reviews in fish biology and fisheries 7,

8/14/2019 Reviews in Fish Biology and Fisheries 7,

1/25

Neuroendocrine regulation of ovulation infishes: basic and applied aspects

R . E . P E T E R 1 3 a nd K.L. YU1 , 2

1 Department of Biological Sciences, The University of Alberta, Edmonton, Alberta T6G 2E9, Canada2 Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong

Contents

Abstract page 174

Introduction 174

Neuroendocrine regulation of ovulation 175

Gonadotrophin-releasing hormone

Dopamine

Other neuropeptides and neurotransmitters

Ovarian hormones

Maturation-inducing steroidsOvarian steroid feedback

Prostaglandins

Other ovarian factors

Ovulation and its associated hormonal changes 179

Patterns of ovarian follicular development

Hormonal changes during periovulatory cycles


Dopamine and other monamines

Pituitary gonadotrophins

Ovarian steroids

Growth hormone

Other hormonesInduction of ovulation 184

Environmental stimuli

Photoperiod

Temperature

Spawning substrate

Social and pheromonal interactions

Environmental stress

Pharmacological manipulation of pituitary receptors

Gonadotrophin-releasing hormone receptors

Dopamine receptors

Reviews in Fish Biology and Fisheries 7, 173197 (1997)

09603166 # 1997 Chapman & Hall

3 Author to whom correspondence should be addressed. E-mail: [email protected]


2/25

Hormonal induction: neuropeptides versus pituitary hormones

Development of superactive fish GnRH analogues

GnRH agonists and dopamine antagonists

Sustained release of GnRH

Routes of GnRH delivery

Future directions in research 190

Acknowledgements 191

References 191

Abstract

This review summarizes the major neuroendocrine mechanisms regulating ovulation, thusproviding a basis for understanding the various environmental and hormonal techniquesfor induction of ovulation of cultured teleosts. The secretion of gonadotrophin-II (GtH-II)is stimulated by gonadotrophin-releasing hormone (GnRH), and, although some teleostshave three different forms of GnRH regionally distributed in the brain, in most speciesinvestigated, only one form is present in the pituitary and apparently involved in GtH-IIsecretion. In nearly all species investigated, dopamine (DA) inhibits GtH-II secretion bydirect actions on gonadotrophs, as well as by inhibition of GnRH release. Sex steroids actat both brain and pituitary levels to regulate GtH- II secretion through a combination of

positive and negative feedback actions; one important positive feedback action is thatsex steroids enhance the responsiveness of the pituitary to GnRH and an importantnegative feedback action is to increase DA turnover, thereby increasing the overall DAinhibitory tone on GtH-II secretion. The preovulatory surge of release of GtH-II isstimulated by a surge release of GnRH. A decrease in DA turnover also occurs todisinhibit GnRH and GtH-II release. Environmental factors including photoperiod,temperature and spawning substrate may cue ovulation and spawning. Social and

pheromonal interactions play a very important role in synchronizing preovulatoryendocrine changes, ovulation and spawning behaviour in many species. A widely usedtechnique for inducing ovulation of cultured fishes is injection of the combination of aGnRH superactive analogue, to stimulate GtH-II release, and a DA receptor antagonist, to

block the inhibitory actions of DA. This is termed the Linpe technique and has provenparticularly useful with those species having synchronous or group synchronous follicular

development and a large preovulatory surge of GtH-II. In other groups of teleosts, particularly those species having asynchronous ovarian development and multiplespawnings over an extended period, treatment with a sustained-release preparation of aGnRH superactive analogue to cause a prolonged, somewhat enhanced release of GtH- IIhas proven highly successful in inducing multiple ovulations and spawnings. However,the lack of specific radioimmunoassays for GtH-II in many of these species has hindered

progress, as the precise pattern of GtH-II release necessary for the recruitment ofvitellogenic oocytes into final maturation and ovulation in these multiple spawnersremains an intriguing neuroendocrine question.

Introduction

Environmental and pheromonal cues are perceived and transduced by the brain intoneuroendocrine signals to regulate secretion of maturational gonadotrophin-II (GtH-II)

174 Peter and Yu


3/25

from the pituitary. Increases in serum GtH-II prior to spontaneous ovulation have beendemonstrated in several teleost species. This preovulatory increase in GtH-II secretiontriggers final oocyte maturation (migration and breakdown of the germinal vesicleaccompanied by completion of meiosis) and ovulation (follicular rupture and discharge ofthe oocyte from the follicle). In the first parts of this review, the neuroendocrineregulatory system underlying the changes in GtH-II secretion and the changes in varioushormones that occur during ovulation will be examined.

