry. Rohini Bose, McGill University
(Page 72)
19. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular
Cholesterol Trafficking in Testicular Leydig Cells. Nancy C. Li, McGill University
(Page 73)
20. Beware of Dogma: Revisiting activin B’s role in follicle-stimulating hormone
synthesis in vivo. Luisina Ongaro Gambino, McGill University (Page 74)
21. FSH and LH downregulate NPR-3 expression in bovine cumulus cells in vitro.
Matheus Pedrotti De Cesaro, McGill University (Page 75)
22. Derivation of putative induced pluripotent stem cells from Mustela vision adipose
tissue. Amanda, B. Trindade, Université de Montréal (Page 76)
23. Heparin-alpha-glucosaminide N-acetyltransferase (HGSNAT) induces
morphological changes in the epididymal epithelium and spermatozoa of adult
mice. Flavia Lorena Carvelli, McGill University (Page 77)
24. Exploring mutations and polymorphism in human binder of sperm homolog 1
gene in idiopathic males. Samin Sabouhi, Université de Montréal (Page 78)
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53
25. Lactate produced during labor exerts anti-inflammatory effects on the uterus via
GPR81 (HCA1). Mathieu Nadeau-Vallée, Université de Montréal (Page 79)
26. Regulation of gene expression in murine granulosa cells. Milena Taibi, McGill
27. Combined effects of DNA methyltransferase 1o-deficiency and ovarian
stimulation on embryonic outcome and epigenetic patterning at mid-gestation.
Josée Martel, McGill University (Page 81)
28. Loss of PORCN induces cellular alterations in the epididymis but is dispensable
for steroidogenesis in the testis and adrenal glands. Adrien Levasseur, Université
de Montréal (Page 82)
29. Impact of Zearalenone mycotoxins on Sertoli cell survival and on their role for the
establishment of the spermatogonial stem cell niche. Christian Savard, Université
de Montréal (Page 83)
30. Regulation of TSPO expression during germ cell differentiation. Gurpreet Manku,
31. Random unilateral chromosome inheritance is a novel explanation for mosaic
embryo aneuploidy. Cayetana Vázquez de Castro Diez, Université de Montréal
(Page 85)
32. Comprehensive Genotype Phenotype Correlations Reveals NLRP7 Role in
Regulating Embryonic Tissue Differentiation and Trophoblastic Proliferation.
Ngoc Minh Phuong Nguyen, McGill University (Page 86)
33. Caractérisation des vésicules extracellulaires produites par les cellules
épithéliales de l’endomètre et de l’épididyme. Yann Becker, Université Laval
(Page 87)
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Établissement d’une lignée de gonocytes fœtaux chez le rat
Fabien Joao et Géraldine Delbès
INRS – Institut Armand Frappier, Laval, Québec
Les gonocytes, précurseurs fœtaux de toute la lignée germinale, passent par des
périodes clés de développement qui, si dérégulées, pourraient être à l’origine de
l’infertilité masculine. Il a été suggéré que les perturbateurs endocriniens affectent la
fertilité masculine surtout si une exposition survient pendant le développement fœtal.
Toutefois, les mécanismes d’action restent mal compris. Il n’existe effectivement pas
de modèle stable d’étude des gonocytes in vitro car ils sont difficiles à purifier et
survivent mal en culture. Notre objectif est d’établir une lignée cellulaire de gonocytes
fœtaux. Nous utilisons un modèle de rats exprimant la GFP dans les gonocytes
permettant de les purifier par FACS à partir de testicules prélevés à 15 ou 16 jours post
conception (jpc). Les cellules obtenues sont maintenues en culture primaire dans des
puits recouverts de laminine ou matrigel. Lorsqu’explantés à 15 jpc, la fluorescence des
gonocytes disparait après 48h de culture et leur morphologie est atypique. Cependant,
le signal GFP est meilleur dans les gonocytes à 16 jpc. La culture sur laminine avec du
choisie car ces conditions permettent le moins d’auto fluorescence et le meilleur
maintien de la prolifération, mesurée en immunofluorescence par l’incorporation de
BrdU. Ces conditions vont être utilisées pour tester différents protocoles de transfection
afin d’immortaliser les gonocytes avec l’antigène large T SV40. L'établissement de
cette lignée nous offrira un modèle unique pour comprendre les effets de polluants
environnementaux sur les de gonocytes fœtaux.
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Analyses épigénétiques et transcriptomiques sur embryons bovins
obtenus à partir d'ovocytes de donneuses en période péripubertaire
Léonie Morin-Doré1, Patrick Blondin2, Christian Vigneault2, Marc-André Sirard1
1
2
Université Laval, Département des Sciences Animales
L’Alliance Boviteq, St-Hyacinthe
Les techniques de reproduction assistée et la forte pression de sélection observée
dans le cadre de la reproduction bovine entraînent l’utilisation de femelles de plus en
plus jeunes à des fins reproductives. Cette situation aura possiblement un impact sur la
qualité de leurs embryons, affectant le taux de succès des procédures et
potentiellement la génisse de la génération suivante. Ce projet de recherche vise à
documenter l'effet de l'âge sur la qualité de l'embryon, au point de vue
transcriptomique, et de caractériser le risque lié à l'utilisation de jeunes animaux à des
fins reproductives.
10 jeunes femelles Holstein ont subi 3 cycles de stimulation ovarienne, permettant 3
collectes d’ovocytes (8, 12, 16 mois). Ces ovocytes ont ensuite été fécondés in vitro
par des spermatozoïdes d'un taureau adulte pour générer 3 lots d’embryons par
animal. Grâce à la plateforme EmbryoGENE, il fut possible de mesurer l'expression
génique au stade blastocyste. L'analyse de contraste selon l'âge, soit 8 vs 16 mois et
12 vs 16 mois, a permis de dénombrer 220 gènes différentiellement exprimés pour le
premier contraste et 274 pour le deuxième. Les résultats suggèrent une cause
métabolique pour expliquer les différences observées entre les sujets immatures et
adultes. Parmi les voies géniques affectées par l'âge, on retrouve notamment les voies
de signalisation mTOR et PPAR, ainsi que la voie de réponse au stress oxydatif
médiée par NRF2.
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Inflammation in human placental trophoblastic cells: a melatonin target?
Lucas Sagrillo-Fagundes, Eugenia Maria Assuncao Salustiano, Cathy
Vaillancourt
INRS-Institut Armand-Frappier and Cinbiose and Biomed research center, Laval, QC,
H7V 1B7, Canada
Melatonin is produced by and plays a protective role in human placental villous
trophoblasts against hypoxia/reoxygenation (H/R)-induced oxidative stress and
apoptosis. Primary villous trophoblasts cultivates under H/R is an in vitro model of
physiopathological
pregnancy
such
as
preeclampsia,
related
with
aberrant
inflammatory response. OBJECTIVE: Determine if melatonin reverses increased
inflammatory response induced by H/R in villous trophoblastic cells. METHODS:
Primary villous cytotrophoblasts isolated from normal term placenta were maintained
under normoxia (8% O2) during 72 h (to allow the differentiation in syncytiotrophoblast)
and then exposed to H/R (0.5% O2 (4 h)) or normoxia. Villous trophoblasts were treated
or not (control) with melatonin (1mM) +/- luzindole (antagonist of melatonin receptors
(1nM)), melatonin +/- LPS (1mg/mL), or siAANAT (limiting melatonin synthetizingenzyme). RESULTS: Melatonin levels (measured by ELISA) are reduced (42%) in
villous trophoblasts under H/R, but increased when treated with melatonin (107%),
luzindole (134%) or both (242%). TNFwith melatonin in normoxia (63%) and H/R (50%). IL-10 is increased by 3-fold in villous
trophoblasts
treated
with
melatonin
compared
with
siAANAT
in
normoxia.
CONCLUSION: Our results suggest that melatonin protects trophoblastic cells against
H/R throughout the modulation of inflammation pathway. Concerning the current
clinical-trials to treat preeclampsia with melatonin, a better understanding of the
protective effects of melatonin in trophoblast cells is required.
Support: NSERC-
discovery grant to CV and FRQNT studentship to LSF and EMAS.
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Comment protéger le testicule pré-pubère de la cytotoxicité des
chimiothérapies?
Amélie Tremblay[1,2], Hermance Beaud[2], Géraldine Delbès[2]
[1]
[2]
Département de Biochimie, Université de Sherbrooke, Sherbrooke, Qc
Centre INRS – Institut Armand-Frappier, Institut national de la recherche scientifique,
Laval, Qc
Les traitements de cancer pendant l’enfance peuvent avoir un impact négatif à long
terme sur la fertilité masculine. En effet, les chimiothérapies ciblent les cellules
testiculaires alors en division, dont les cellules de Sertoli et les spermatogonies,
indispensables
pour
la
future
spermatogenèse.
La
cryoconservation
de
spermatozoïdes n’étant pas réalisable chez les garçons pré-pubères, d’autres avenues
de
préservation
de
la
fertilité
doivent
être
envisagées.
Nous proposons de tester l’hypothèse selon laquelle certains composés décrits comme
cytoprotecteurs permettraient de réduire la cytotoxicité associée à la doxorubicine et la
vincristine (0,01-
Nous avons mesuré la cytotoxicité de la doxorubicine et de la
vincristine, en co-traitement avec un cytoprotecteur, sur des lignées de cellules de
Sertoli (SER-W3) et de spermatogonies (GC-SPG6) grâce au test MTT (methylthiazolyldiphenyl-tetrazolium bromide). Les cinq cytoprotecteurs choisis sont la
denylate Cyclase-Activating
cytoprotecteurs n’a permis de diminuer la cytotoxicité dose dépendante de la
doxorubicine ou de la vincristine dans les GC-SPG6. La carnitine, l’amifostine ou le
PACAP n’ont pas eu d’effet ou ont significativement augmenté la cytotoxicité de la
doxorubicine ou de la vincristine sur les SER-W3. Par contre, nous avons observé une
rès 24h de cotraitement avec le curcumin et la vitamine C chez les SER-W3. Les propriétés
d’antioxydants communes à ces deux cytoprotecteurs pourraient jouer un rôle clé dans
les mécanismes de protection des cellules de Sertoli contre la doxorubicine.
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Melatonin activates NRF2 and autophagy in human placental cells
Josianne Bienvenue-Pariseault, Lucas Sagrillo-Fagundes, Cathy Vaillancourt
INRS-Institut Armand-Frappier and Biomed research center, Laval, QC, H7V 1B7
Melatonin protects placenta against hypoxia/reoxygenation (H/R) oxidative damage.
Autophagy is an important pathway to degrade cellular content. Nrf2, is a transcription
factor that induces the expression of cytoprotective genes. H/R and hypoxia are
involved in physiopathological pregnancy disorders like preeclampsia. The objective of
this study is to determine how melatonin modulates autophagy and Nrf2 in both normal
and tumor human trophoblastic cells under H/R and hypoxia. BeWo cells
(choriocarcinoma line) and primary villous cytotrophoblasts (vCTB) isolated from term
placenta were maintained under normoxia during 24 h (BeWo) or 72 h (vCTB) (to allow
the differentiation in syncytiotrophoblast (STB)) and then exposed to H/R (0.5% O2 (4
h)), hypoxia (0.5% O2 (24h h)) or normoxia. H/R and concomitant treatment with
melatonin generate a two-fold increase (p<0.05) of ATG7 expression in STB. Nrf2 is
doubled under H/R in STB when treated with melatonin (p<0.034). In BeWo cells,
melatonin and H/R generated a 1.73-fold (p<0.01) increase in Beclin-1, BeWo were
transfected with siRNA to MT1 and MT2 (melatonin receptors). siMT1 and siMT2
decreased ATG7 (p<0.05). Under hypoxia, MT1/MT2 knockdown decreased ATG7
levels (p<0.01) compared to all other treatments. Melatonin treatment induces Nrf2
degradation in BeWo cells (-58%). These results demonstrate that melatonin regulates
autophagy and Nrf2 in part through its receptors. Nrf2 and autophagy have
cytoprotective effects in normal and tumor cells. The different results obtained in tumor
vs normal trophoblasts suggest that melatonin has deleterious effects in cancer cells
and beneficial effects in normal cells.
