# The endocrinology of teleosts reproduction: an approach to the control of fish reproduction

Bernard Breton, Roland Billard

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## To cite this version:

Bernard Breton, Roland Billard. The endocrinology of teleosts reproduction: an approach to the control of fish reproduction. Archiv für Fischereiwissenschaft, 1984, 35 (1), pp.55-74. hal-01600708

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<table><tr><td>Arch. FischWiss.</td><td>35</td><td>Beih. 1</td><td>55-74</td><td>Berlin, Dezember 1984</td></tr></table>

From the Fourth Congress of European Ichthyologists, Hamburg, September 1982.

The endocrinology of teleosts reproduction: an approach to the control of fish reproduction

by B. BRETON and R. BILLARD

Institut Nationale de la Recherche Agronomique, Rennes, France

with 6 figures and 2 tables

## Kurzfassung

Die Endokrinologie der Reproduktion von Teleosteern: ein Vorschlag zur Beeinflussung und Überwachung von Fortpflanzungsvorgängen bei Fischen.

Neuere Untersuchungen auf dem Gebiet der Fisch-Endokrinologie haben zu einem besseren Verständnis hormonaler Faktoren während der Gametenentstehung geführt: ihre Art zu wirken und die Sekretion während der verschiedenen Stadien des Fortpflanzungszyklus zu regulieren.

Die grundlegenden Ergebnisse werden zusammenfassend dargestellt und Anwendungsmöglichkeiten bei der Überwachung der Fortpflanzung und der Geschlechtsbestimmung aufgezeigt. Besondere Aufmerksamkeit ist dem Reife-Gonadotropin (GTH) gewidmet, von dem die meisten Stadien der Gametokinese abhängen.

Die neuesten Entwicklungen auf dem Gebiet der Biochemie dieser Hormone und die Untersuchung ihrer Wirkung auf die Gonaden sind besonders wichtig bei deren Einsatz in der Hypophysationstechnik nach exogenen Injektionen unter spezifischen zoologischen Bedingungen. Es ist nun möglich, die Sekretion des endogenen Gonadotropins auf drei Weisen zu Beeinflussen:

- durch Veränderung der Umweltbedingungen

- durch Beeinflussung des Hypothalamus

- oder direkt durch die Beeinflussung der Hypophyse.

Dieser Überblick wird die Techniken von einem praktischen Blickwinkel aus darstellen, um zu zeigen, auf welchem Stand (Labor - Pilotprojekt - Fischfarm) sie z.Z. jeweils genutzt werden.

Einige weitere wichtige Forschungsbereiche, die direkt oder indirekt Auswirkung auf unsere Fähigkeiten haben werden, die Überwachung der Fortpflanzung bei in Gefangenschaft gehaltenen Fischen zu verbessern, werden ebenfalls vorgestellt.

## Abstract

Recent research in the field of fish endocrinology have led to a better knowledge of the hormonal factors involved in the control of gamete production, of their mode of action and of the regulation of their secretion during the different stages of the reproductive cycle. These fundamental results are reviewed in correlation with their possible applications to the control of fish reproduction and sex determination. Special attention is paid to the maturational gonadotropin (GTH) on which most of the stages of the gametokinetic cycle depend. The latest developments in the biochemistry of these hormones and in the study of their effects on the gonads are especially important in terms of zoological specificity for their use in hypophysation techniques after exogenous injections.

It is also possible now to modulate the secretion of endogenous gonadotropin at three levels by:

- modifying the environmental factors

- acting on the hypothalamus

- or directly on the pituitary.

This review will consider the techniques from a practical point of view to know at what scale (laboratory - pilot - fish farm), they are actually used.

Some further important aspects of research that are directly or indirectly relevant to increasing our ability to control reproduction of fishes kept in captivity will also be presented.
## Introduction

The control of reproduction at all stages of the life cycle has always been one of the main problems in fish culture. In some cases, as in milkfish, gametogenesis does not occur in captivity, and most of the time the final steps of that process, i.e. gamete release, do not occur spontaneously. At the moment, in order to control all stages of the fish reproductive cycle, an aquaculturist must choose between two strategies:

1. the capture of mature breeders, fry or fingerlings from the wild, upsetting the equilibrium of natural populations,

2. the development of broodstock management. Controlled reproduction in the second strategy has been limited for a long time to artificial induction of spawning in species which do not spontaneously ovulate in captivity, using the hypophysation technique first developed by von IHERING in 1937. This procedure has been extensively employed for many cultured species and empirically developed because of the paucity of basic information on the reproductive physiology of fishes (for review see HARVEY and HOAR, 1980).