The control of ovulation is essential for aquaculture of fish. Development of

techniques to control the timing of egg and fry production would enable more efficientand effective use of resources in the hatchery. Although there has been a number ofearly reviews on hormonal control of fish ovulation (Donaldson and Hunter, 1983;Goetz, 1983; Stacey, 1984), the last 10 years have seen a significant advance in ourunderstanding of the basic and applied aspects of the neuroendocrine regulation ofovulation in teleost fishes. We will review this information, using the perspectives

provided by the advances provided in basic studies.

Neuroendocrine regulation of ovulation

G O N A D O T R O P H I N - R E L E A S I N G H O R M O N E

It is well documented in teleosts, as in other vertebrates, that brain gonadotrophin-

releasing hormone (GnRH) plays a major role in the regulation of GtH-II secretion fromthe pituitary. The first GnRH identified in the brain of a fish, chum salmon(Oncorhynchu s keta, Salmonidae), was determined to be [Trp7, Leu8]-GnRH(Sherwood et al., 1994); the basic structure of the GnRH decapeptide originallyidentified in the brain of mammals is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2(mGnRH). Other molecular forms of immunoreactive GnRH identified in brains ofteleosts are [His5, Trp7, Tyr8]-GnRH (chicken GnRH-II or cGnRH-II), mGnRH, [His5,Leu7, Asn8]-GnRH (catfish GnRH or cfGnRH) and [Ser8]-GnRH (seabream GnRH orsbGnRH; Sherwood et al., 1994; King and Millar, 1995; Yu et al., 1997).

The existence of multiple molecular GnRH forms in the brain of teleosts has beenwell demonstrated (Sherwood et al., 1994; King and Millar, 1995). The primarystructures of the multiple GnRH forms have been recently confirmed by molecular

biology methods (amino acid sequencing and= or cDNA analysis) in a number of teleostspecies including African catfish (Clarias gariepinus, Clariidae), Thai catfish (Clariasmacrocephalus, Clariidae), African cichlid ( Haplochromis burtoni, Cichlidae), giltheadsea bream (Sparus auratus, Sparidae), striped bass ( Morone saxatilis, Moronidae),goldfish (Carassius auratus, Cyprinidae) and several salmonid species (Sherwood et al.,1994; Gothilf et al., 1995; King and Millar, 1995; Yu et al., 1997). In African cichlid,gilthead sea bream and striped bass, three forms of GnRH (sGnRH, cGnRH-II andsbGnRH) were characterized, while two forms (sGnRH and cGnRH-II) have beenconfirmed in the brain of catfishes and goldfish. The coexistence of multiple GnRHforms in the same teleost species has led to studies on their respective bioactivities,distribution, regulation and physiological functions.

In salmonid species, only sGnRH is present in the pituitary (Sherwood et al., 1994;Gothilf et al., 1995; King and Millar, 1995; Yu et al., 1997). In African cichlid,

gilthead sea bream and striped bass, all three forms of GnRH stimulate GtH- II secretionin sexually mature females. Although cGnRH-II is apparently not present in the

Neuroendocrine regulation of ovulation in fishes 175


4/25

pituitary, cGnRH-II is the most active among the three native GnRH peptides instimulating GtH-II secretion in gilthead sea bream and striped bass (Gothilf et al.,1995). In pituitary of goldfish and African catfish, cGnRH-II coexists in the pituitarywith a second native GnRH form, sGnRH and cfGnRH respectively (Schulz et al.,1995; Yu et al., 1997). In African catfish, cGnRH-II is much more potent (150 fold)than the second native form in the stimulation of GtH-II secretion (Schulz et al., 1995).In goldfish, while cGnRH-II is significantly more potent than sGnRH in stimulatingGtH-II release, the magnitude of difference in potency is relatively small (Peter et al.,

1991).