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Myoid cells of the rodent epididymis: implications of functions linked to
sperm motility
Regiana Oliveira1, Adam Parent1, Daniel G. Cyr2, Mary Gregory2, C.E. Smith3,
Louis Hermo3
1
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec,
Canada
2
INRS-Institut Armand-Frappier, Pointe-Claire, Quebec, Canada
3
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec,
Canada
Myoid cells wrap around the seminiferous tubules of the testis and epididymis. While
their role in contractility providing movement of sperm down the duct is documented,
little is known of functions related to sperm maturation. By electron microscopy, the
flattened myoid cells formed 3-4 concentric layers. While close approximations of the
edges of myoid cells of one layer were randomly encountered, small foot-like processes
emanated from myoid cells along their length from one layer to that of the layer above
or below. At times, myoid cells of adjacent layers ran closely parallel to each other for
long distances. The size of the intervening space between adjacent myoid cells at
these close approximation sites was attenuated, with connexin 43 being prominent.
Tight junctions were not a conspicuous feature of adjacent myoid cells. Aside from the
abundance of actin filaments, these cells also contained caveolae, the clathrin
independent vesicles made up of caveolin proteins, with localization of caveolin 1 being
visualized, but not caveolin 2 and 3. Morphometric analysis of Cav1-/- mice revealed
that the size of tubule and epithelial profile areas in the testis was enlarged. In the
proximal epididymis, these parameters slightly shrunk compared to Cav1+/+ mice.
Statistical analysis of sperm counts of Cav1-/- mice revealed no changes. Velocity
parameters of motility of Cav1-/- sperm suggested that sperm moved slower than
normal. Thus by their contractile nature, myoid cells may agitate epididymal tubules to
allow proper interactions of sperm and luminal contents for sperm maturation.
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Analysis of the association between maternal NODAL polymorphisms and
preterm birth
Lisa M. Starr, Taghreed Heba and Daniel Dufort
Research Institute McGill University Health Centre
Nodal, a morphogen of the transforming growth factor-beta (TGF-)-super family, plays a
critical role during embryonic development. Recently, we showed that maternal
decidual-specific Nodalknockout mice have preterm birth. The aim of the current study
was to investigate the association of NODAL single nucleotide polymorphisms (SNPs)
and risk of preterm birth in humans. Data from Canadian women from a premature birth
cohort was analyzed to determine genotype distributions of NODAL SNPs and their
association with gestational age. Preliminary results suggest that NODAL SNPs do not
independently associate with preterm birth. In order to assess whether this relationship
is mediated by another factor, effect of environmental factors on the relationship
between NODAL SNPs and the risk of preterm birth will be analyzed. Our findings
could be investigated as a possible genetic marker for the risk of preterm birth.
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In vitro cell reprogramming using porcine oocyte extract
Werner G. Glanzner1; Eliza R. Komninou2; Ashwini Mahendran3; Karina
Gutierrez3; Rodrigo C. Bohrer3; Naomi Dicks3; Vilceu Bordignon3.
1
Biotechnology and Animal Reproduction, BioRep, UFSM, Brazil;
Departamento de Biotecnologia, UFPEL, Brazil;
3
Department of Animal Science, McGill, Canada;
2
Nuclear transfer experiments have shown that the cytoplasm of oocytes has the
capacity to reprogram the chromatin of differentiated cells to a totipotent state. There is
also evidence suggesting that oocyte cytoplasmic extract can be used to produce
induced pluripotent cells (iPS) in vitro. Epigenetic modifications including DNA and
histone methylation, and histone acetylation are important components affecting the
reprogramming process. The aim of this study was to evaluate the effect of swine
germinal vesicle phase oocyte extract (OE) in association with inhibition of histone
deacetylases (HDACi) for reprogramming porcine fibroblast cells in vitro. Cell
morphology and mRNA expression of genes involved in epigenetic modifications
(DNMT1, EZH1, EZH2, SUZ12, KDM6A and KDM6B) and cell pluripotency (Rex1, cMyc, Oct4, Sox2 and Nanog) were evaluated at different periods after cell treatment.
Embryonic-like (ESC-like) stem colonies formed in average 16 days after treatment with
OE or OE plus the HDCAi Scriptaid. There was no difference between the two
treatments in the number of ESC-like colonies. Pluripotency genes Rex1 and c-Myc
were upregulated after treatment, but Oct4, Sox2 and Nanog expression was not
altered. Decreased mRNA levels of DNMT1 and EZH2 were observed after 3 days of
treatment with OE. There was no significant difference in gene expression between
ESC-like colonies derived by OE and OE+Scriptaid on day 21 post-treatment. These
findings suggest that porcine cells treated with OE or OE+Scriptaid undergo similar but
incomplete reprogramming towards pluripotency in vitro.
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La concentration lipidique du liquide folliculaire des femmes suivant une
fécondation in vitro est associée à une augmentation de la production
intra-ovarienne d’androgènes
J. Faubert1, A. Gervais1, M.-C. Battista1, B. Carranza-Mamane2,3, H.B. Lavoie3, J.-P.
Baillargeon1
1
Service d’endocrinologie, Département de Médecine
Département d’Obstétrique-Gynécologie, FMSS, Université de Sherbrooke,
Sherbrooke, Canada ;
3
Procréa Cliniques Montréal, Montréal, Québec
2
Objectif : Chez des femmes en processus de fertilisation in vitro (FIV), démontrer
l’impact des lipides du liquide folliculaire sur les niveaux de testostérone, des
métabolites des lipides, des marqueurs de l’inflammation et de la fertilité. Méthode : Le
liquide folliculaire a été conservé suite au prélèvement d’ovule et a été évalué pour les
niveaux de : testostérone (LC-MS/MS), lipides (acides gras non-estérifiés (AGNE) plus
triglycérides; colorimétrie), métabolite
-
oxydation; LC-MS/MS) et marqueur de l’inflammation (IL-6). Résultats : Les patientes
(n=80) avaient 33±4 ans et un IMC de 26 [23-31] kg/m2. Les concentrations folliculaires
de testostérone étaient de 0,44 [0,19-2,37] nM et de lipides, de 0,43 [0,36-0,54] nM.
Les niveaux de testostérone étaient positivement associés à ceux des lipides (r=0,38,
-6 (r=0,30, p=0,03) (Pearson).
Les prédicteurs indépendants des niveaux de testostérone étaient les niveaux de
lipides (R2 partiel=18%) et d’IL-6 (R2 partiel=5%) (régression linéaire multivariée par
étape). Les niveaux folliculaires de testostérone étaient aussi associés au pourcentage
-0,23 (Spearman); p=0,04). Conclusion : Nos résultats montrent
que la production ovarienne d’androgènes est influencée par l’exposition ovarienne aux
-oxydation des acides gras, et
secondairement à l’inflammation. La fertilité des patientes pourrait aussi être affectée
par cette lipotoxicité, ce qui devrait être testé dans de futures études évaluant
directement les cellules de la granulosa.
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63
Angiogenic activity of PGF2A in women with endometriosis
Halima Rakhila1, Marie-Eve Bergeron2, Marleen Daris2, Mathieu Leboeuf2,
Madeleine Lemyre2, Caroline Rhéaume2, Marc Pouliot1
1
Department of Microbiology and Immunology, Centre Hospitalier Universitaire de
Québec, Faculty of Medicine, Université Laval, Québec City, QC, Canada
2
Department of Obstetrics and Gynecology, Centre Hospitalier Universitaire de
Québec, Faculty of Medicine, Université Laval, Québec City, QC, Canada
We investigate interleukin-8 (CXCL8) and vascular endothelial growth factor(VEGF)
expressions in endometrial stromal cells of women with endometriosis compared with
controlsin response to Prostaglandin F2a (PGF2a). Primary cultures of eutopic and
ectopic endometrial stromal cells isolated from endometrial biopsies of endometriosis
patients, as well as eutopic endometrium of control patients in proliferative and
secretory cycle phases exposed to PGF2a, with assessment of CXCL8 and VEGF
expression. Cells grown to confluence were preincubated for 1 h with PGF2a-FP
receptor antagonist (AL8810) or cyclo-oxygenase-2 (cox-2) inhibitor (NS398) and
stimulated with different concentrations of PGF2a or PGF2a analog (Fluprostenol) for
different periods of time. PGF2a biosynthetic pathwayswere analyzed by RT-qPCR and
Western blot.While, CXCL8 and VEGF secretions were analyzed by ELISAs. Our study
showed that PGF2a stimulated CXCL8 and VEGF expressions in ectopic endometrial
stromal cells of women with endometriosis in the both phases of the menstrual cycle,
without noticeable change in eutopic endometrial stromal cells of women with or without
endometriosis. Cell exposure to Fluprostenol confirmed these observations.After 24h of
stimulation, diminutions of CXCL8 and VEGF were significant when AL8810 was added
with either PGF2a or Fluprostenol.Meanwhile, we observed that PGF2a increased
significantlycox-2expression while its other biosynthetic enzymes showed no difference.
We, therefore, used NS398 which correlated a decrease for both VEGF and IL-8
secretions. This study showed that angiogenicpathways are involved in PGF2amediated activation through FP and Cox-2 inectopic endometrial stromal cells.
Expression of CXCL8 and VEGFin ectopic endometrial cells throughout the menstrual
cycle of women with endometriosis may, considering the role of these cytokines in cell
growth and angiogenesis, play an important role in the capability of endometrial cells to
develop and to survive in ectopic locations.
64
e
th
In Vitro Embryo Production from Holstein Calf Oocytes Recovered by
Laparoscopic Oocyte Pick-up
L. Currin, L. Michalovic, W. Glanzner, K. Gutierrez, R. Bohrer, P. da Rosa, M. De
Cesaro, N. Dicks, Y. Schuermann, H. Baldassarre, V. Bordignon
Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, Quebec,
Canada
Oocyte competence and reproductive biology in prepubertal heifer calves are not fully
understood. Multiple publications have reported high oocyte yields recovered from
calves aged 2-6 months old but low embryo development rates following in vitro
embryo production. The objective of this study is to characterize the developmental
competence of oocytes from young calves. We report herein the oocyte/embryo yields
obtained from 6 Holstein calves that were subjected to gonadotropin stimulation and
laparoscopic ovum pick-up (LOPU) every 2 weeks, starting at 2 and ending at 5 months
of age. A total of 766 follicles were aspirated (avg. 17/calf/session) resulting in 625
cumulus oocyte complexes (COCs) recovered (avg. 14/calf/session; 82% recovery
rate). A total of 457 (73%) COCs were graded eligible for IVM, of which 353 cleaved
(77%) and 109 (24%) reached a viable blastocyst stage at the end of IVC, of which 42
(38.5%)
were
graded
as
freezable.
In
balance,
approximately
two
viable
blastocysts/calf/session were produced. No adhesions or sequels were observed in the
ovaries at the end of the experiment. Analyses of embryos fixed after fertilization or
culture are now being conducted to determine normal fertilization and cell integrity.
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65
Integrative
meta-analysis
compartments
reveals
of
the
shared
transcriptome
pathways
of
three
associated
with
follicular
oocyte
competence in bovine ovaries
Daulat Raheem Khan*, David Landry*, Christian Vigneault¤, Patrick Blondin¤ and
Marc-André Sirard*
* Centre de Recherche en Biologie de la Reproduction, Département des Sciences
Animales, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval,
Québec, Canada
* L'Alliance Boviteq, Saint-Hyacinthe, Québec, Canada.
Ovarian follicle contains variety of cells which interact closely to presage oocyte
developmental competence. Classically the gene expression profiles of different
follicular cells have been studied separately, beside the importance of interaction
between these cells. This may be potential caveat since enrichment of cell-type-specific
transcriptomic profiles may obscure the underlying shared pathways which may
actually be implicated in oocyte competence acquisition. Therefore, the integrative
transcriptome meta-analysis of different follicular cells remains crucial to understand
the physiology of the whole tissue. Previously, in cows it has been shown that after
super-stimulation with FSH, appropriate FSH starvation (FSH-coasting) improves
oocyte quality. Using this model, we analysed transcriptomic profiles of granulosa cells,
cumulus cells and the oocytes following FSH-coasting of 20h, 44h, 68h or 92h.
Interestingly, the coasting period of 44h after FSH stimulation yielded the best quality
oocytes. Here, we performed co-regulation analysis of these studies to identify gene
networks across all three distinct follicular cells that are enriched during oocyte
developmental competence. Taking highest competence period as reference, networkbased meta-analysis of differentially expressed genes identified highly connected hub
genes. Biofunction analysis highlighted changes in regulation of mitochondrial function
and metabolic pathways particularly fatty acid (FA) synthesis and its beta-oxidation.
Moreover, nutrient-sensing pathways were also differentially regulated in different
conditions of FSH-coasting. We conclude that suitable ovarian stimulation and FSHcoasting interval appropriate follicular metabolism and mitochondrial function which
herald better oocyte competence.
