SCRS/1999/106 Rev. Col.Vol.Sci.Pap. ICCAT, 51 (3) : 860

SCRS/1999/106 Rev.
Col.Vol.Sci.Pap. ICCAT, 51 (3) : 860-864 (2000)
RÉSUMÉ
Il est critique, pour une gestion efficace des pêches, de définir l’âge de maturité
des espèces de poisson qui sont exploitées. Cette tâche peut toutefois s’avérer
malaisée du fait que, dans de nombreuses pêcheries, le poisson est traité en mer, puis
débarqué éviscéré. L’accès aux tissus de gonades étant ainsi limité, il est difficile de
déterminer le sexe et la maturité. Nous avions traité de cette question dans un travail
antérieur sur la pêche au mérou au sud-est des Etats-Unis. Le présent travail nous a
amenés à élaborer des méthodes biochimiques pour identifier les composantes qui
sont spécifiques des mérous mâles et femelles en processus de maturité. Les premiers
objectifs biochimiques ont été la vitellogénine (VTG), protéine de la formation du
jaune d’oeuf, la testostérone (T), hormone stéroïde de la reproduction, la 11kétotestostérone (11-KT) et l’estradiol-17$ (E2 ). Nous avons trouvé que, chez le
mérou, les calendriers de maturité découlant des mesures biochimiques de la maturité
étaient identiques dans l’ensemble à ceux qui fournissaient les techniques
traditionnelles d’analyse histologique des gonades. Le présent document décrit nos
tentatives d’application de cette technologie au thon rouge (Thunnus thynnus).
Etant donné qu’il est difficile d’accéder aux thons rouges en captivité, nous
avons élaboré un antisérum VTG pour l’albacore (Thunnus albacares) , et un
antisérum de la composante lipovitelline (Lv) de l’oeuf du thon rouge. Ces antisérums
ont servi de base pour l’élaboration d’un test d’inmunoabsorption pour la VTG du
thon. Les test d’inmunoabsorption définis antérieurement pour T, 11-KT et E2 ont été
retenus pour être utilisés avec les tissus de thon rouge. Ces tests ont servi à analyser
les prélèvements sanguins et musculaires relevés sur des thons rouges à différents
stades du cycle de reproduction.
Nous avons élaboré des méthodes biochimiques pour déterminer le sexe et le
degré de maturité du thon rouge, d’après la détection des composantes spécifiques du
sexe dans les tissus sanguins et musculaires du poisson en processus de maturité. Un
échantillonnage plus étendu comprenant plusieurs classes de taille sur tout le cycle
annuel de reproduction nous permettra de compléter la validation de ces tests de
maturité, définir entièrement la biologie de la reproduction du thon rouge, et définit
par les moyens biochimiques l’âge de maturité du thon rouge de l’Atlantique.
RESUMEN
Para conseguir una ordenación efectiva de pesquerias, tiene una importancia
crítica poder identificar la edad de madurez de las especies de peces explotados. Esta
tarea, sin embargo, puede resultar difícil de realizar debido al hecho de que en
muchas pesquerías la captura se procesa en la mar, y los peces se desembarcan
eviscerados. Esta situación limita el acceso a los tejidos gonadales, dificultando la
determinación del género o de la madurez. En trabajos anteriores tratamos este tema
en relación a la pesquería de mero (Serranidae) en el sudeste de Estados Unidos.
Durante el curso de esta tarea desarrollamos métodos bioquímicos para identificar
componentes específicos de la madurez en machos y hembras. Los objetivos
bioquímicos principales eran el VTG, (vitellogenin), proteína precursora del vitelo , y
la hormona esteroide reproductora testosterona (T), 11-ketotestosterona (11-KT) y
estradiol-17 β (E2). Se determinó que, para Serranidae, los esquemas de madurez
generados a partir de mediciones bioquímicas eran en esencia idénticos a los
esquemas de madurez generados a partir de técnicas tradicionales de análisis
histológicos de las gónadas. En este documento se describen los esfuerzos que se
siguen desarrollando para aplicar esta tecnología al atún rojo (Thunnus thynnus).
A causa del limitado acceso a atunes rojos cautivos, hemos generado un
antiserum VTG para rabil (Thunnus albacares), así como un antiserum del
componente lipovitelin del huevo (Lv). Estos antisera se utilizaron como base para
desarrollar un ensayo inmunoabsorbente relacionado con la enzima de los túnidos,
VTG. Se adaptaron los inmunoensayos de T, 11-KT y E2, previamente caracterizados,
para utilizarlos con tejidos de atún rojo. Estos ensayos se aplicaron para analizar
sangre y extractos de músculos de atún rojo recolectados durante diferentes períodos
del ciclo reproductor.