<table><caption>Figure 1</caption><thead><tr><th>OBJECTIVE</th><th>POTENTIAL APPLICATIONS</th></tr></thead><tbody><tr><td>MONOSEXUAL CULTURE</td><td><ol><li>To wear out proliferation and dwarfism</li><li>To avoid reproduction for introduction of new species</li><li>To eliminate precocious reproduction</li><li>To increase reproductive potential</li><li>To select a sex growing more rapidly</li><li>To reduce size of hatchery</li><li>To reduce number of breeders</li><li>To increase the commercial values of catches when ovaries has a higher values than testis</li></ol></td></tr><tr><td>STERILIZATION</td><td><ol><li>Same as ①②③</li><li>Weight gain suppressing gonadal development</li><li>Best food conversion</li><li>To extend the life cycle in species which dead after first spawning</li><li>To prevent precocious return of salmon to freshwater</li></ol></td></tr></tbody></table>
Fish culture has been especially limited to developing countries where it represents a necessary source of protein, but more recently this activity has been extended to developed countries which have studied mainly salmonids and new undomesticated species such as sea bream, sea bass, turbot and others; the successful culture of these fish depends upon man's ability to control the entire life cycle. To do this, an important research effort has been made in the last ten years, and our knowledge of reproductive physiology has progressed considerably. Several methods have been proposed to improve the new hypophysation technique as well as practical ways of controlling most stages of the reproductive cycle. One method of control is *sexual differentiation* in order to produce sterile or monosex fishes, as summarized on figure 1.

There are several advantages to sterilizing fishes. First, suppressing gonadal development eliminates the retarded growth often observed at the onset of sexual maturity and weight loss at evisceration owing to excision of the gonads which may represent up to 30% of body weight. Moreover, the food conversion ratio is better in juvenile salmonids, and sterile fishes may be commercialized at any time of the year according to market demand without having to bother about the hypothesis of sexual maturity inducing depreciation of meat quality and postreproductive mortality. Sterilization also eliminates the inconvenience of precocious reproduction which causes dwarfism, and generally favors the introduction of new species without risking demographic explosion. Lastly, it prevents precocious salmonids from returning to freshwater.

The rearing of monosexual lines consisting only of males or females has some of the same advantages as the rearing of sterile fish but, in addition, there is no fertilization when gametogenesis occurs because the sexual partner is absent. This resolves the problem of dwarfism in *Tilapia*, for example, or of introducing new species in nature. Other benefits in extensive culture are: faster growth in one sex (female turbot or male *Tilapia*) and later maturity in one sex, usually the female. Moreover, it is interesting to increase female production in the hatchery because this reduces the size of the broodstock and the hatchery, or increases egg production, especially in salmonids which have relatively low fecundity.

The second step is to control the reproductive cycle from puberty to spawning, as summarized on figure 2. There are several degrees of complexity of control, ranging from the control of one phase to that of the whole gametogenetic cycle. The easiest way is to achieve sexual maturation, which involves oocyte maturation and ovulation in the female and induction or enhancement of spermiation in the male at the time of the year normal to species which do not ovulate spontaneously in captivity. A second way is to advance or retard reproduction by several months or weeks. The most complex way is to develop the capability of a particular species to mature and spawn at any time of the year.
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<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

most teleost fish in the temperate zone are seasonal breeders and the reproductive cycle is an adaptation to variations in environmental cues in order to deliver the fry at the most appropriate time. This is the case especially in spring-spawning fish that have small eggs and therefore very short autonomy over the larvae. The environmental stimuli are integrated in neuroendocrine and endocrine structures, some species having developed stimulatory and inhibitory control over gonadotropic secretion since it is more directly influenced by environmental factors. The aim of reproductive endocrinology is to determine what structures are involved, their nature and the biological action of their secretory products and how they are secreted and modulated by endogenous and exogenous factors at each step of the gametokinetic cycle.