66
e
th
Maternal Casein-Based Diet Sensitizes Male Offspring to in-Utero
Endocrine Disruptor (ED) Induced Reproductive Toxicity
Steven Jones1,2, Annie Boisvert1,3, Sade Francois1,4, Andrada Naghi1, 4, Liandong
Zhang1,5 Martine Culty1,4
1
The Research Institute of the McGill University Health Centre
Division of Experimental Medicine of McGill University.
3
Departments of Medicine of McGill University, Montreal, Canada.
4
Pharmacology & Therapeutics of McGill University.
5
Department of Urology, Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong
University, China.
2
Perinatal exposure to endocrine disruptors (EDs) is believed to predispose males to
reproductive abnormalities. Although males are exposed to EDs from conception to
adulthood, few studies have evaluated the effects of ED mixtures. Our previous work
demonstrated that fetal exposure to a mixture of Genistein (GEN) and DEHP, via
gavage of pregnant dams fed a soy-based diet, induced short and long term alterations
in rat testis gene expression and histology different from single EDs. In the present
study, pregnant dams fed with a casein-based diet devoid of phytoestrogens, were
gavaged with control corn oil, 0.1 or 10 mg/kg GEN, DEHP alone or combined. Male
offspring reproductive functions and testicular gene expression profiles were assessed
at different ages.
While casein diet alone did not alter any of the end-points examined compared to
regular soy diet, fetal ED exposure induced significant alterations in testis histology,
decreased expression of Leydig and germ cell markers, impaired germ cell migration
and changes in REDOX mediators in inflammatory macrophages in PND6 pups. Adult
male offspring showed higher rates of infertility than controls and rats fed soy-diet.
Serum estradiol levels were reduced in all treatment groups at PND90, but not
testosterone. This study unveiled more severe reproductive toxicity in rats exposed to
EDs under casein diet, than with soy-based diet. Treated animals did not follow
classical dose response effects, at perinatal and adult ages, highlighting the importance
of assessing a range of doses during appropriate windows of exposure and under
varying baseline dietary conditions.
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67
Deciphering the role of the Nodal signaling pathway in preterm birth
Taghreed Heba, Craig Park and Daniel Dufort
Department of Obstetrics and Gynecology, Research Institute of the McGill University
Health Centre, Montréal, Québec, Canada
Preterm birth, defined as delivery before 37 weeks of gestation, is the single leading
cause of perinatal mortality in developed countries, affecting up to 12-13% of
pregnancies. Despite the prevalence and severity of premature delivery, the
mechanisms and causes that underlie spontaneous and idiopathic preterm births are
still unknown. One contributing factor that has been identified is genetic predisposition,
which increases the susceptibility of preterm birth. Our lab has been studying the role of
Nodal signaling pathway during pregnancy. Nodal is a morphogen that belongs to the
Transforming Growth Factor-beta (TGF-B) superfamily. Nodal has been shown to play
critical roles during embryonic development and our lab has recently shown that it is
also required during gestation. Indeed, we have demonstrated that Nodal signaling is
active in the uterus during early pregnancy and is required for both embryonic
implantation and for the proper timing of parturition. A uterine specific deletion of Nodal
results in a 75% reduction in the rate of pregnancy due to implantation defects.
Furthermore, the Nodal mutant females that did become pregnant gave birth two days
prior to the term. Interestingly, our results demonstrate that heterozygous females have
a 30% incidence of spontaneous preterm birth demonstrating that these females have a
predisposition to giving birth preterm. Remarkably, an injection of low dose of an
inflammatory mediator lypopolysaccharides (LPS) on day 15.5 of pregnancy leads to
premature delivery 24 hours after LPS injection in 30% of heterozygous females.
Moreover, LPS administration results in abnormal histological placental morphology
and an acute inflammatory response in Nodal heterozygous mice but not in controls.
Our results show that Nodal heterozygous females may have an increase acute
inflammatory response to low doses of LPS which may lead to defects in the timing of
parturition.
68
e
th
Wnt4 and Wnt5a play redundant roles in the development and function of
the uterus, but not of the ovaries
St-Jean G, Paquet M, Boerboom D
Faculté de Médecine Vétérinaire, Université de Montréal (all authors)
Previous studies have shown that granulosa cell-specific ablation of either Wnt4 or
Wnt5a results in similar ovarian phenotypes, suggesting possible functional redundancy
between these secreted signaling molecules. To test the latter hypothesis, a double
conditional knockout (cKO) of Wnt4 and Wnt5a was created using the Amhr2[cre]
strain. A breeding trial conducted with Wnt4/5a cKO females revealed an almost
complete loss of fertility that was much more severe than the subfertility phenotypes
observed in both Wnt4 and Wnt5a single cKOs. Unexpectedly however, histopathology
and follicle counting analyses failed to show the increase in follicular atresia and loss of
antral follicles that characterizes the Wnt4 and Wnt5a single cKOs. Furthermore,
ovulatory efficiency in the Wnt4/5a cKO females was comparable to controls,
suggesting that the fertility phenotype was of extra-ovarian origin. As the Amhr2[cre]
strain also targets the uterine stroma, we conducted histopathologic evaluations of the
uterus, which revealed an almost complete absence of uterine glands. Additionally,
posterior part of the reproductive tract. This defect was characterized by absence of the
vaginal opening, agenesis of the posterior vagina, lack of continuity between the uterus
and external genitalia, and severe mucometra. Our results therefore indicate redundant
roles of Wnt4 and Wnt5a in reproductive tract development and uterine physiology.
Ongoing experiments are aimed at defining the cause of infertility in the Wnt4/5a cKO
model, as well as better defining the roles of Wnt4 and Wnt5a in the ontogeny of the
reproductive tract.
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69
Nuclear YAP does not regulate oocyte development in mammals
Laleh ABBASSI1, Safia MALKI2, Katie COCKBURN3, Angus MACAULAY4, Claude
ROBERT4, Janet ROSSANT3, Alex BORTVIN 2 and Hugh CLARKE1
1
Research Institute of the McGill University Health Center; McGill University, Montreal,
Canada
2
Carnegie Institution for Science, Baltimore, USA
3
Hospital for Sick Children; University of Toronto, Toronto, Canada
4
Animales, Université Laval, QC, Canada.
Recent studies have shown that subjecting ovarian fragments to mechanical or
pharmacological
interventions
that
activate
YAP
(Yes-associated
protein),
a
transcriptional co-activator, can trigger primordial follicles to enter the growth phase.
However, in the absence of knowledge of the normal function of YAP in the follicle, our
understanding of the mechanistic basis of these strategies remains very limited. We
found that YAP is expressed and largely excluded from the nucleus at all stages of preand post-natal mouse oocyte development. Phosphorylation of S112 enables YAP, in
other cell types, to associate with 14-3-3 proteins, which anchors it in the cytoplasm.
We detected S112-phosphorylated YAP in both growing and fully grown oocytes and
showed that this phosphorylation is regulated by a cyclic AMP-protein kinase Adependent mechanism in oocytes. Using the nuclear export inhibitor, Leptomycin B, we
observed that a subpopulation of YAP, likely dephosphorylated, can enter the nucleus
in growing oocytes. However, this YAP is rapidly exported back to the cytoplasm
suggesting that growing oocytes are unable to retain dephosphorylated YAP in the
nucleus. We also found that YAP failed to accumulate in the nuclei of fully grown
oocytes even when we induced its dephosphorylation and blocked nuclear export. Our
results demonstrate that multiple mechanisms cooperate to ensure that YAP canot
accumulate in the oocyte nucleus. We conclude that nuclear YAP does not play a
physiological role during oocyte development in mouse and therefore the mechanism
by which activators of YAP induce follicle growth is unlikely to involve activation of YAPdependent transcription in the oocyte.
70
e
th
Expression of two Sperm Adhesion Molecule 1 (SPAM1) isoforms in bull
testis and epididymis
Andrée-Anne Saindon 1,2, Serge Goupil 1,2, Pierre Leclerc 1,2,3
1
Reproduction, santé de la mère et de l’enfant, Centre de recherche du CHU de
Québec (CHUL), Québec, Canada, G1V 0A6
2
Centre de recherche en biologie de la reproduction (CRBR)
3
Faculté de médecine, Département d’obstétrique, gynécologie et reproduction,
Université Laval, Québec, QC, Canada, G1V 4G2
Sperm Adhesion Molecule 1 (SPAM1) is a protein involved in the fertilization process.
SPAM1 is comprised of an N-terminal domain possessing a hyaluronidase activity that
disperses the cumulus cells and a C-terminal domain involved in the secondary binding
of spermatozoa to the zona pellucida. Previous studies using antibodies against the Nand C-terminal domains showed the presence of two isoforms weighing 70 and 80 kDa
that differ in their C-terminus, and are expressed in the post-acrosomal and acrosomal
region, respectively, of permeabilized spermatozoa. We hypothesize that one isoform
(80 kDa) is synthesized during spermatogenesis and is located in the acrosomal region
of spermatozoa, while the other (70 kDa) is synthesized in the epididymis and would
bind to the post-acrosomal region of spermatozoa during the epididymal transit.
3’RACE experiments on RNA isolated from the testis, and the caput and cauda
epididymis revealed the presence of 2 transcript variants in all 3 tissues. The open
reading frames of the 2 variants differ by the presence or absence of 90 nucleotides,
corresponding to the transcript of one exon. These variants would result in 2 isoforms,
one lacking a stretch of 30 amino acids which includes a putative transmembrane
domain. Our objective is to obtain the nucleotide sequence of SPAM1 in spermatocytes
and spermatids and compare them to the testicular and epididymal sequences to
determine whether the difference in the transcript splicing occurs pre- or post-meiosis.
We also aim to determine if SPAM1 is part of a protein complex and identify possible
interacting partners.
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71
Ubiquitin ligase Huwe1 modulates male germ cell development by
regulating spermatogonial differentiation and meiotic entry
Rohini Bose, Simon S. Wing
Department of Medicine, Research Institute of the McGill University Health Centre
Spermatogenesis involves crucial, highly regulated transitions between developmental
programs such as mitotic proliferation, meiosis and differentiation. The ubiquitin
proteasome system plays a significant role in protein turnover and cellular remodeling
and may be involved in these transitions. We previously identified ubiquitin ligase
Huwe1 in the testis and showed that inactivating it in gonocytes results in a delay in
their mitotic re-entry and leads to spermatogonial depletion. Here we inactivated it in
differentiating spermatogonia by expressing Cre recombinase using the Stra8
promoter. Huwe1-/Y males (KO) were infertile. The average testes weight of adult KO
was only 30% of the WT. Morphological analysis of adult testis revealed a
heterogeneous
phenotype
with
tubules
displaying
fewer
spermatocytes
and
spermatids. To identify the specific stage(s) at which the spermatogenic defect was
occurring, the tubules were synchronized using WIN 18,446/retinoic acid. With this
approach, there were 84% fewer pre-leptotene spermatocytes in the KO along with a
44% decrease in BrdU incorporation indicating a defect in meiotic entry. The KO testes
showed a down regulation in early meiotic markers (Spo11, Smc1b, Sycp1, Sycp3).
Chromosome spread analysis using SCP3 (marker of meiotic progression) revealed
severe degeneration of spermatocytes (5.6% WT vs. 56.8% KO) with percentage of
zygotene and pachytene spermatocytes falling by 91% and 96% respectively. When
sacrificed at a later time point after completion of the first wave of spermatogenesis, a
down-regulation of markers of spermatogonial differentiation (Stra8, Dazl, Sohlh1,
Sohlh2) was observed. Collectively, these results indicate Huwe1’s crucial role in
regulating key switches in spermatogenesis.
72
e
th
Sterol
Carrier
Protein-2,
a
Nonspecific
Lipid-Transfer
Protein,
in
Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Nancy C. Li1,2, Jinjiang Fan1,3, and Vassilios Papadopoulos1
1
The Research Institute of the McGill University Health Centre, Center for Translational
Biology, 1001 Boul. Décarie, Montreal, QC H4A 3J1, Canada
2
Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec,
H3G 1A4, Canada
3
Department of Medicine, McGill University, Montreal, Quebec, H3G 1A4, Canada
4
Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1A4, Canada
Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought
to play a major role in intracellular lipid transport and metabolism, and it has been
associated with diseases involving abnormalities in lipid trafficking. The Scp2 gene
encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins,
both of which contain a 13k Da SCP2 domain in their C-termini. Using 22-NBDcholesterol, a fluorescent analog of cholesterol, we found that 22-NBD-cholesterol was
not localized in the peroxisomes. This raises questions about previous reports on the
localization of the SCPX/SCP2 proteins and their relation to the peroxisomes, as well
as to the mitochondria in intracellular cholesterol transport. Immunofluorescence
staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells
showed that SCPX/SCP2 are present in both mouse testicular interstitial tissue and in
MA-10 cells. Fluorescent fusion proteins of SCPX/SCP2, as well as confocal live-cell
imaging, were used to investigate the subcellular targeting of these proteins and the
function of the putative mitochondrial targeting sequence in MA-10 cells. The results
obtained showed that SCPX/SCP2 are targeted to the peroxisomes by the C-terminal
PTS1 domain, but the putative N-terminal mitochondrial-targeting sequence alone is
not potent enough to localize SCPX/SCP2 to the mitochondria. Homology modeling
and molecular docking studies indicated that the SCP2 domain binds cholesterol, but it
lacks the specificity of binding and/or transport. These findings further our
understanding of the role of SCPX/SCP2 in intracellular cholesterol transport/trafficking.
e
th
73
Beware of Dogma: Revisiting activin B’s role in follicle-stimulating
hormone synthesis in vivo
Luisina Ongaro1, Xiang Zhou1, Ying Wang1, Thomas B. Thompson2, Ulrich
Boehm3, Gloria Su4, and Daniel J. Bernard1
1
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec,
Canada.