Hemos desarrollado métodos bioquímicos para determinar el género y estados
de madurez del atún rojo, basados en la detección de componentes específicos del
sexo en la sangre y músculos de los peces que están alcanzando la madurez. Una
recolección más extensa que incluya múltiples clases de tallas durante el ciclo
reproductivo annual nos permitirá completar la validación de estos tests de madurez,
caracterizar en su totalidad la biología reproductiva del atún rojo e identificar con
aplicaciones bioquímicas la edad de madurez del atún rojo del Atlántico.
BACKGROUND
In prior research on temperate basses (genus Morone) and gag grouper (Mycteroperca
microlepis), funded by the National Marine Fisheries Service (NMFS) MARFIN program, we verified
that the gender and maturation status of the fish could be ascertained by measuring sex-specific hormones
and protein in frozen blood and muscle samples (Heppell 1998; Heppell and Sullivan 1999a,b; Heppell et
al. 1999). Our approach was based on radioimmunoassay (RIA) of androgens (testosterone, T; and 11ketotestosterone, 11-KT) and estrogen (Estradiol-17β; E2 ) as well as an enzyme-linked immunosorbent
assay (ELISA) of the circulating yolk protein precursor, vitellogenin (VTG).
Radioimmunoassays for the androgens and E2 that we have used to study reproductive physiology
in scores of teleost fishes were rigorously validated for use with gag blood plasma and saline extracts of
fresh or frozen gag muscle tissue. Vitellogenesis was induced in captive, juvenile gag by injecting them
with E2 and their plasma was later harvested as a source of VTG for purification. Gag VTG was isolated,
purified,
biochemically characterized and used as an antigen to raise a specific antiserum in rabbits. The antiserum
and purified VTG were used to develop a sensitive, antibody-capture, disequilibrium-type ELISA for
measuring VTG in plasma and muscle extracts.
Levels of the sex steroids and VTG in gag muscle co-vary with blood plasma levels of these compounds
and increase at particular stages of ovarian or testicular maturation during the annual reproductive cycle
(Heppell and Sullivan 1999b).
Specifically, VTG levels rise continuously during the main phase of secondary oocyte growth
(vitellogenesis) in females, whereas T and E2 levels rise late in the reproductive cycle. Both T and 11-KT
levels are elevated during late spermatogenesis and spermiation in males. It was verified that
measurement of VTG and E2 in muscle samples could be used to identify maturing females as well as
standard histological procedures applied to gonad samples taken from killed fish. Maturity schedules for
female gag prepared using the two different approaches were virtually identical in their predictions of
sexual demographics. Radioimmunoassay of T and 11-KT levels could be used to distinguish maturing
males from maturing females or immature fish.
These tests were useful for several months of the year, during most of the period of gonadal
recrudesence. They provide an important opportunity to obtain information critical for stock assessment
through shore-based sampling of fish as they are landed, even of fillets from the marketplace. For species
like gag and other grouper that are not sexually dimorphic and often landed eviscerated, this approach is
especially convenient and appropriate.
In developing maturity schedules for Atlantic bluefin, it has been logistically difficult to obtain
sufficient numbers of high quality gonad samples for routine histological analyses. The histological
approach is also slow to generate usable data and, if age-at-maturity is strongly effected by fishing
pressure, maturity schedules derived in this manner may be obsolete before completion of the histological
analyses. Therefore, over the last two years, we have extended our new method for assessing maturity to
studies of bluefin tuna (Thunnus thynnus). Our biochemical tests of maturity are potentially ideal for use
on bluefin, as they can produce results in short order and be applied to muscle samples that are easily
obtained in good condition at landing or in the marketplace. They could easily be combined with data
generated on population genetics from analyses of the same muscle samples. We envision that these tests
would be compatible with radiotelemetry or other tagging studies, using a non-lethal, harpoon- or darttype sampling device fitted with a muscle biopsy needle. With a properly designed implantation device,
muscle biopsy could occur simultaneously with tag implantation.
RESULTS
Our standard suite of sex steroid RIAs were first validated for use with bluefin plasma (or serum)
and saline extracts of frozen muscle. Because we lacked facilities or collaborators to maintain bluefin in
captivity, it was not possible to induce vitellogenesis in captive juveniles to generate VTG-rich blood
plasma as a source of VTG for purification. It was also difficult to obtain fresh blood samples from
strongly vitellogenic wild-caught females for this purpose. As an alternative, we designed a dual
approach to generating the antiserum necessary for constructing a tuna VTG ELISA. In collaboration
with the NMFS-Honolulu laboratory, we induced vitellogenesis in juvenile yellowfin tuna (Thunnus
albacares) in a manner similar to that previously described for gag. Subsequently, we collected the
plasma, purified and characterized the yellowfin VTG, and used the plasma as a source for generating
tuna VTG-specific antiserum. In addition, we isolated, purified and biochemically characterized the egg
yolk protein, lipovitellin (Lv) from samples of ovary obtained from gravid female bluefin.