## Gametogenesis and its endocrinological control

Figure 3 shows the general organization of the endocrine reproductive axis. Three main structures can be defined. Environmental stimuli first reach the central nervous system where information is directed to the hypothalamus that activates the pituitary gland by means of chemical messengers, the releasing hormones. These messengers then induce the secretion of pituitary hormones; among these are gonadotropins stimulating gonadal steroid hormone secretion that acts directly on gonadal activity as well as in feedback on the hypothalamo-hypophysial axis.
Other structures directly or indirectly involved in the control of reproduction may be liver, adrenal glands, pineal gland, chromaffin tissue, the ultimobranchial body and thyroid. But, except for hepatic vitellogenin synthesis, the role of these organs is not clear (see review by FONTAINE, 1976).

### The hypothalamus

The regulatory influence of the hypothalamus on the reproductive cycle was first determined after pituitary transplant which only maintained prolactin cell activity (BALL et al., 1965). Histochemical, immunohistochemical and electric stimulation and lesion experiments have shown two hypothalamic areas controlling the gonadotropic activity of the pituitary gland — the preoptic nucleus and the lateralis tuberis nucleus (NLT) — and one extra-hypothalamic area, the dorsalis pars medialis (ADPM) (see review by PETER, 1982). An immunoreactive material, similar to the synthetic LH-RH that

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
stimulates LH and FSH secretion in other vertebrates, has been demonstrated in all three areas but never at the same time in the same species. Synthetic LH-RH and its analogs also stimulate *in vitro* and *in vivo* secretion of gonadotropin in salmonids and cyprinids as does hypothalamic extract from the same species (BRETON et al., 1972; BRETON and WEIL, 1973). Thus, in teleosts there is stimulatory hypothalamic control of pituitary gonadotropic activity by Gn-RH, which is believed to be similar to mammalia LH-RH when the biological activity of LH-RH in fish and mammals is compared. Further biochemical, immunochemical and biological studies have led to the hypothesis that fish Gn-RH would certainly differ from the LH-RH decapeptide by at least one, and perhaps two, amino acids in positions 8 and 6 or 7 (KING and MILLAR, 1980). These positions are not essential for determining biological activity but they might be involved in determining zoological specificity; this would explain why GtH stimulation requires more LH-RH in fish than in mammals. It should also be noted that pituitary gland responsiveness to the same LH-RH assay varies according to species and within the same species with the season (WEIL, 1981).

Besides this stimulatory effect, large lesions in the NLT of gravid female goldfish induce ovulation and a surge of plasma gonadotropin levels in conditions where ovulation would never occur normally. This has been linked to the existence of gonadotropin-releasing inhibiting factor (GRIF) now shown to be a dopamine (see review by PETER, 1982).

## The pituitary gland and gonadotropins

The pituitary gland always plays a role in initiating and sustaining gametogenesis. Hypophysectomy induces gametogenesis to disappear quickly but it is restored by pituitary extracts containing gonadotropic hormones whose number has not been definitively determined.

A glycoprotein gonadotropin (GtH) has been isolated from the pituitary glands of various teleosts (Table 1). Contrary to mammalian LH and FSH, which are always purified using the same two bioassays, fish gonadotropins, even of the same species, are separated using the bioassays of different authors; this means that pure GtH biological activity cannot be standardized and thus that the crude pituitary extracts used for hypophysation cannot be standardized either. However, all glycoprotein GtH's have been demonstrated to induce maturation and ovulation and that is why they have been called "maturational" GtH's in opposition to putative vitellogenic GtH (see review by BURZAWA-GERARD, 1980). Maturational hormones are species-specific and have a molecular weight of 30 000; they can be dissociated into two subunits, one corresponding to their zoological specificity and the other to their biological specificity (BURZAWA-GERARD, 1980). This recent finding may have an important meaning in the development of immuno-
<table><thead><tr><th>SPECIES</th><th>ASSAYS</th><th></th></tr></thead><tbody><tr><td rowspan="5">Cyprinus carpio</td><td>● Frog maturation</td><td>Burzawa-Gerard, 1973</td></tr><tr><td>● In vitro trout oocyte maturation</td><td>Breton-unpublished</td></tr><tr><td>● In vitro stimulation of androgen secretion by carp testis</td><td>Huang & al. 1981</td></tr><tr><td>● In vitro stimulation of immature trout oocyte c-AMP</td><td>Idler & NG 1979</td></tr><tr><td>● Ovulation of the loach Misgurnus anguillicaudatus</td><td>Fish Reproductive Group (China) 1978</td></tr><tr><td rowspan="2">O.Tschawytscha</td><td>● Stimulation of Hypophysectomised goldfish maturation</td><td>Donaldson & 1972</td></tr><tr><td>● In vitro trout oocyte maturation</td><td>Breton & 1978</td></tr><tr><td rowspan="3">O.Keta</td><td>● In vitro stimulation of immature trout oocyte c-AMP production</td><td>Idler & 1975</td></tr><tr><td>● 32P uptake by chick testis</td><td>Yoneda and Yamazaki 1976</td></tr><tr><td>● Stimulation of Hypophysectomised goldfish maturation</td><td>Yoneda and Yamazaki 1976</td></tr><tr><td rowspan="2">O.Gorbuscha</td><td>● Ovulation of the loach Misgurnus anguillicaudatus</td><td>Zenkevich-Lace 1982</td></tr><tr><td>● In vitro trout oocyte maturation</td><td>Breton-unpublished</td></tr><tr><td>S.Gairdneri</td><td>● In vitro trout oocyte maturation</td><td>Breton & 1976</td></tr><tr><td>S.Mossambica</td><td>● In vitro stimulation of testosterone production by rat Leydig cells</td><td>Farmer and Papkoff 1977</td></tr><tr><td>Hypoglossoides platessoides</td><td>● In vitro maturation of Hypoglossoides oocytes</td><td>NG & Idler 1979</td></tr></tbody></table>