2
Department of Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati, Cincinnati, Ohio
3
Department of Pharmacology and Toxicology, University of Saarland School of
Medicine, Homburg, Germany
4
Department of Otolaryngology and Head and Neck Surgery, Columbia University,
New York, USA
The activins were discovered and named based on their abilities to stimulate folliclestimulating hormone (FSH) secretion from the pituitary gland. According to current
dogma, activin B produced by pituitary gonadotropes stimulates transcription of the
FSH
subunit gene (Fshb). Data from our laboratory challenge this model. Inhbb
knockout mice, which cannot make activin B, exhibit elevated rather than reduced FSH
levels in vivo. Similarly, gonadotrope-specific deletion of the canonical activin type I
receptor, Acvr1b, does not impair FSH synthesis in vivo. Nonetheless, mice lacking the
activin type II receptor (Acvr2) exhibit FSH deficiency. Other ligands in the family can
bind ACVR2. Thus, we hypothesize that a TGF protein, other than the activins, is the
primary intra-pituitary regulator of FSH synthesis. Growth and differentiation factor
11(Gdf11) is highly expressed in purified murine gonadotropes. In a gonadotrope cell
line model, GDF11 regulates Fshb transcription similarly to activins, however, GDF11
uses TGFBR1 rather than ACVR1B as its type I receptor. GASP-1 inhibits GDF11- but
not activin-stimulated Fshb transcription/expression in L T2 cells and primary pituitary
cells. GASP-1, however, does not inhibit basal Fshb expression, which is dependent
upon an endogenous TGF ligand. These results suggest that: 1)exogenous GASP-1
cannot access endogenous GDF11 before its binds its receptors in culture, 2)GDF11
may not play a significant role in Fshb expression in vitro or in vivo. To address the
latter possibility, we are generating mice with gonadotrope-specific deletions of Gdf11
or, its type I receptor, Tgfbr1. These results will redefine our understanding of
mechanisms of FSH synthesis.
74
e
th
FSH and LH downregulate NPR-3 expression in bovine cumulus cells in
vitro
Matheus Pedrotti De Cesaro1, Mariana Priotto de Macedo1, Joabel Tonellotto dos
Santos1, Paulo Roberto Antunes da Rosa1, Andressa Minussi Pereira Dau1,
Ricardo Della Méa1, Raj Duggavathi2, Paulo Bayard Dias Gonçalves1, Vilceu
Bordignon2
1
Laboratory of Biotechnology and Animal Reproduction, Federal University of Santa
Maria, Santa Maria, RS, Brazil.
2
Department of Animal Science, McGill University, Sainte Anne de Bellevue, QC,
Canada.
The aim of this study was to evaluate the expression of natriuretic peptide receptors
(NPR-1, NPR-2 and NPR-3) during in vitro maturation of bovine cumulus-oocyte
complexes (COCs). Bovine COCs were cultured for 0, 3, 6, 9 or 12h in TCM 199 in
absence or presence of FSH (0.5mg/ml) and LH (5.0 g/ml). Cumulus cell expansion
( m2/COC) was evaluated in 10 COCs from each time point and treatment. Thirty
oocytes from each time-point were striped from cumulus cells to assess meiotic
progression, and cumulus cells were used to quantify transcript abundance of NP
receptors. Meiotic progression after 9 and 12h of maturation was higher in oocytes
matured in the presence (86.9 and 99%) than absence (56 and 70%) of FSH and LH,
respectively. Cumulus cell expansion did not change in COCs matured in the absence
of gonadotropins, but was 3-folds greater after 12h of culture in the presence of FSH
and LH. Transcripts abundance of NPR-1 and NPR-2 was similar between treatments
and time points of culture. However, NPR-3 mRNA decreased in cumulus cells of
COCs cultured with FSH and LH. The highest mRNA abundance of NPR-3 was
detected at 12h of maturation in the absence of gonadotropins. These findings indicate
that the increase in cumulus expansion and oocyte meiotic progress induced by FSH
and LH is associated with a decrease inNPR-3 but not NPR-1 and NPR-2 expression in
cumulus cells of bovine COCs.
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th
75
Derivation of putative induced pluripotent stem cells from Mustela vision
adipose tissue
Amanda B. Trindade1,2, Jacinthe Therrien1, Joaquim M. Garcia2; Lawrence C.
Smith1
1
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire,
Université de Montréal, St-Hyacinthe, Canada and 2 Faculdade de Ciências Agrárias e
Veterinárias, UNESP, Jaboticabal, Brazil.
The American Mink, Mustela vision, has been used as a research model in many fields,
particularly in reproduction due to the phenomenon of delayed implantation. For
instance, it has been reported that mink embryonic cells show different patterns of
proliferation during the reactivation and implantation stages (Desmarais et al., 2003).
Therefore, a source of mink pluripotent stem cells could be useful to investigate the
mechanisms of embryonic diapose in this species. Thus, adult adipose cells were
transfected with a piggyBac (PB) transposon system with doxycycline-inducible
transcription of the murine reprogramming factors Oct4, Sox2, Klf4 and c-Myc. After 4
weeks, embryonic stem cell-like colonies appeared and were cultured in the presence
of doxycyclin as putative induced pluripotent stem cells (miPSC) for further analysis.
Isolated miPSC were small, rounded and underwent rapid self-renewal for more than
30 passages. Apart from intensive alkaline phosphatase activity, miPSC showed
significant expression of endogenous OCT4, SOX2 and NANOG by RT-PCR and
stained strongly for SOX2, TRA 1-60 and NANOG by immunohistochemistry. Next, we
investigated whether these putative miPSC were dependent on the continuous
expression of exogenous reprogramming factors to maintain pluripotency. After culture
in the absence of doxycycline for six passages, we observed that the miPSC couldn’t
sustain the expression of OCT4, SOX2 and NANOG, suggestion of partial and/or
unstable reprogramming. Although dependent on doxycycline, these miPSC lines will
be useful experimental model to study mink early embryo development and the
phenomenon of delayed implantation in this species.
76
e
th
Heparin-alpha-glucosaminide
N-acetyltransferase
(HGSNAT)
induces
morphological changes in the epididymal epithelium and spermatozoa of
adult mice
Carvelli L.1, Pshezhetsky A. V.2, Oliviera R.1, Hermo L.1, Morales C.R.1
1
2
Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.
Department of Pediatrics and Biochemistry, McGill University, Montreal, Canada.
The epithelial cells lining the epididymis provide a proper environment for sperm
maturation controlled in part, by the endocytosis of substances from the lumen.
Heparan sulfate (HS) is a component of basement membranes and the apical cell
surface. In many cell types, HS is degrated after endocytosis in a stepwise fashion in
lysosomes by the action of several enzymes, such as Heparin-alpha-glucosaminide Nacetyltransferase (HGSNAT). In mice, inactivation of the Hgsnat gene shows defects in
sperm motility and in vitro fertilization. The objectives of this investigation were to
determine the morphological effects of Hgsnat inactivation on the epithelial cells of
epididymis and epididymal spermatozoa of adult mice by electron microscopy (EM). In
wild type mice, the epithelial principal cells contained several small to medium sized
dense spherical lysosomes. This contrasted the Hgsnat deficient mice, where
numerous large to gigantic sized looking vacuoles of different shapes appeared both
supranuclearly and infranuclearly; they were confirmed to be lysosomes by
immunohistochemical analysis. Basal cells also became highly vacuolated and greatly
increased in size, as did halo and myoid cells. Occasional huge cells occupied the mid
to basal region of the epithelium which were suggested to be detached principal cells.
In the lumen, numerous spermatozoa presented abnormal bent or multiple tails. These
observations although evident in all the epididymal regions, were especially prominent
in the corpus region. Our results reveal that upon Hgsnat inactivation, epididymal
epithelial cell morphology is altered that may affect sperm structure and maturation.
Supported by NSERC and CIHR.
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Exploring mutations and polymorphism in human binder of sperm
homolog 1 gene in idiopathic males
Samin Sabouhi Zarafshan, Bruno Prud’homme, Puttaswamy Manjunath
Maisonneuve-Rosemont Hospital Research Center, Department of Biochemistry and
Molecular Medicine, University of Montreal, Quebec, Canada
The latest statistics in Canada shows that ~8% of human male adults in a population
will seek medical attention for infertility problems. Genetic abnormalities are thought to
be responsible for many of those idiopathic infertility cases. Capacitation, sperm
maturation step, is a key step for fertilization.Our laboratory has demonstrated that a
group of proteins from bovine seminal plasma called Binder of Sperm (BSP proteins)
bind to the sperm membrane and promote capacitation. Recently, one BSPhomologous gene has been identified in the human (BSPH1) and is expressed in the
epididymis. In vitro experiments using a recombinant BSPH1 revealed that it is able to
promote sperm capacitation similar to the bovine proteins. Our working hypothesis is
that, in the population, mutations exist in the BSPH1 gene, which cause defect in sperm
capacitation and these mutations could be the origin for certain cases of idiopathic
infertilities. The goal of this project is to identify new mutations or polymorphisms in the
BSPH1 gene and to investigate the impact of these genetic differences on fertility. We
would focus on three objectives: (1) produce BSPH1 recombinant proteins with known
mutations and study their effect of BSPH1 mutations already identified on sperm
capacitation, to sort out which mutation is good to study, we analyse them by SNAP 2
software ranging from tolerant to intolerant. (2) identify new mutations in the genomic
region coding for BSPH1 and verify if there is a correlation with male infertility and (3) to
find problems linked with the expression or regulation of the BSPH1 gene that would
not be identified in the sequencing of coding regions.Identification of infertility due to
mutation(s) related to BSP genes could provide a diagnostic tool and therapeutic
applications.
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Lactate produced during labor exerts anti-inflammatory effects on the
uterus via GPR81 (HCA1)
Mathieu Nadeau-Vallée*1, Ankush Madaan*1, Jose Carlos Rivera2, Dima Obari3,
Xin Hou1, Sylvie Girard4, Sylvain Chemtob1
1
Departments of Pediatrics, Ophthalmology and Pharmacology, CHU Sainte-Justine
Research Center, Montréal, Canada, H3T 1C5;
2
Maisonneuve-Rosemont Hospital, Research Center, Montreal, Canada, H1T 2M4.
3
Department of Pharmacology, Université de Montréal, Montréal, Canada H3C 3J7
4
Departments of Obstetrics and Gynecology, CHU Sainte-Justine Research Centre,
Montréal, Canada, H3T 1C5.
Inflammation inside the uterus triggers pro-labor pathways and orchestrates on-time
labor onset, but must be resolved at postpartum. During labor, glycogen and glucose
are utilized by myometrial smooth muscle cells (mSMC) to produce ATP, leading to the
accumulation of intermediates of carbohydrate metabolites, including lactate. Recently,
lactate has been shown to activate a G protein-coupled receptor: GPR81 (HCA1).
GPR81 has been suggested to regulate inflammation. We hypothesize that the lactate
produced during labor could act on its receptor in the uterus to exert feedback antiinflammatory effects. Using GPR81-/- mice, we investigated the expression of GPR81
in uterus and the pharmacological role of lactate during labor via GPR81.
Immunohistological analysis revealed expression of GPR81 in the uterus specifically in
mSMC. We found that GPR81 expression increases during gestation to peak near
labor, and that lactate levels increase from 2mM up to 9mM during labor. In primary
mSMC and ex vivo uteri from WT mice stimulated with the pro-inflammatory cytokine
interleukin-1, lactate reduced the transcription of key pro-inflammatory genes; this was
not observed in cells and tissues from GPR81-/- mice. GPR81 was found to act
specifically during labor, as we found that GPR81-/- mice had unaltered gestation
length, albeit having increased inflammation during labor. Collectively, our data show
that the lactate produced during labor has anti-inflammatory effects on the uterus via
GPR81. This discovery may represent a novel feedback mechanism to control the
inflammation during labor and establishes a rationale for the usage of GPR81 agonists
for the prevention of PTB associated with inflammation.