Lv is the dominant yolk protein derived from VTG in tuna and other teleosts and bears the main
antigenic determinants of the parent VTG molecule. Briefly, the ovary samples were extracted with
buffered saline solution and the extract was subjected to affinity chromatography on hydroxylapatite
(Sigma, HA-Ultrogel) followed by gel filtration on Superose-6 (Pharmacia) during high-pressure liquid
chromatography (HPLC). Bluefin Lv in the chromatography extracts was detected by single radial
immunodiffusion using the antiserum raised against yellowfin tuna VTG.
Bluefin Lv was purified as two polypeptides exhibiting very similar apparent mass (~100 kDa) in
sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) that were identified as Lv's by
N-terminal amino acid microsequencing and Western blotting experiments using the antiserum to
yellowfin tuna VTG. The duality of Lv's was not surprising as many teleosts produce two VTG isoforms
(Matsubara et al. 1999; Specker and Sullivan 1994). The antisera to bluefin tuna Lv(s) or yellowfin tuna
VTG were used in conjunction with the respective, purified Lv(s) or VTG to develop sensitive ELISAs.
These assays were then validated for measuring VTG in plasma or muscle samples from either species.
Either ELISA can detect VTG in plasma or muscle extracts at extraordinarily low levels (~10 ng/ml). In
studies on juvenile yellowfin tuna injected with either E2 or saline solution, it was verified that VTG
levels in plasma and muscle are highly correlated.
DISCUSSION AND FUTURE WORK
We have developed sensitive immunological assays of sex-specific compounds for evaluation of
bluefin tuna maturity. These compounds can be detected in fresh or frozen muscle samples in the same
manner as our biochemical maturity tests for gag and other grouper. The cross-reactivity of our two
antisera and ELISAs with VTG from both bluefin and yellowfin tuna, evident during the assay
validations, suggest that these tests may be widely applicable to other scombrid fishes. We made similar
observations on our ELISA for gag VTG, which is applicable to multiple grouper species, even across
genera. Final validation, field testing and implementation of these assays for management of bluefin tuna
are needed.
In order to obtain an accurate maturity estimate for bluefin tuna, it is required that an archive of
muscle samples be obtained with adequate numerical representation of mature males, females, and
juveniles from each quarter of the year for each putative stock of interest. To complete validation of the
new maturity tests, a small proportion of the muscle samples from fish in each category will need to be
accompanied by frozen blood plasma or serum samples as well as gonad samples fixed in neutral buffered
formalin for histological analyses. These multiple sample sets (muscle, plasma and gonad) will be
required from at least N=6 fish in each of the following groups: mature (spermiating) male, mature
(gravid) female, immature male, and immature female.
REFERENCES
Heppell. S.A. 1998. The reproductive physiology of gag grouper (Mycteroperca microlepis). Ph.D. Dissertation.
Department of Zoology and Physiology Program.. North Carolina State University. 189 p.
Heppell, S.A., and C.V. Sullivan. 1999a. Gag (Mycteroperca microlepis) vitellogenin: purification, characterization
and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper.
Fish Physiol. Biochem. 20:361-374.
Heppell, S.A., Jackson, L.F., Weber, G.M., and C.V. Sullivan. 1999. Enzyme-linked immunosorbent assay (ELISA)
of vitellogenin in temperate basses (genus Morone): plasma and in vitro analyses. Trans. Amer. Fish. Soc.
128:532-541.
Heppell, S.A., and C.V. Sullivan. 1999b. Identification of gender and reproductive maturity in the absence of
gonads: muscle tissue levels of sex steroids and vitellogenin in gag (Mycteroperca microlepis). Can. J. Fish.
Aquat. Sci. (in press).
Matsubara, T., Ohkubo, N., Andoh, T., Sullivan, C.V., and A. Hara. 1999. Two forms of vitellogenin, yielding two
distinct lipovitellins, play different roles during oocyte maturation and early development of barfin
flounder, Verasper moseri, a marine teleost spawning pelagic eggs. Devel. Biol. (in press).
Specker, J.L., and C.V. Sullivan. 1994. Vitellogenesis in fishes: status and perspectives. pp. 304-315, in:
Perspectives in Endocrinology (K.G. Davey, R.E. Peter and S.S. Tobe, eds.), National Research Council,
Ottawa, Canada.