logical systems directed against a subunit and capable of recognizing GtH in species in which they are not presently purified; their structural data and direct stimulatory effect on gonads are characteristic of all vertebrate gonadotropins purified up to now.

These gonadotropins induce or restore total spermatogenesis, including maturation in immature or hypophysectomized mature fish. Thus, only one GtH in males controls all the stages of gametogenesis, even before the onset of puberty (BILLARD et al., 1982). The discrepancy in the number of GtH's arises in females. Maturational GtH has been demonstrated to induce all stages of oogenesis, including exogenous vitellogenesis, that is, yolk incorporation in goldfish, coho salmon, Indian catfish and European eel (BURZAWA-GERARD, 1982). On the contrary, a pure preparation of s-GtH failed to induce yolk deposition in juvenile rainbow trout, whereas a total pituitary extract did, indicating the presence in the pituitary extract of a factor stimulating yolk incorporation (UPADHYAY et al. 1978). In chum salmon, carp and flounder pituitaries, IDLER et al. (1980) have isolated a second gonadotropin having a very low carbohydrate content and stimulating yolk incorporation. But it was further demonstrated that maturational GtH also stimulated yolk incorporation in these as well as in other species (IDLER, 1982). We shall have definite proof when we can totally regenerate
vitellogenesis up to maturation and ovulation using hormonal preparations
in hypophysectomized females. Thus, the situation remains confused, although
most fish endocrinologists think that the regulation of vitellogenesis (a
very complex process) probably requires at least one other pituitary factor
besides maturational GtH. Whether this factor will be a gonadotropin in
the strict sense of the word, that is from a biological and biochemical
point of view, is a question as long as its glycoprotein nature and subunit
structure, (essential in defining a gonadotropin) have not been demon-
strated.

Biological effects and mode of action

It would be too long to review in detail all the morphological and bioche-
mical changes induced by GtH in fish gonads. We shall restrict this paper
to general results which can be applied to attaining practical control of
the cycle.

As already mentioned for the male, GtH controls all the stages of sperma-
togenesis up to spermiation, even in juvenile fish. This action is probably
mediated by androgen steroids whose secretion is strongly stimulated by
GtH (NG and IDLER, 1980), perhaps by progestagens, and especially by 17 α-
hydroxy-20 β-dihydroprogesterone that is found in great quantities in trout
blood plasma at the onset of spermiation (SCOTT and BAYNES, 1982). These
steroids induce the strongest enhancement of spermiation in pike.

Via steroid hormones, GtH in the female regulates directly or indirectly
some events of oocyte development. Whether or not GtH is implicated in the
maintenance and mitotic division of oogonia, as well as in initial oocyte
growth, is unclear, but it does stimulate the initiation and further de-
velopment of exogenous vitellogenesis by the intermediary of hepatic syn-
thesis of vitellogenin (CAMPBELL and IDLER, 1976; VAN BOHEMEN, 1981).
Lastly, GtH activates oocyte maturation by stimulating the release of a
maturation-inducing steroid (FOSTIER et al., 1973; FOSTIER et al., 1981).