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Regulation of gene expression in murine granulosa cells
Ejimedo Madogwe, Milena Taibi, Yasmin Schuermann, and Raj Duggavathi
McGill University, Department of Animal Science
The three LH-induced genes, Star, Ptgs2 and Pgr are critical for ovulation; because
failed expression of any one of these genes results in abrogation of ovulation.
Epigenetic modifications such as histone acetylation and methylation have been
reported to regulate gene expression by altering chromatin accessibility. However, how
such modifications alter the expression of ovulatory genes is still largely unknown. We
investigated chromatin accessibility and histone acetylation at the promoter regions of
the
aforementioned
genes.
Granulosa
cells
were
collected
from
immature
superovulated mice at different time points of follicular development and ovulation.
First, deoxyribonuclease (DNase) was used to digest the open chromatin. Quantitative
PCR analysis showed reduced amplification of the promoter regions of Star, Ptgs2 and
Pgr at 4h post-hCG than 0h and 48h post-eCG indicating that the chromatin was more
accessible at 4h post-hCG. Formaldehyde-assisted isolation of regulatory elements
(FAIRE) assay revealed higher nucleosome-depleted areas (open chromatin) in the
promoter regions of Ptgs2 and Pgr at 4h post-hCG. Finally, we used chromatin
immunoprecipitation (ChIP) assay to determine histone acetylation status in the
promoter regions of Star, Ptgs2 and Pgr. In all three genes, there was a significant
increase in acetylation of histone H3 at lysine 27 at 4h post-hCG compared to 48h posteCG. These results demonstrate that LH increases chromatin accessibility and histone
acetylation at the promoter region of Star, Pgr and Ptgs2. Our planed studies include
assessing the dynamics of repressive histone markers on the promoters of these genes
and evaluating the role of signaling pathways in histone modifications.
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Combined effects of DNA methyltransferase 1o-deficiency and ovarian
stimulation on embryonic outcome and epigenetic patterning at midgestation
Martel, J, Whidden, L, Chan D, Trasler JM.
McGill University Health Center- Research Institute, Montréal, Québec
The use of assisted reproductive technologies (ART) has been linked with an increased
incidence of growth and genomic imprinting disorders in children. DNA methylation,
catalyzed by DNA methyltransferases (DNMTs), is the major epigenetic mark
associated with imprinting. Mouse studies have shown reduced expression of DNMTs
in aging oocytes. We propose that factors related to underlying infertility (i.e. reduced
expression of DNMTs) will increase offspring susceptibility to aberrant DNA methylation
patterning, exacerbated by ART. We assessed the effects of maternal deficiency in
DNMT1o, in combination with ovarian stimulation on offspring DNA methylation and
development. Blastocysts were collected from superovulated control and Dnmt1o+/females and transferred non-surgically to recipients. Mid-gestation embryos and
placentas were collected, assessed for developmental delay and morphological
abnormalities,
and
DNA
methylation
was
examined
at
imprinted
genes
(pyrosequencing) and globally (Reduced Representation Bisulfite Sequencing -RRBS).
Similarly, mid-gestation embryos were collected from naturally cycling (NAT) control
and Dnmt1o+/- females. A significant increase in the proportion of abnormal embryos in
Dnmt1o+/- females was observed in the ART group only. DNMT1o deficiency resulted
in significantly increased variability in DNA methylation at imprinted H19 and significant
loss of methylation at Peg1 in ART-placentas, with little change in embryos. ART-RRBS
analysis revealed an increased sensitivity to DNMT1o deficiency in placenta compared
to embryo, with sex-specific trends apparent. Thus, in contrast to the NAT group, ART
female placentas appear to be particularly susceptible to hypomethylation at intergenic
regions. These results indicate that placenta could be a good indicator of genes
affected by ART and infertility factors, that could lead to embryo defects.
Acknowledgments: Supported by CIHR, RQR and the RI-MUHC.
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Loss of PORCN induces cellular alterations in the epididymis but is
dispensable for steroidogenesis in the testis and adrenal glands
Adrien Levasseur 1, Marilène Paquet 1, Derek Boerboom 1, Alexandre Boyer 1
1
Centre de Recherche en Reproduction Animale (CRRA), Faculté de médecine
vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada
It was demonstrated that WNT signaling could regulate steroidogenesis in both ovaries
and adrenal glands, whereas deletion of Wnt5a in a mouse model caused testicular
hypoplasia and bilateral cryptorchidism. Despite these results, potential roles for WNT
signaling in testicular steroidogenesis have never been evaluated. In order to study the
roles of the WNT proteins secreted by Leydig cells in vivo, a transgenic mouse model
targeting the acetyltransferase protein Porcupine (PORCN) in steroidogenic cells
(Porcn[tm1.1Vdv/Y];Nr5a1[cre/+]) was generated to eliminate the secretion of WNTs by
Leydig cells. Preliminary results demonstrated robust recombination of the Porcn allele
in the testes and adrenal glands and a 4-fold (P < 0.0001) decrease in Porcn
expression in those tissues. A decrease in the gonadosomatic (90% of control, P <
0.0002), and the adrenosomatic indexes (84%, P = 0.004) was observed in 4 month-old
mutant animals. A 2.5-fold (P = 0.008) decrease in total sperm count and a 2.0-fold (P
= 0.0096) decrease in sperm density were also observed in the epididymides of these
animals. However, no difference was observed in intratesticular testosterone levels or
in the expression of steroidogenic genes in the testes or in the adrenal glands of mutant
animals. Finally, unilateral increased testis weight was observed in 43% (3/7) of 4
month-old mutant mice. This increase was associated with liquid retention in the
seminiferous tubules, which could be caused by a spermatocele or a blockage further
down the reproductive tract. Histopathological analyses of the epididymides showed the
presence of mucinous metaplastic cells blocking the cauda of the epididymides. This
blockage was associated with the liquid retention observed in the testes. These
preliminary data suggest that WNT secretion by Leydig cells is dispensable for
steroidogenesis, but is necessary for the differentiation of epididymis cauda epithelial
cells. Studies are underway to determine if the phenotype observed in the epididymis is
caused by the downregulation of Porcn expression in the Leydig cells or by
recombination of the Porcn allele in the epididymis.
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Impact of Zearalenone mycotoxins on Sertoli cell survival and on their role
for the establishment of the spermatogonial stem cell niche
Christian Savard, Perrine Nogues, Younes Chorfi, and Alexandre Boyer.
Animal reproduction research center (CRRA), Faculté de médecine vétérinaire,
Université de Montréal, Saint-Hyacinthe, Québec, Canada
The worldwide contamination of grains designated to human and animal feeding with
Fusarium mycotoxins is a significant problem. Among Fusarium mycotoxins,
zearalenone (ZEA) is one of the most prevalent mycotoxins found in cereals and has
been linked to male reproduction problems. In this study, the impact of ZEA on the
Sertoli cell line TM4 was evaluated.
Dose-dependent (1 M-100 M) studies, demonstrated that ZEA at the concentrations of
40 M or higher significantly decreased the viability of TM4 cells. ROS production was
also exacerbated by ZEA and occured concomitantly with the activation of JNK and
p38. Inhibition of JNK and p38 with SP600125 and SB203580 respectively was able to
prevent the increase of ROS observed in cells treated with ZEA demonstrating that the
activation of MAPK signaling pathway is responsible for this increase. The impact of
ZEA on mRNA expression of junction-associated molecules, cell receptors and
secreted factors regulating the establishment of the spermatogonial stem cell niche was
also evaluated. TM4 cells were treated with the highest concentration of ZEA (20 M)
not affecting cell viability. Among the genes tested, ZEA significantly decreased the
expression of three genes, Ccl9, Fgf2 and Kitl, necessary for spermatogonial stem cell
migration and proliferation/differentiation of spermatogonia. Interestingly, treatment with
MAPK inhibitor did not restore gene expression of Ccl9 and Fgf2 suggesting that the
observed decrease of expression of these genes depend on a different pathway.
These results suggest that exposition of pre pubertal animals to dietary ZEA could
affect the establishment of the spermatogonial stem cell niche, spermatogenesis and
eventual sperm production in these animals.
Funded by Mitacs (Accelerate Program) and MediVet
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Regulation of TSPO expression during germ cell differentiation
Gurpreet Manku1, 2, 3 and Martine Culty1, 2, 3
(1)
The Research Institute of the McGill University Health Centre, and Departments of (2)
Pharmacology & Therapeutics and (3) Medicine, McGill University, Montreal, Quebec,
Canada.
Translocator protein 18kDa (TSPO) is a high affinity cholesterol- and drug-binding
protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a
role in cholesterol mitochondrial transport (among other functions). We have previously
shown that TSPO is expressed in postnatal day (PND) 3 rat gonocytes, precursor cells
to spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and
migration, followed by retinoic acid (RA)-induced differentiation. Understanding these
processes is important since their disruption may lead to the formation of carcinoma in
situ, precursor of testicular germ cell tumors (TGCTs). Previously, we showed that
TSPO is expressed in gonocytes and in some adult germ cells, and it was not involved
in gonocyte proliferation. In the present study, we found that TSPO expression is
downregulated between PND3 gonocytes and PND8 spermatogonia, and in gonocytes
undergoing RA-induced differentiation. Similarly, in F9 embryonal carcinoma cells, a
TGCT cell line with embryonic stem cell properties, there was a significant decrease in
TSPO expression during RA-induced differentiation. Furthermore, in normal human
testes, one could locate TSPO not only in Leydig cells, but also in discrete phases of
germ cell development such as the forming acrosome of round spermatids. By contrast,
seminomas, the most common type of TGCT, presented strong expression of TSPO
mRNA and protein. Thus, TSPO appears to be tightly regulated during germ cell
differentiation, and its high levels in seminomas suggest that its dysregulation might
have deleterious results in germ cells. These data suggest that TSPO has an important
role in germ cell development.
84
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Random unilateral chromosome inheritance is a novel explanation for
mosaic embryo aneuploidy
Cayetana VÁZQUEZ DE CASTRO DIEZ (1), Shardul TRIVEDI (3), Kazuo YAMAGATA
(4)
, Jenna HAVERFIELD (1), Greg FITZHARRIS (1,2,3)
1
Centre de Recherche du CHUM, Université de Montréal, Canada
Departement d’Obstetrique et Gynécologie, Université de Montréal, Canada
3
Department of Cell and Developmental Biology, University College London, UK
4
Kinki University, Japan
2
Whilst aneuploidy in the preimpantation embryo is considered a major cause of
infertility, the mechanisms responsible are poorly understood. Human preimplantation
embryos commonly possess micronuclei (MN), small nucleus-like structures containing
small amounts of DNA. However, the impact of micronuclei has remained elusive. Here
we show that MN commonly occur in mouse embryos both in vitro and in vivo, 70% of
embryos typically possessing 1-5 MN. Using fixed and live time-lapse imaging we find
that encapsulation of chromosomes in MN leads to reduced nuclear envelope integrity
and function, accompanied by high levels of DNA damage, as determined by
immunofluorescence analysis of markers; LaminB1, LSD1 and H2AX, respectively.
Strikingly, during the next cell cycle, MN usually fail to be segregated correctly along
with the rest of the chromosomes and are inherited by one of the daughter cells (94%).
MN chromosomes lacked CREST kinetochore staining did not interact with spindle
microtubules in mitosis. We show that MN have no impact on the timings of cell
divisions. Using 4D tracking of MN during mitosis in H2B:RFP embryos co-injected with
inert fluorescent microbeads we show that MN movement is random resulting from
normal cytoplasmic dynamics. Consistent with this, MN distribution is similar between
the first two embryonic lineages. We thus conclude that loss of kinetochore function
within MN leads to repeated random unilateral chromosome inheritance. This
unexpected pattern of chromosome inheritance is a previously undescribed form of
chromosome missegregation, and provides a novel explanation for the high levels of
aneuploidy in early embryos.
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Comprehensive Genotype Phenotype Correlations Reveals NLRP7 Role in
Regulating
Embryonic
Tissue
Differentiation
and
Trophoblastic
Proliferation
Ngoc Minh Phuong Nguyen1,2, Li Zhang2, Ramesh Reddy1,2, Christine Déry1,2,
Jocelyne Arseneau3, Annie Cheung4, Urvashi Surti5, Lori Hoffner5, Muhieddine
Seoud6 , Ghazi Zaatari7, Rashmi Bagga8, Radhika Srinivasan9, Philippe Coullin10,
Asangla Ao1,2, Rima Slim1,2*.