DOP AMINE

Dopamine (DA) acts as a GtH-II release inhibitory factor in goldfish and a wide range ofother teleosts, and has been thoroughly reviewed recently (Peter et al., 1986, 1991;Trudeau and Peter, 1995). DA has been demonstrated to directly inhibit basal, as well asGnRH-stimulated GtH-II release (Peter et al., 1986, 1991; Trudeau and Peter, 1995).Evidence indicates that removal of dopaminergic inhibition, together with increasedGnRH stimulation, constitute important neuroendocrine mechanisms leading to the

preovulatory GtH-II surge and ovulation in many species (Peter et al., 1991). However,Atlantic croaker (Micropogonias undulatus, Sciaenidae) has been demonstrated to be anexception in that no evidence of DA inhibition of GtH secretion has been found, in spiteof thorough investigation (Copeland and Thomas, 1989).

O T H E R N E U R O P E P T I D E S A N D N E U R O T R A N S M I T T E R S

Pituitary GtH-II secretion is stimulated by a number of neuroendocrine factors in additionto GnRH, including neuropeptide Y (NPY), -aminobutyric acid (GABA), norepinephr-ine (NE), serotonin (5-HT), cholecystokinin (CCK) and excitatory amino acids (EAAs),and have been reviewed elsewhere (Peter et al., 1991; Trudeau and Peter, 1995; Yu et al.,1997). A model is presented in Fig. 1 to illustrate the relationship of theseneuroendocrine factors to GnRH and DA, and GtH-II secretion.

O VA R I A N H O R M O N E S

Maturation-inducing steroids

GtH-II causes final oocyte maturation by inducing ovarian maturational competence and by stimulating the follicle cells to synthesize maturation-inducing steroids (MIS ormaturation-inducing hormone; Nagahama et al., 1995). Studies in a variety of teleostsincluding cyprinid and salmonid species have shown that the most potent steroid ininducing oocyte final maturation is 17,20 -dihydroxy-4-pregnen-3-one (17,20 -P;

Nagahama et al., 1995). In contrast, 17,20 ,21-trihydroxy-4-pregnen-3-one (17,20 ,21Por 20 -S) has been identified as the MIS in the Atlantic croaker and other perciformfishes including spotted sea trout (Cynoscion nebulosus, Sciaenidae; Thomas and Trant,1989), striped bass (King et al., 1995), toadfish (Halobatrachus didactylus, Batrachoi-didae; Modesto and Canario, 1995), gilthead sea bream (Canario et al., 1995) and turbot(Scophthalmus maximus, Scophthalmidae; Muginier et al., 1995). Also, 20 -S has been

found in free and conjugated form in the blood of ovulating sea bass (Dicentrarchuslabrax, Moronidae; Scott et al., 1990).

176 Peter and Yu


5/25

GONADOTROPH

DOPAMINE

GABA

NPY

NE

5-HT GnRHNEURONAL

SYSTEM

OLFACTORY SYSTEM

PHERMONES

BRAIN

PITUITARY

GtH-II

OVARYT

E2

OOCYTES

1 2

Fig. 1. A model for the neuroendocrine regulation of GtH-II secretion in the female goldfish. Lines

with arrowheads indicate known stimulatory actions; lines with bars indicate known inhibitory actions.

Abbreviations: E2, oestradiol; GABA, -aminobutyric acid; GnRH, gonadotrophin-releasing hormone;

NPY, neuropeptide Y; NE, norepinephrine; 5-HT, serotonin; T, testosterone. Adapted from Peter et al.(1991).



6/25

Ovarian steroid feedback

The feedback effects of sex steroids are presumably exerted at the brain and pituitarylevels to allow integration with environmental cues (e.g. temperature, photoperiod,rainfall, vegetation) to ultimately trigger the preovulatory GtH-II surge in cyprinids (Aida,1988). Studies in goldfish suggest that elevated levels of both testosterone (T) andoestradiol (E2) are important for the induction of the preovulatory GtH-II surge(Kobayashi et al., 1989). Kobayashi and co-workers (1989) showed that an increase in

temperature induces a preovulatory surge-like increase in GtH-II levels in T and E2implanted ovariectomized (OVX) or sexually regressed female goldfish, but not in OVXfish. Pankhurst and Stacey (1985) found that ovulation and serum GtH-II levels wereunaffected by E2 injection or implantation in female goldfish. These findings, togetherwith the observation that high T and E2 levels were found in the goldfish undergoingovulation, suggest that elevated levels of sex steroids may be of importance for theinduction of the preovulatory GtH-II surge.