Fish GtH's stimulate the production of cyclic AMP (IDLER et al., 1975) and
increase adenyl cyclase activity in testis and ovary (FONTAINE et al.,
1972); this is the first step in gonadotropic action after the gonadotropin
binds to a specific receptor. The cyclic AMP relay in the action of vi-
tellogenic GtH has not been demonstrated yet. It is only very recently
that GtH receptors were reported in trout ovary and testis (VAN DE KRAAK
and DONALDSON, 1982).

Gonadotropin secretion

It is also necessary to study the profiles of GtH secretion to evaluate
GtH requirements during each stage of the reproductive cycle. This can now
be done owing to the development of specific and sensitive radioimmuno-
assays for cyprinid and salmonid species.
Plasma GtH levels are generally undetectable in juvenile immature salmonids and cyprinids. We have no indication of the putative variation of plasma GtH at the onset of puberty, perhaps because we lack an exact definition of this period. But suppressing GtH secretion by methallibure, an antigo-nadotropic substance, before spermatogenesis begins in the Atlantic salmon parr, inhibits further development of gametogenesis, indicating that GtH might be required at the onset of puberty (GOSSLING, personal communication).

In trout reproducing for the second time or more, a rise in GtH occurs at the beginning of spermatogenesis and exogenous vitellogenesis. Further-more, until spermiation there is no marked variation in GtH secretion in males (BILLARD et al., 1978; BILLARD et al., 1982); this profile is general in all studied species (salmon, trout, carp, tench, goldfish). In males as in females, GtH secretion reaches maximal values during the last phase of the reproductive cycle (FOSTIER et al., 1982). In goldfish there is a periovulatory surge of GtH, depending on the photoperiod, but GtH returns to basal level after ovulation (STACEY et al., 1979). In trout, there is also a daily increase in GtH around the time of ovulation, but the GtH level continues to rise after ovulation, especially when the eggs remain in the body cavity. The significance of this phenomenon is unknown. Although long-term variations in plasma GtH are physiologically important, recent works have demonstrated that circadian and ultradian short-term variations might be more important (ZOHAR et al., 1982). For example, the spring GtH rise in trout consists of short GtH pulsations of high amplitude, while during vitellogenesis, even if basal levels remain constant, GtH secretion is pulsatile; this secretion pattern is necessary in order to induce cir-cadian estradiol secretion for the synthesis of vitellogenin. On the con-trary, in cyprinids there is a daily cycle of GtH (HONTELA and PETER, 1978; GILLET et al., 1978) and all the factors suppressing these rhythms, such as underoxygenation and starvation, also suppress gonadal recrudescence (GILLET et al., 1981).

If these rhythms are not taken into account during hormonal treatment, it might make a difference in the results; therefore, most GtH biological action should be reconsidered in view of the existence of these rhythms since they might explain the lack of response to hormonal treatment.

## Gonads and steroid hormones

Most of the gonadal steroids are produced under gonadotropic stimulation. The main steroids secreted by the testis are androgens and especially 11-ketotestosterone which is essentially produced by interstitial cells. The levels of these steroids increase during spermatogenesis and are maxi-mum at spermiation (FOSTIER et al., 1982). A high level of progestagen (17α-OH-20β-P) has also been described in trout just prior to spermiation
(SCOTT and BAYNES, 1982). The main biological role of androgens is to mediate the promotion of spermatogenesis and spermiation by gonadotropin, but the role of progestagen is not known although it seems to play a role in the chemical composition of milt, and especially in its potassium concentration; high concentrations of this mineral inhibit sperm motility (see review by BILLARD et al., 1982).

In the female, the two main steroids secreted are 17α-estradiol and 17α-OH-20β-P. In salmonids, at least estradiol is directly involved in trout vitellogenesis due to its stimulating role in hepatic vitellogenin biosynthesis. The level of estradiol remains high throughout vitellogenesis, but maturation and ovulation can only occur if this level drops while, on the contrary, 17α-OH-20β-P levels increase (FOSTIER and JALABERT, 1982). The nature of the maturation-inducing steroid (MIS) is dichotomous. In some species and in salmonids, walleye and killfish MIS, and probably 17α-OH-20β-P produced by follicular cells, are progestins. In other fish species, MIS, being secreted by the adrenal glands (Indian catfish and yellow perch), would have an extraovarian origin and be a corticosteroid. But in all species except zebrafish, in which the maturating potency of steroids has been tested, 17α-20β-P has always been found to be the most potent. A possible explanation would be that the MIS is a progestagen whose action is modulated by corticosteroids (JALABERT, 1975, 1976).