1
2
3
Departments of Human Genetics and Obstetrics and Gynecology, Department of Pathology,
4
McGill University Health Centre, Montreal, Quebec, Canada, Department of Pathology,
5
University of Hong Kong, China, Department of Pathology, Magee-Women’s Hospital,
6
7
Pennsylvania, USA, Department of Obstetrics and Gynecology, Department of Pathology,
8
9
American University of Beirut, Lebanon, Department of Obstetrics & Gynecology, Cytology &
10
Gynecological Pathology, Post Graduate Institute of Medical Education and Research, India
INSERM U 782, Endocrinologie et Génétique de la Reproduction et du Développement, France.
Hydatidiform mole is a human pregnancy with excessive trophoblastic proliferation and
abnormal embryonic development. NLRP7 was identified to be a major gene
responsible for recurrent hydatidiform moles. To better understand its role, we used five
approaches to comprehensively characterize the parental contribution to 103
hydatidiform moles from patients with or without NLRP7 mutations and variants. We
demonstrate that all 36 molar tissues from patients with two NLRP7 defective alleles
are diploid biparental except for one mole found to be mosaic with two cellular
populations. In these 36 moles, we investigated the expression of the imprinted cell
cycle regulator gene,CDKN1C (p57KIP2), and the proliferation marker, Ki-67. We found
that missense mutations are associated with p57KIP2 expression, presence of embryonic
tissues of inner cell mass origin, mild trophoblastic proliferation, and low Ki-67
expression. However, protein-truncating mutations in the coding region were
associated with the lack of p57KIP2 expression, absence of embryonic tissues,
excessive trophoblastic proliferation, and high expression of Ki-67. In these 36 diploid
biparental moles caused by mutations in NLRP7, the level of trophoblastic tissue
proliferation was inversely correlated with the presence of embryonic tissues of inner
cell mass origin. Our data suggest that NLRP7 acts upstream of p57KIP2and Ki-67
during early development and regulates, directly or indirectly, a tight balance between
embryonic tissue differentiation and trophoblastic proliferation.
86
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Caractérisation des vésicules extracellulaires produites par les cellules
épithéliales de l’endomètre et de l’épididyme
Yann Becker, Nicolas Lacroix-Pepin, Olivia Jerczynski, Michel A. Fortier,
Clémence Belleannée
Centre de recherche du CHU de Québec, Département d'obstétrique, gynécologie, et
reproduction, Université Laval, Québec, Canada
La maturation du spermatozoïde et l’acquisition de sa capacité à féconder se produit
tout au long des tractus mâle et femelle, jusqu’à son interaction avec l’ovocyte. Le
génome des spermatozoïdes étant extrêmement condensé et inapte à synthétiser de
nouvelles protéines, toutes les modifications spermatiques observées au niveau posttesticulaire sont dépendantes des facteurs présents dans leur fluide environnant. Parmi
ces facteurs, les vésicules extracellulaires (VEs) jouent des rôles importants dans la
communication intercellulaire via le transfert de protéines et de microARNs. Notre
étude vise à caractériser les VEs libérées par les cellules principales de l’épididyme et
par les cellules épithéliales de l’endomètre à partir des lignées cellulaires immortalisées
DC2 et HIEEC-22. La production de VEs a été étudiée par cytométrie après marquage
des cellules au CMFDA et stimulation ou non avec du calcium ionophore. Les tailles de
ces VEs ont été évaluées par Nanosizer après purification des exosomes et
caractérisées au niveau de leur expression des marqueurs protéiques d’exosomes et
de leur contenu en microRNAs. Ces lignées cellulaires non permettront ainsi d’étudier
les mécanismes de libération des VEs le long des tractus mâles et femelles et
d’évaluer, in vitro, leur rôle dans la communication intercellulaire et le contrôle du
pouvoir fécondant.
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Conférencier invité - Invited Speaker
Dr. Christopher Geyer
Dr. Christopher Geyer completed a B.S. in 1995 in
Biology at the Virginia Polytechnic Institute and State
University (Virginia Tech), and then worked for two
small biotechnology companies (PPL Therapeutics and
CropTech Biosciences). In 2000, he left to pursue a
Ph.D. at The University of Texas Health Science
Center at San Antonio in the laboratory of Dr. John
McCarrey. After graduating in 2005, he joined Dr. E.
Mitch Eddy’s laboratory as a postdoctoral fellow at the
National Institute of Environmental Health Sciences
(NIEHS) in Research Triangle Park, NC. In 2010, he became an assistant professor in
the Department of Anatomy & Cell Biology at the Brody School of Medicine at East
Carolina University.
His laboratory uses mouse spermatogenesis as a model system to investigate
mechanisms regulating cellular differentiation. Spermatogenesis begins in the neonatal
mouse testis with the segregation of prospermatogonia into distinct undifferentiated and
differentiating populations. A proportion of undifferentiated spermatogonia retain stem
cell potential (as spermatogonial stem cells, or SSCs), and the remainder becomes
progenitor spermatogonia that proliferate and differentiate in response to retinoic acid
(RA). Currently, the members of his laboratory are working hard to answer three basic
questions about this critical cell fate decision. First, how do spermatogonia differentially
respond to RA? Second, what molecular pathways does RA activate to direct
differentiation? Third, what cellular changes occur during differentiation? The answers to
these questions will provide foundational information about not only spermatogonial
differentiation, but also about the processes that precede (stem cell self-renewal and
progenitor proliferation) and follow (meiosis).
On Wednesday, Dr. Geyer will give a talk entitled: “Defining the essential role for
retinoic acid (RA) in spermatogonial differentiation”.
88
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Présentations – Presentations : Session III
Présidente – Chair : Jacquetta Trasler, McGill University
Co-présidente – Co-chair : Vanessa Théberge, Université Laval
I. Exercise training prevents intrauterine growth restriction in mice lacking
p57kip2 gene
Aida Kasaei Roodsari, Université de Montréal (Page 90)
11h30 – 11h45
II. Female mice lacking SMAD3 DNA binding activity and SMAD4 in
gonadotropes are FSH deficient and infertile
Yining Li, McGill University (Page 91)
11h45 – 12h00
III. Protecting the Genetic Diversity of the Honey Bee (Apis mellifera):
Preservation Methods of Drone Semen
Marilène Paillard, Université Laval (Page 92)
12h00 – 12h15
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Exercise training prevents intrauterine growth restriction in mice lacking
p57kip2 gene
Aida Kasaei Roodsari1,2, Olga Asaftei 1,2, Ms. Crina Solomon1 and Julie L.
Lavoie1,2
1
2
Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM)
Department of Kinesiology of the Université de Montréal, Montréal, Québec, Canada.
Intrauterine growth restriction (IUGR) affects 5-10% of human pregnancies and is a
major cause of perinatal morbidity and mortality worldwide. In our previous studies on
preeclampsia mouse models, we found that exercise training (ExT) before and during
pregnancy reduces maternal blood pressure, and prevented IUGR by improving
placental development.
The aim of this study was to investigate the beneficial effects of ExT on IUGR in an
animal model without preeclampsia.
In this study, we used p57kip2+/- mice, a model of IUGR. To investigate the role of ExT
we placed mice in cages with free access to an exercise wheel four weeks prior to and
throughout pregnancy. At the end of gestation, mice were sacrificed to harvest and
weigh fetus and placentas. We confirmed the presence of IUGR in sedentary p57+/mice, reduced placental mass, increased placental alterations as well as smaller litter
size with an increased number of necrotic pups. ExT prevented IUGR as well as
normalized litter size and placental mass and alterations. The expression of the
vascular endothelial growth factor was reduced in knockout sedentary mice and was
normalized by ExT. In contrast to trained mice, we found elevated inflammatory
markers in the placenta (interleukin-1 and MCP-1) in sedentary knockout mice, which
suggests that placental inflammation may contribute to the placental pathology in this
model.
These results propose that ExT prevents IUGR by improving angiogenesis, placental
alterations and inflammation.
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Female mice lacking SMAD3 DNA binding activity and SMAD4 in
gonadotropes are FSH deficient and infertile
Yining Li1, Ulrich Boehm2, Chu-Xia Deng3, Jonathan Graff4 and Daniel J. Bernard1
1
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec,
Canada
2
Department of Pharmacology and Toxicology, University of Saarland School of
Medicine, Homburg, Germany
3
Faculty of Health Sciences, University of Macau, Macau SAR, China
4
Department of Developmental Biology, University of Texas Southwestern, Dallas,
Texas, USA
Pituitary follicle-stimulating hormone (FSH) is an essential regulator of gonadal function
and fertility in females and of quantitatively normal spermatogenesis in males. Pituitaryderived activins directly stimulate FSH synthesis by regulating transcription of the FSH
subunit gene (Fshb) in gonadotrope cells. According to the in vitro studies, activininduced murine Fshb transcription essentially requires SMAD4 binding to a SMADbinding element (SBE) in the proximal Fshb promoter. Surprisingly, mice lacking
Smad4 gene in gonadotropes retain residual FSH production and fertility. We
hypothesize that this is due to the compensation of SMAD3, which is also capable to
bind to SBE at Fshb promoter region. To test this hypothesis, using the Cre-lox system,
we generated mice lacking both SMAD3 DNA binding domain as well as SMAD4 in
their gonadotropes (hereafter, S3/4cKO). Our results show that both basal and activinstimulated Fshb mRNA expression is significantly impaired in primary pituitary cultures
from male S3/4cKO mice. Furthermore, both S3/4cKO males and females are
hypogonadal. S3/4cKO females have thread-like uteri and their ovaries lack preovulatory follicles and corpora lutea, suggesting severe FSH deficiency and
anovulation. Indeed, S3/4cKO females are sterile after 6-months breeding trials. In
conclusion, our study suggests that SMAD3 is required for FSH synthesis in vivo and
the residual FSH synthesis in Smad4 deficient mice likely reflects compensatory
actions of SMAD3.
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Protecting the Genetic Diversity of the Honey Bee (Apis mellifera):
Preservation Methods of Drone Semen
Marilène Paillard1,2, Andrée Rousseau2, Pierre Giovenazzo2, Janice L. Bailey1
1
Animales, Université Laval;
2
Équipe de Recheche en Apiculture, Centre de recherche en sciences animales de
Deschambault
In the last decades, there have been dramatic losses of honey bee colonies in many
countries. Conservation of honey bee sperm is an effective strategy to protect the
species and their genetic diversity. Sperm storage is possible at room temperature, but
for many species, cryopreservation is the preferred method for the long-term storage of
gametes. However, cryopreservation of honey bee drone semen is not optimized. Our
overall objective is to develop a method of drone semen cryopreservation, therefore,
two experiments were conducted. Hypothesis #1 was that cryopreservation of drone
semen is more effective for long-term storage than at above-freezing temperatures. We
therefore compared the efficiency, based on sperm viability, of two honey bee semen
preservation temperatures: frozen (cryopreservation is, in fact, necessary to conserve semen. However, the cryoprotectant
used for drone sperm freezing, DMSO (dimethyl sulfoxide), is toxic to queens after
instrumental insemination. Hypothesis #2, therefore, was that centrifugation of
cryopreserved semen to remove DMSO prior to insemination improves queen fertility.
Our results indicate that centrifuging semen does not affect sperm viability (78.2±2.9 vs
74.8±3.8; P>0.05). After queen insemination, both spermathecae and brood production
were evaluated, but the results varied greatly due to queen mortality, possibly due to
the mucus present in the semen. Therefore, we cannot yet confirm that centrifugation
improves queen health after insemination. Nonetheless, our study confirms that
cryopreservation of honey bee sperm is possible and necessary for long-term
conservation.
92
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Conférencière invitée - Invited Speaker
Dr. Rebecca Krisher
Dr. Krisher received her M.S. at North Carolina State
University, and then worked at Granada BioSciences
research division in College Station, Texas for
several years prior to completing her Ph.D. at Virginia
Tech. She conducted post-doctoral research with Dr.
Barry Bavister at the University of Wisconsin. She
was Assistant and then Associate Professor at
Purdue University and the University of Illinois before
moving to her current role as Research Director at
the National Foundation for Fertility Research in Lone
Tree, Colorado.