The turnover rate of DA increases during vitellogenesis in goldfish (Trudeau et al.,1993b) and rainbow trout (Oncorhynchus mykiss, Salmonidae; Saligaut et al., 1992),reaching a maximum in sexually mature prespawning fish. This indicates that the DAinhibitory tone is at its maximum in prespawning fish. The DA turnover rate in goldfish(Trudeau et al., 1993b) and rainbow trout (Linard et al., 1995b) is increased by E2

treatment, constituting a steroid negative feedback mechanism. In support of thismechanism, co-localization of oestrogen receptors with a group of preoptic tyrosinehydroxylase-positive neurons, most likely dopaminergic, has been demonstrated inimmunohistochemical studies in rainbow trout (Linard et al., 1995a). Regulation ofhypothalamic NE turnover by E2 has also been described in goldfish (Trudeau et al.,1993a).

Evidence for gonadal steroid action involving GnRH neurons in teleosts comes primarily from studies on immature fish in which positive steroid feedback on GtH-IIsecretion is predominant (Goos, 1987). For example, using specific radioimmunoassaysfor each native GnRH form, a recent study in immature female European eel (Anguillaanguilla, Anguillidae) demonstrated the differential regulation of the two GnRHforms by gonadal steroids (Montero et al., 1995). Results of these studies suggestthat positive feedback of E2 on GtH-II secretion in immature eels and in juvenile

rainbow trout may be mediated, at least in part, through brain GnRH neurons, aswell as directly on the pituitary (Yu et al., 1997). However, a preliminary report ofan in situ hybridization study in masu salmon (Oncorhynchus masou, Salmonidae)suggests that biosynthetic activity of the sGnRH neurons in the preoptic regionis positively regulated by gonadal steroids, whereas the sGnRH neurons in theventral telencephalon are under negative steroid control (Amano et al., 1996).Steroid binding sites or oestrogen receptor expressing neurons in the brain of goldfishand rainbow trout have a distribution in proximity to, but not overlapping that of theGnRH neurons, suggesting that the effects of sex steroids on the GnRH neuronalsystem may be mediated by other neurotransmitters or neuropeptides, similar to thesituation in mammals (Linard et al., 1995a; Trudeau and Peter, 1995). In addition tothese actions on the GnRH neuronal system, T, through aromatization to E2, potentiates

the action on GnRH in GtH-II release both in vivo and in vitro in goldfish (Trudeau andPeter, 1995).

178 Peter and Yu


7/25

Prostaglandins

Ovulation is mediated by prostaglandins (PGs) of the F series in teleosts (Goetz, 1983).Increased synthesis of PGs, induced by either GtH-II or MIS, appears to be a generalrequirement for the GtH-induced ovulation in teleosts (Goetz et al., 1991). It has beendemonstrated that 17,20 -P and PGF2 enhanced the activity of two of the five

proteolytic enzymes produced by the ovarian follicles in goldfish (Goetz et al., 1987).This PG may also serve as a releaser pheromone to induce spawning behaviour in male

goldfish (Stacey and Sorensen, 1991). In mammalian studies, it was suggested that PGmediates the luteinizing hormone-induced rise in ovarian collagenolytic activity (Tsafririet al., 1991).

Other ovarian factors

A whole series of ovarian paracrine and autocrine factors have been shown to influencethe actions of GtHs in the ovulatory process in mammals. In goldfish, mammalianinhibin-A and activin-A, as well as goldfish gonadal extracts, stimulate GtH-II releasefrom the pituitary in vitro (Ge et al., 1992; Ge and Peter, 1994).

Using an in vitro whole ovarian follicle assay system, recent studies have shown thatan ovarian kallikrein-like protease may be involved in the induction of follicle wallcontraction of brook trout (Salvelinus fontinalis, Salmonidae) by stimulating the

conversion of angiotensinogen to angiotensin (Goetz et al., 1995). The expression of akallikrein gene family mRNA in the brook trout ovary has recently been demonstrated(Goetz et al., 1995).