Other biological effects of steroid hormones are their negative feedback on the hypothalamo-pituitary complex as demonstrated after castration. In plant experiment and correlative studies on the secretion rates of both GtH and steroids. The steroid negative feedback inducing plasma GtH surges, and consequently, ovulation, can be lifted either by the injection or implantation of antisteroidal substances such as clomiphene or tamoxifen (BRETON et al., 1975; see review by PETER, 1982). Positive feedback has also been demonstrated in immature prepuberal salmonids in which aromatizable steroids or estrogens (injected or implanted) induce pituitary synthesis and release of GtH, if appropriate therapy is used initiating the onset of precocious development in males, but not in females (CRIM and EVANS, 1979, 1982). The central nervous system is thus probably involved in the control of puberty, possibly via an inhibitory system that disappears at puberty.

Other actions of steroids are their involvement in the determination of secondary sex characters and their partial control of reproductive behavior in some species.

Concerning sexual differentiation (see review by FOSTIER et al., 1983), it is not definitively demonstrated that steroid hormones are sex inducers. Fish germ cells remain bipotential during at least part of sexual differ-
entiation, but undifferentiated germ cells (primary cells) have been found
in the testis of some gonochoric species. That steroids can determine final
sex during this time does not mean that they are natural sex inducers since
other compounds could have untypical or indirect effects, and external
factors such as temperature might play an important role, as in the African
catfish, *Clarias lazera*, i.e. when the temperature increases from 30 to 39°C
within 3 h at day 9 of development, an all-male brood results (VAN DEN HURK
and CAMBERT, 1982). However, YAMAMOTO (1969) has proposed a dual model in
which androgens are andro-inducers and estrogen are gyno-inducers. Although
some data in the literature do not agree with this hypothesis, it has been
the basis of numerous practical developments in sex control in fish culture.

## Environmental factors and endocrine components

Depending on the species, photoperiod and temperature can modulate the dynamics of the gametokinetic cycle. These factors presumably do so partly due to influences on GtH secretion and on the modulation of target organ responsiveness to hormonal stimulation. There is no doubt that better knowledge of these mechanisms would help us to make better use of the combination of environmental factors controlling the cycle. I wish to cite some examples showing the importance of such studies.

During the first part of the salmonid cycle, the GtH rise inducing exogenous vitellogenesis is independent of the photoperiod, but further occurrence of the pulsatile mode of GtH secretion, which benefits vitellogenetic development most, occurs when the photoperiod decreases, and especially around 14 h of light/day, under which both pituitary responsiveness to Gn-RH stimulation and gonad responsiveness to GtH become maximal (BRETON and BILLARD, 1977; BRETON et al., 1984).

As already mentioned, there is a daily cycle of blood GtH in goldfish, resulting from the interaction between high temperature and long period. We have already underlined the importance of the existence of these rhythms of secretion in the maintenance of gametogenesis. What seems to matter is not the environmental factors stimulating overall GtH secretion, but the short-term chronobiological effects of these environmental factors. For example, maintaining goldfish for a long time at high temperature results in gonadal regression, although plasma GtH levels are strongly stimulated (GILLET et al., 1977). This is probably due to suppression of the daily GtH cycle and also to a direct effect of temperature on the gonad affecting steroidogenic activity, for example. It is more important at present to study chronobiological effect than hypothetical combinations of light, temperature and other factors to determine the best conditions for practical control of the reproductive cycle.
## Practical applications

What are the practical possibilities of applying these theoretical data? Except for sex control, maturational GtH is involved in most of the processes occurring during the cycle; but GtH requirements differ both quantitatively and chronobiologically according to the considered stages. In our opinion, this last notion must be kept in mind as a guide to future development and research in the field; understanding the regulatory mechanisms of GtH receptors would also be very helpful in the light of our first results.