Dr. Krisher’s research program focuses on defining physiological processes within
mammalian oocytes and embryos that are critical for subsequent embryonic and fetal
development. Specifically, Rebecca focuses on metabolic pathways that affect viability
and competence. Her laboratory primarily uses the mouse and bovine model in their
basic research. She is currently working to elucidate the metabolic role of specific amino
acids on metabolism and proliferation. She is also collaborating with colleagues at the
metabolomics facility at Colorado State University to determine the overall metabolic
profile of embryos, and to elucidate potential non-invasive metabolic biomarkers of
embryo competence. Rebecca is also interested in the consequences of maternal age
on oocyte quality. Her laboratory is investigating methods to enhance the metabolism
and thus the quality of maternally aged oocytes and the resulting embryos. Her
laboratory is also investigating new approaches for human in vitro maturation.
In her current role, she is translating these research findings into clinical application via
improved culture media and new clinical treatments and assays to advance human
assisted reproduction.
On Wednesday, Dr. Krisher will give a talk entitled: “The Mammalian Oocyte:
Metabolic Challenges and Opportunities”.
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Présentations – Presentations : Session IV
Président – Chair : Patrick Blondin, Boviteq
Co-président – Co-Chair : Samuel Leblanc, Université de Sherbrooke
I. Sustained activation of the canonical WNT pathway in spermatogonial stem
cells induces their differentiation and causes progressive germ cell loss in
the transgenic mouse model Ctnnb1 [tm1Mmt/+];Ddx4[cre/+]
Adrien Levasseur, Université de Montréal (Page 95)
14h45 – 15h00
II. The Role of ERK1/2 in bovine ovulation
Yasmin Schuermann, McGill University (Page 96)
15h00 – 15h15
III. Modulation de la réponse inflammatoire des macrophages et cellules
trophoblastiques par la cytokine gestationnelle leukemia inhibitory factor
(LIF)
Jovane Hamelin-Morrissette, UQTR (Page 97)
15h15 – 15h30
IV. High-dose folic acid supplementation alters the human sperm methylome
and is influenced by the MTHFR C677T polymorphism
Mahmoud Aarabi, McGill University (Page 98)
15h30 – 15h45
V. The importance of the Homologous Recombination and the Nonhomologous
End-Joining pathways for DNA repair during early embryo development
Rodrigo C. Bohrer, McGill University (Page 99)
15h45 – 16h00
94
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Sustained activation of the canonical WNT pathway in spermatogonial
stem cells induces their differentiation and causes progressive germ cell
loss in the transgenic mouse model Ctnnb1 [tm1Mmt/+];Ddx4[cre/+]
Adrien Levasseur 1, Xiangfan Zhang 2, Sabrina Sicilia 1, Marilène Paquet 1, Makoto
Nagano 2, Alexandre Boyer 1, Derek Boerboom 1
1
Centre de Recherche en Reproduction Animale (CRRA), Faculté de médecine vétérinaire,
Université de Montréal, St-Hyacinthe, Québec, Canada
2
Department of Obstetrics and Gynecology, McGill University, Montréal, Québec, Canada
WNT signaling is involved in the regulation of multiple biological processes, such as
embryogenesis, cell fate determination and differentiation, and stem cell regulation. In
vitro spermatogonial stem cell (SSC) culture suggests that canonical WNT signaling,
through
-catenin (CTNNB1) is involved in SSC differentiation while non-canonical
WNT signaling is involved in SSC maintenance and proliferation. To demonstrate the
role(s) of canonical WNT signaling in SSC differentiation in vivo, transgenic
Ctnnb1[tm1Mmt/+];Ddx4[cre/+] mice were generated to obtain sustained activation of
the WNT/CTNNB1 pathway in SSCs. Sustained activation of CTNNB1 was readily
detectable in pro-spermatogonia from 5-day-old Ctnnb1[tm1Mmt/+];Ddx4[cre/+] mice
and progressive atrophy of the testes was observed starting at 4 months of age. At the
histological level, seminiferous tubules of 8-month-old Ctnnb1[tm1Mmt/+];Ddx4[cre/+]
mice and older showed clear signs of degeneration. Transplantation of germ cells from
8-month-old Ctnnb1[tm1Mmt/+];Ddx4[cre/+] mice into testes of germ cell-depleted wildtype recipient mice demonstrated a 49% (p=0.02) reduction in total functional SSC
numbers in mutant testes compared to control testes. To determine the effects of
CTNNB1 on proliferation and differentiation of SSCs, SSCs from 5-day-old
Ctnnb1[tm1Mmt/+];Ddx4[cre/+] and Ctnnb1[tm1Mmt/+] mice were cultured and RNA
was extracted from these SSCs (passage 5). A 4.5, 7.8 and 1.6 (p<0.005) fold
decrease in the expression of the SSC proliferation markers Oct4, Plzf and GFR 1,
respectively, was observed in SSCs from Ctnnb1[tm1Mmt/+];Ddx4[cre/+] compared to
control SSCs. A 2.5 (p<0.005) fold increase in the differentiation marker Kit was also
observed in mutant SSCs compared to control SSCs. Taken together, these results
suggest that CTNNB1 can direct SSC toward differentiation, causing progressive
depletion of the SSC pool in Ctnnb1[tm1Mmt/+];Ddx4[cre/+] mice.
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95
The Role of ERK1/2 in bovine ovulation
Yasmin Schuermann1, Monique T. Rovani2, Bernardo Gasperin3, Rogério
Ferreira4, Juliana G. Ferst2, Gustavo Ilha2, Paulo B. Goncalves2, Vilceu
Bordignon1, Milena Taibi1, Ejimedo Madogwe1 and Raj Duggavathi1.
1
Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Canada
Laboratory of Biotechnology and Animal Reproduction, BioRep, Veterinary Hospital,
Federal University of Santa Maria, Santa Maria, Brazil
3
Laboratory of Animal Reproduction-ReproPEL, Federal University of Pelatos, Capão
do Leão, Brazil
4
Department of Animal Science, Santa Catarina State University, Santa Catarina,
Brazil
2
The most common reason a dairy cow would be removed from the Canadian herd is
reduced reproductive performance, which accounts for 15.6% of culls. Therefore,
understanding the fundamental mechanisms of ovarian functions in cattle is necessary
to address infertility due to ovarian dysfunction. It was established from studies using
mice that lack of the extracellular-regulated kinase 1 and 2 (ERK1/2) leads to ovulation
failure and infertility due to abnormal gene expression in granulosa cells of ovulating
follicles. Bovine model offers the advantage of examining gene expression in both
granulosa and theca cells of follicles of interest. We synchronized seventeen cows and
treated (intra-follicular microinjection) their dominant follicles with vehicle or PD0325901
(ERK1/2 inhibitor) at three different doses. Cows treated with 50 M PD0325901 failed
to ovulate in response to GnRH stimulus, while others ovulated normally. In the second
experiment, we treated the dominant follicles of ten synchronized cows with vehicle or
PD0325901 (50 M). Cows were ovariectomized at 6h post-GnRH to collect granulosa
and theca cells of ovulating follicles. Relative mRNA levels EGR1, PGR and TNFAIP6
were significantly lower in granulosa cells. Intriguingly, STAR mRNA abundance was
higher in theca cells of inhibitor treated cows. Taken together, these data indicate that
similar to mice, inhibition of ERK1/2 signals abolishes ovulation in cows. However, the
underlying molecular mechanisms appear to be different. Theca cells appear to exhibit
compensatory expression of the steroidogenic enzyme STAR potentially to escape
ovulation failure. Further analysis will be directed towards protein analysis and next
generation sequencing of granulosa cells.
96
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Modulation de la réponse inflammatoire des macrophages et cellules
trophoblastiques par la cytokine gestationnelle leukemia inhibitory factor
(LIF)
Jovane Hamelin Morrissette(1,2), Julie Girouard(1,2), Denise Belgorosky(3), Carlos
Reyes-Moreno(1,2)
1
Centre de recherche BioMED
Université du Québec à Trois-Rivières
3
Instituto de Oncologia “Ángel H. Roffo” Área Investigación, Ciudad de Buenos Aires,
Argentina
2
Le système immunitaire utérin est exceptionnel dans sa capacité à protéger la
muqueuse d’infections tout en supportant l’implantation de la semi-allogreffe qu’est le
fœtus. Toutefois, l’activation aberrante de voies pro-inflammatoires dans les
-maternelle peut affecter la survie et la fonction
du trophoblaste, induisant des complications gestationnelles comme l’avortement
spontané précoce chez les humains. La cytokine LIF, essentielle à l’implantation
embryonnaire chez la souris, a été proposée comme modulatrice de la réponse
inflammatoire tant chez l’humain que chez la souris. Le rôle de LIF la régulation de la
réponse immune en période de gestation, par contre, reste partiellement compris. Ces
études visent à déterminer les mécanismes moléculaires par lesquels agit LIF pour
moduler la
-macrophage colony-
stimulating factor (GM-CSF) et la toxine bactérienne lipopolysaccharide (LPS). Nos
résultats montrent d’abo
-
CSF/Stat5 dans les macrophages humains. De plus, LIF, lorsqu’injecté dans la cavité
péritonéale des souris, peut réduire la production d’oxyde nitrique induite par le LPS
dans les macrophages subséquemment isolés. Ces résultats montrent que LIF agit
pro-inflammatoires. De plus, dans la lignée cellulaire BeWo, ainsi que dans les cultures
primaires de syncytiotrophoblastes, LIF réduit la production de gonadotrophine
CSF/Stat5.
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97
High-dose folic acid supplementation alters the human sperm methylome
and is influenced by the MTHFR C677T polymorphism
Mahmoud Aarabi1,2,3, Maria C. San Gabriel3,4, Donovan Chan2,3, Nathalie A.
Behan5, Maxime Caron1,6,7, Tomi Pastinen1,6,7, Guillaume Bourque1,6,7, Amanda J.
MacFarlane5, Armand Zini3,4, Jacquetta Trasler1,2,3,8
1
2
Department of Human Genetics, McGill University Montreal Children’s Hospital
Research Institute of the McGill University Health Centre, Montreal, QC, Canada
4
5
Division of Urology, Department of Surgery, McGill University, Nutrition Research Division,
6
7
Health Canada, Ottawa, ON, Canada 7McGill University Genome Quebec Innovation Centre,
8
Montreal, QC, Canada Departments of Pediatrics and Pharmacology & Therapeutics, McGill
University.
3
Dietary folate is a major source of methyl groups required for DNA methylation, an
epigenetic
modification
that
is
actively
maintained
and
remodelled
during
spermatogenesis. While high dose folic acid supplementation (up to ten times the daily
recommended dose) has been shown to improve sperm parameters in infertile men,
the effects of supplementation on the sperm epigenome are unknown. To assess the
impact of six months of high dose folic acid supplementation on the sperm epigenome,
we studied 30 men with idiopathic infertility. Blood folate concentrations increased
significantly after supplementation with no significant improvements in sperm
parameters. Methylation levels of the differentially methylated regions of several
imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal
both before and after supplementation. Reduced representation bisulfite sequencing
(RRBS) revealed a significant global loss of methylation across different regions of the
sperm genome. The most marked loss of DNA methylation was found in sperm from
patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T
polymorphism, a common polymorphism in a key enzyme required for folate
metabolism. RRBS analysis also showed that most of the differentially methylated tiles
were located in DNA repeats, low CpG density and intergenic regions. Ingenuity
Pathway Analysis revealed that methylation of promoter regions was altered in several
genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6,
COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal
alterations of the human sperm epigenome associated with high dose folic acid
supplementation, effects that were exacerbated by a common polymorphism in
MTHFR.
98
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The
importance
of
the
Homologous
Recombination
and
the
Nonhomologous End-Joining pathways for DNA repair during early
embryo development
Rodrigo C. Bohrer, Naomi Dicks, Karina Gutierrez, Werner G. Glanzner, Luke
Currin, Laura Michalovic, Matheus Pedrotti de Cesaro, Raj Duggavathi and Vilceu
Bordignon
Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, Quebec,
Canada
DNA double-strand breaks (DSBs) are known to affect early embryo development. In
previous studies, we observed that DSBs affect embryo cleavage kinetics, blastocyst
formation, gene expression and somatic cell reprogramming in porcine embryos. The
homologous recombination (HR) and the nonhomologous end-joining (NHEJ) are the
main pathways activated in response to DSBs to promote DNA repair in somatic cells.
However, the importance of each pathway for DSBs repair in early embryo
development has not been determined. In this study we used specific inhibitors of HR
and/or NHEJ pathways to investigate their specific and combined effects on embryo
development, accumulation of DSBs and cell death. We observed that inhibition of both
pathways significantly reduced embryo development to blastocyst stage in either
control or embryos having increased DNA damage induced by UV exposure. HR
inhibition had more significant effects on embryo development compared to NHEJ
inhibition. The inhibition of each pathway alone or combined increased the
accumulation of DSBs in either control or UV-exposed embryos. Cell apoptosis rate
was increased in blastocysts cultured with inhibitors of HR or both pathways. These
results indicate that both HR and NHEJ pathways are activated to preserve genome
integrity in developing embryos before blastocyst stage. It also suggests that the HR
pathway is more important than the NHEJ pathway for DSB repair during early embryo
development.