We must also remember that endocrinological interrelations are so complex that every modification introduced into an equilibrated system modifies many components of that system, sometimes leading to results opposite to those expected.

Nevertheless, there are two general ways in which the reproductive cycle can be controlled (Fig. 4). The first is to modulate the plasma GtH levels either by changing the endogenous endocrinological equilibrium or by injecting exogenous gonadotropin.

<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
This can be accomplished in several ways:

- by acting on environmental factors;

- by inducing or overcoming the inhibition of GtH secretion after injection of neuromediators or some antagonists;

- by modifying steroid hormone feedback on the hypothalamo-pituitary axis by inhibiting either positive or negative feedback on GtH secretion. This can be accomplished by either using biologically active steroids directly (the so-called negative or positive feedback), or by ineffective or aspecific competition using antisteroidal substances, such as anti-estrogens, clomiphene and tamoxifen, which bind on steroid receptors and inhibit their action;

- by injecting pituitary homogenate or partially purified gonadotropin.

The second way of controlling the reproductive cycle is to act directly on the gonads after the injection of a gonadotropic preparation or steroids in order to induce the last phase of maturation. At this stage, the possible direct action of external factors, such as temperature, must not be underestimated.

It is impossible to review here all the methods proposed in the literature for each species; we shall give only a synopsis of the effectiveness of these methods at each stage of gonadal development.

### Sex reversal and sterilization
(see review by DONALDSON and HUNTER, 1982). The effects of sex steroid treatments during sexual differentiation depend on the nature of the steroid, its dose, the duration of treatment, the time at which it is applied and whether the sex steroid is administered in the diet or in the water. For example, in the Cichlid, *Sarotherodon mosambica*, estrogenic treatment brings about complete feminization only if applied from days 6 to 25, that is, if the treatment begins before morphological differentiation. This feminization must include both physiological and morphological differentiation (NAKAMURA and TAKAHASHI, 1983). The same is true of masculinization treatments, but sterilization generally needs higher doses and longer treatments. A hatchery technique developed for rainbow trout recommends 100 days of treatment with 10 mg of methyltestosterone/kg of food from hatching day to obtain sterilization, while masculinization is obtained after a 75-day treatment using only 3 mg of methyltestosterone/kg of food; complete feminization is reached after a 30-day treatment with 20 mg of estradiol/kg of food.

As in the case of chicken or calf, hormonal treatment with various drugs may result in consumer refusal, even if no residues are detected in the commercialized fish. There are several ways to solve this problem; for instance, the gynogenetic approach and a future field technique in which the eggs of normal females are fertilized using cryopreserved spermatozoa of androgen-reversed females.
## Puberty

As already mentioned, we do not understand the mechanism involved in the determinism of the onset of puberty, but precocious maturity can be induced in males (but not in females) in the laboratory using gonadotropic preparations. Gametogenesis has always been inhibited at the same stage using methallibure in Atlantic salmon parr, and further research is necessary to determine the signals inducing the initiation of puberty. They might be hormonal as well as nutritional or environmental, for example a constant period of 24 h of light applied at 0+ blocks reproduction in two-years old female rainbow trout (BOURLIER, unpublished).

## Gametogenesis (Figure 5)

Gametogenesis can be advanced in puberal males (but not females) by giving gonadotropin, but this technique is not practical because repeated injections cause stress and are time-consuming; even when hormonal preparations are administered after cholesterol pellet implantation, the technique is costly and is used only when it is not possible to do otherwise, i.e. in special species because the zoological species-specificity of the gonadotropin is more strict as regards the induction of gametogenesis than the induction of spawning. Modification of environmental factors is the most

<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
common technique used to either advance or delay gametogenesis. At present, no inhibitory technique is available, although androgens have been successfully used (BILLARD et al., 1980) in puberal rainbow trout in the laboratory.

Control of ovulation (Fig. 6)

(see reviews by SUNDARARAJ and LAM, 1982; FOSTIER and JALABERT, 1982).

While pituitary preparations are still the most widely used technique to induce ovulation, new chemicals could be used in hypophysation and new strategies, such as hypothalamic lesions, might be applied directly via chemicals (mono-sodium glutamate) or after modification of the endogenous endocrine balance.

Table 2 presents some products now available and the level of their effectiveness with comments. They include gonadotropic preparations (crude and purified), progestagens, LH-RH and analogs, the antiestrogens-clomiphen and tamoxifen, and combinations of these. Other possibilities have been discovered recently; dopamine, a gonadotropic inhibiting-releasing factor, does not act directly on pituitary secretion, but inhibits Gn-RH action at the pituitary level. Thus, in species such as common carp and goldfish,

<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
MAIN HORMONES USED IN THE PRACTISE OF HYPOPHYSIS

<table><thead><tr><th>Hormones</th><th>Doses mg/kg bw</th><th>Species</th><th>Use in fish farm</th><th>Biological action</th><th>Comments</th></tr></thead><tbody><tr><td>Pituitary extraits (PE)</td><td>3.5</td><td></td><td></td><td></td><td></td></tr><tr><td>Partially purified GTH (P.P)</td><td>0.1</td><td>ALL</td><td>+++</td><td>exogenous supply</td><td>increasing doses if distant related species. biological activity unknown</td></tr><tr><td>PE+17α20β (P)</td><td>PE 0.3+P3</td><td>carps salmonids</td><td>++</td><td>exogenous supply and direct gonadal action</td><td></td></tr><tr><td>P alone</td><td>2.3</td><td>salmonids</td><td>+</td><td>direct gonadal action</td><td>depend on stage of maturation</td></tr><tr><td>LH RH and LH RHa</td><td>0.001-0.1</td><td>phyto fish tench</td><td>++</td><td>direct stimulation of endogenous secretion</td><td>ovulation need natural environment</td></tr><tr><td>LH RHa a PP+LH RHa</td><td>0.2 0.1+0.01</td><td>salmonids</td><td>pilot</td><td></td><td></td></tr><tr><td>HCG</td><td>300.50000</td><td>sea bass mullet sea bream</td><td>++</td><td></td><td>efficient only in species easy to spawn</td></tr><tr><td>Antisteroids clomiphen</td><td>1-10</td><td>cyprinids</td><td>-</td><td>modification of steroid feed-back</td><td></td></tr><tr><td>Tamoxifen</td><td></td><td>salmonids</td><td>-</td><td></td><td></td></tr></tbody></table>

which are refractory to LH-RH action, a combined treatment using specific competitors for dopamine receptors (pimozide followed by LH-RH) induces ovulation, even in an environment devoid of spawning substrates (CHANG and PETER, 1982).

Female response to each of these preparations varies, depending on the individual, species, type of hormone used and stage of ovarian development; other problems are related to gonadotropins. The gonadotropic preparations are not standardized and their biological activity compared to an hormonal reference. To develop an easy, reliable, rapid and inexpensive test to determine the gonadotropic content of pituitary extracts is a priority.

Crude pituitary extracts also contain other factors which could potentiate or inhibit gonadotropic activity. The use of partially purified GtH is an improvement since it does not seem to induce antibody formation, while crude pituitary extracts do and the fish become less responsive to further treatment. On the other hand, highly purified gonadotropins are fragile and difficult to use in fish farms and they would be even more expensive. Therefore, a partially purified preparation of known biological activity would be the ideal solution; such preparations are already available from private companies.
The possibilities offered by LH-RH are certainly most interesting, and it is imperative to isolate and determine the structure of fish Gn-RH so that it can be synthesized and used on a large scale in fish culture.

There are many problems in defining optimal ovarian development in fish, such as the initial gonadal stage. Not only response would depend on the annual reproductive sequence, but it might also be a daily variation in gonadal responsiveness to hormonal treatment. This may imply treatment at a given time and even sequential treatment at a precise time.

Many other problems related to physical and chemical environments, social factors and endocrinological components of reproductive behavior might also influence response to hormonal treatment.

In conclusion, we now begin to understand the reproductive physiology of fish; some progress has already been accomplished by our approach to the control of the reproductive cycle and many new possibilities are now open. We must pursue our investigations in order to define the best ways to apply the results. This needs field investment since most of the new techniques are still at the laboratory stage.

## Acknowledgements

This work was supported by INRA (Institut National de la Recherche Agronomique) and CNEXO (Centre National d'Exploitation des Océans). We wish to thank Mrs. BOUIX for typing the paper, Mr. SAMBRONI for drawing the figures and tables and Mrs. DAIFUKU for editing the manuscript.

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First authors address: Dr. B. Breton, INRA, Laboratoire de Physiologie, Campus de Beaulieu, F-35042 Rennes.