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99
Notes:
100
e
th
Partenaires financiers – Financial Partners
Autres partenaires – Other Partners
e
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101
Participants RQR 2015
Nom - Last Name Prénom - Name Organisation - Organization
Courriel - E-mail
Aarabi
Mahmoud
McGill University
[email protected]
Abbassi
Laleh
McGill University
[email protected]
Akoury
Elie
McGill University
[email protected]
Alnoman
Abdullah
[email protected]
Aminmarshi
Fatemeh
[email protected]
Asaftei
Olga
[email protected]
Assaf
Roxane
McGill University
[email protected]
Asselin
Eric
UQTR
[email protected]
Bagheri
Negar
McGill University
[email protected]
Bailey
Janice
Université Laval
[email protected]
Baldassarre
Hernan
McGill University
[email protected]
Baldoceda
Luis
Fertilys
[email protected]
Baumholtz
Amanda
RI-MUHC
[email protected]
Beaud
Hermance
INRS-Institut Armand-Frappier
[email protected]
Becker
Yann
CHUL
[email protected]
Belleannee
Clemence
Université Laval
[email protected]
Benoit
Gabriel
[email protected]
Bergeron
Francis
Université Laval
[email protected]
Bernard
Dan
McGill University
[email protected]
Bienvenue-Pariseault
Josianne
INRS - Institut Armand-Frappier
[email protected]
Blondin
Patrick
Semex
[email protected]
Blouin
Julie
Réseau Québécois en reproduction
[email protected]
Boerboom
Derek
[email protected]
Bohrer
Rodrigo
Mcgill University
[email protected]
Boissonneault
Guylain
Université de Sherbrooke
[email protected]
Boisvert
Annie
RI-MUHC
[email protected]
Bolduc
Nathalie
LifeLabs Genetics
[email protected]
Bose
Rohini
McGill University
[email protected]
Courriel - E-mail
Bouchard
Marie France
Boudali
Samia
Boufaied
Ines
Research center of Ste-Justine
[email protected]
Boyer
Alexandre
[email protected]
Campioli
Enrico
RI-MUHC
[email protected]
Cao
Mingju
CHU Ste-Justine Research Centre
[email protected]
Carbonaro
Adriana
Centre de recherche CHU Ste-Justine
[email protected]
Carvelli
Flavia Lorena
McGill University
[email protected]
Cesar
Valerie
[email protected]
Chan
Donovan
MUHC-RI CHHD Program
[email protected]
Chung
Yuri
MUHC-RI
[email protected]
Clarke
Hugh
McGill University
[email protected]
Connolly
Alexandre
[email protected]
Coutinho
Ana Rita
Fertilys
[email protected]
Culty
Martine
McGill University, RI-MUHC
[email protected]
Currin
Luke
McGill University
[email protected]
de Lamirande
Eve
McGill / MUHC
[email protected]
De Lima
Paula
Sao Paulo University State
[email protected]
de Oliveira
Regiana Lúcia
McGill University
[email protected]
Delbes
Geraldine
[email protected]
Desmarais
Joelle
McGill University
[email protected]
Diaw
Mouhamadou
Université de Montréal - FMV
[email protected]
Di-Luoffo
Mickael
CHU de Québec
[email protected]
Ditisheim
Agnes
CHU Ste-Justine
[email protected]
Dolbec
Catherine
Semex
[email protected]
Downey
Anne Marie
McGill University
[email protected]
Dufort
Daniel
McGill University
[email protected]
El Husseini
Nazem
McGill University
[email protected]
Eskandari-Shahraki
Marzieh
[email protected]
Esmael
Mostafa
UQAM
[email protected]
Essagian
Charles
RI-MUHC
[email protected]
Université Laval
[email protected]
[email protected]
Courriel - E-mail
Fagundes
Lucas
[email protected]
Fan
Jinjiang
The MUHC-RI
[email protected]
Farah
Omar
McGill University
[email protected]
Faubert
Joanie
[email protected]
FitzHarris
Greg
[email protected]
Fortin
Chloé
Université Laval
[email protected]
Fulton
Debra
McGill University
[email protected]
Gagnon
Hugo
PhenoSwitch Bioscience
[email protected]
Gamero Estevez
Enrique
McGill University
[email protected]
Gaye
Bintou
[email protected]
Gévry
Nicolas
[email protected]
Geyer
Christopher
East Carolina University
[email protected]
Girard
Sylvie
Sainte-Justine Research Centre
[email protected]
Glanzner
Werner
McGill University
[email protected]
Guerrero-Netro
Hilda M.
[email protected]
Gutierrez
Karina
McGill University
[email protected]
Hamelin Morrissette
Jovane
[email protected]
Han
Peng
[email protected]
Haverfield
Jenna
CRCHUM
[email protected]
Heba
Taghreed
McGill University
[email protected]
Hermo
Louis
McGill University
[email protected]
Herrero
Belen
MUHC Reproductive centre
[email protected]
Herst
Pauline
Université Laval
[email protected]
Jerczynski
Olivia
Université Laval
[email protected]
Joao
Fabien
INRS - Institut Armand Frappier
[email protected]
Joly
Carol-Ann
UQTR
[email protected]
Kasaei Roodsari
Aida
CRCHUM
[email protected]
Keser
Vafa
McGill University
[email protected]
Khan
Daulat Raheem
Université Laval
[email protected]
Khatir
Hadj
UDM
[email protected]
Khawajkie
Yassemine
McGill University
[email protected]
Courriel - E-mail
Khudhari
Adhwaa
[email protected]
Kimmins
Sarah
McGill University
[email protected]
Krisher
Rebecca
National Foundation for Fertility Research
[email protected]
L. Charest
Phanie
Université Laval
[email protected]
Labrecque
Rémi
Boviteq
[email protected]
Lambert
Simon
Université Laval
[email protected]
Lambrot
Romain
McGill University
[email protected]
Landry
Mylène
MUHC-RI
[email protected]
Landry
David
Université Laval
[email protected]
Larrivée
Jean-François
Immune Biosolutions
[email protected]
Lassonde
Guylaine
[email protected]
Lau
Matthew
McGill University
[email protected]
Lavoie
Julie
Université de Montréal/CRCHUM
[email protected]
Leblanc
Samuel
[email protected]
Leclerc
Pierre
Université Laval
[email protected]
Leduc
Frédéric
Immune Biosolutions
[email protected]
Lee
Blanche
McGill University
[email protected]
Lee
Sunghoon
Research Institute of the MUHC
[email protected]
Lefevre
Pavine
McGill University
[email protected]
Legault
Lisa-Marie
Centre de recherche CHU Ste-Justine
[email protected]
Lessard
Maryse
Université Laval
[email protected]
Levasseur
Adrien
[email protected]
Li
Nancy C.
McGill University
[email protected]
Li
Yining
McGill University
[email protected]
Li
Xinfang
RI MUHC
[email protected]
Liu
Xueqing
McGill University
[email protected]
Lopez
Rosalba
McGill University
[email protected]
Lounas
Amel
Université Laval
[email protected]
Lusignan
Marie-France
RI-MUHC
[email protected]
Lussier
Jacques
[email protected]
Ly
Lundi
Research Institute of the MUHC
[email protected]
Courriel - E-mail
Macaulay
Angus
[email protected]
Madogwe
Ejimedo
McGill University
[email protected]
Manjunath
Puttaswamy
[email protected]
Manku
Gurpreet
McGill University
[email protected]
Maréchal
Loïze
Centre de Recherche du CHU de Québec
[email protected]
Martel
Josée
MUHC Research Institute
[email protected]
Meinsohn
Marie-Charlotte
[email protected]
Michalovic
Laura
McGill University
[email protected]
Moawad
Adel
McGill University
[email protected]
Mondadori
Rafael
McGill University
[email protected]
Morales
Carlos R.
McGill University
[email protected]
Morin-Doré
Léonie
Université Laval
[email protected]
Murphy
Bruce
[email protected]
Nadeau-Vallée
Mathieu
[email protected]
Nagano
Makoto
McGill University
[email protected]
Nakagawa
Shoma
Centre Recherche CHUM, Université de Mo [email protected]
Ndiaye
Kalidou
[email protected]
Nguyen
Ngoc Minh Phuong
McGill University
[email protected]
Noblanc
Anaïs
Université McGill
[email protected]
Ongaro Gambino
Luisina
McGill University
[email protected]
Opu
Md Touhidur Rashid McGill University
[email protected]
Pagé-Larivière
Florence
Université Laval
[email protected]
Paillard
Marilène
Université Laval
[email protected]
Paquet
Marilene
[email protected]
Pedrotti De Cesaro
Matheus
McGill University
[email protected]
Pepin
Émilie
CRCHUM
[email protected]
Piché
Marie-Lou
UQTR
[email protected]
Pilon
Nicolas
UQAM
[email protected]
Portela
Valerio
[email protected]
Price
Chris
[email protected]
Prud'homme
Bruno
CRHMR
[email protected]
Courriel - E-mail
Racowsky
Catherine
Brigham and Women's Hospital
[email protected]
Rahimi
Sophia
McGill University
[email protected]
Rakhila
Halima
CHU de Québec
[email protected]
Reyes-Moreno
Carlos
UQTR
[email protected]
Richard
Francois
Université Laval
[email protected]
Rico
Charlene
Université de Montréal, CRRA/Semex
[email protected]
Robert
Nicholas
Université Laval
[email protected]
Roy
Simon
Civic Bioscience Limitée
[email protected]
Ruchat
Stephanie-May
UQTR
[email protected]
Rwigemera
Arlette
[email protected]
Sabouhi Zarafshan
Samin
[email protected]
Saindon
Andrée-Anne
Université Laval
[email protected]
Savard
Christian
[email protected]
Schang
Gauthier
McGill University
[email protected]
Schuermann
Yasmin
McGill University
[email protected]
Shafiei
Shiva
McGill University
[email protected]
Sheng
Kai
McGill University
[email protected]
Shojaei Saadi
Habib A.
Université Laval
[email protected]
Shuchat
Sholom
Concordia University
[email protected]
Siklenka
Keith
McGill University
[email protected]
Slim
Rima
RI-MUHC
[email protected]
Starr
Lisa
RI-MUHC
[email protected]
St-Jean
Guillaume
[email protected]
Sullivan
Robert
Université Laval
[email protected]
Taguibao
Candice
[email protected]
Taibi
Milena
McGill University
[email protected]
Taketo
Teruko
McGill University
[email protected]
Théberge
Vanessa
Université Laval
[email protected]
Therrien
Jacinthe
[email protected]
Torabi
Ali
[email protected]
Toufaily
Chirine
McGill University
[email protected]
Courriel - E-mail
Trasler
Jacquetta
McGill, MUHC
[email protected]
Tremblay
André
CHU Ste-Justine
[email protected]
Tremblay
Amélie
[email protected]
Tremblay
Jacques J.
Université Laval
[email protected]
Tremblay
Roch
[email protected]
Tremblay
Patricia
Université Laval
[email protected]
Trevors
Christopher
LifeLabs Genetics
[email protected]
Trindade
Amanda
[email protected]
Tsoi
Mayra
[email protected]
Turgeon
Marc-Olivier
McGill University
[email protected]
Vaillancourt
Cathy
[email protected]
Van Themsche
Céline
UQTR
[email protected]
Vaz
Brandon
McGill University
[email protected]
Vazquez de Castro Di
Cayetana
CR CHUM
[email protected]
Vieux
Karl-Frederic
McGill University
[email protected]
Viger
Robert
Université Laval
[email protected]
Vigneault
Christian
Semex
[email protected]
Vincent
Patrick
Semex
[email protected]
Wang
Kevin
McGill University
[email protected]
Wang
Ying
McGill University
[email protected]
Whidden
Laura
McGill University
[email protected]
Wing
Simon
McGill University & MUHC
[email protected]
Xiao Yun
Zhang
MUHC Reproductive Center
[email protected]
Yang
QIN
RI-MUHC
[email protected]
Yatsyna
Anna
[email protected]
Yu
Alex
McGill University
[email protected]
Zamberlam
Gustavo
[email protected]
Zhang
Xiang Fan
RI-MUHC
[email protected]
Zhang
Li
McGill University
[email protected]
Zhu
Jiaqiao
McGill University
[email protected]

participant booklet of the 2015 RQR Symposium

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