Ovulation and its associated hormonal changes

P A T T E R N S O F O V A R I A N F O L L I C U L A R D E V E L O P M E N T

Most teleosts have distinct ovarian cycles, with ovulation often being limited to particulartimes of the year. Three patterns of ovarian follicular development are generally observedin teleosts: synchronous, group synchronous and asynchronous (Wallace and Selman,1981). Anadromous salmonids (e.g. Pacific salmon, Oncorhynchus) and catadromousanguillid eels, which die after spawning, have a short, well-defined spawning season andsynchronous follicular development. For teleosts such as goldfish and medaka (Oryziaslatipes, Adrianichthyidae) exhibiting group synchronous follicular development, two ormore distinct populations (or clutches) of oocytes, each at a different developmentalstage, exist concurrently in the ovary. In this case, ovulation and spawning may occuronce or several times in a season, depending on the species and environmentalconditions. In multiple spawners such as gilthead sea bream and killifish (Fundulusheteroclitus, Fundulidae) with asynchronous follicular development, oocytes at all stagesof maturity (without pronounced clutches) are found in the ovary. Ovulation andspawning occur at intervals over a period of a prolonged spawning season.

H O R M O N A L C H A N G E S D U R I N G P E R I O V U L A T O R Y C Y C L E S


In many teleost species, ovulation is associated with a surge of GtH-II secretion,

triggering ovulation of a large number of oocytes, perhaps the entire oocyte population(Peter et al., 1991; Kime, 1993). This suggests that GnRH may have important roles in



8/25

the regulation of ovulation in fish. Despite extensive studies of the structure andbioactivities of GnRH peptides, there is little information available on the physiology ofthe brain GnRH neuronal system during ovulation in teleosts. Changes in brain GnRHlevels associated with the stimulation of pituitary GtH-II secretion during spawning have

been described in only brown trout (Salmo trutta, Salmonidae), goldfish and roach(Rutilus rutilus, Cyprinidae; Peter et al., 1991; Yu et al., 1997). In female roach, aninverse relationship between brain GnRH and serum GtH-II levels has been observedduring the spawning period. However, a significant change in the total brain GnRHcontent was not found in brown trout during the periovulatory period.

In goldfish, spontaneous ovulation can be induced by increasing the water

temperature and adding artificial vegetation to serve as a spawning substrate (Staceyet al., 1979). The preovulatory surge in blood levels of GtH-II, lasting about 12 h,

a b c d

13:00 13:00 13:0006:00

Day 1 Day 2 Day 3

Time

20

60

100

140

c b d ac a d ba d b c

SerumG

H

(ng/mL)

Serum GH

*

*

*

*

*

*

pre-exposure

control

nonovulatory

ovulatory

d c a b

a b c d

a b d c

Serum GtH

0

100

200

300

S

erumG

tH

(ng/mL)

*

*

* *

Pituitary

Hypothalamuspre-exposurecontrol

nonovulatory

ovulatory

b c d ab c d ac b d a

d c a bd a b cb c a d

0.2

0.4

0.6

4.0

3.0

2.0

1.0

GnRH

concentration(ng/mgprotein)

dc

13:0006:00

Day 3Day 2

Time

Day 1

13:0013:00

ba

Fig. 2. Temporal changes in serum GtH-II and growth hormone levels, and in the total GnRH

concentrations in different brain regions at various times during the periovulatory period in female

goldfish. Female goldfish were induced to ovulate by addition of spawning substrate and increase of

water temperature (Stacey et al., 1979). As well, pheromonal interactions of female and male goldfish in

the same aquarium would have contributed to synchronization of ovulation in the females (Fig. 3;

Sorensen et al., 1988; Stacey and Sorensen, 1991). Values represent mean 6 SEM. Number of fish in

each group at different sampling times: n

16 for control female group, 1622 for non-ovulatory

180 Peter and Yu


9/25

begins before the onset of dark, resulting in ovulation and s pawning the followingmorning at the onset of light (Stacey et al., 1979; Yu et al., 1987, 1991; Fig. 2). This

precise timing of the periovulatory events allows a temporal correlation of the changesin GnRH concentrations in discrete brain areas and the pituitary with the changes inserum GtH-II levels during the periovulatory period. Coincident with the preovulatorysurge in serum GtH-II levels, female goldfish also have a surge in serum growthhormone levels (Fig. 2). Coincident with the onset of the surge in serum GtH-II levels,there is a depletion of total immunoreactive (ir)-GnRH concentrations in the olfactory

bulbs, telencephalon plus preoptic region, hypothalamus, and pituitary, with repletion back to starting concentrations occurring by the end of the surge of GtH-II some 12 hlater (Fig. 2). It is, however, not known whether sGnRH or= and cGnRH-II neuronal

activities are differentially affected during spawning, because of the limitations of theradioimmunoassay system used in these studies. Despite the observed coordinated

*

*

* *

Telencephalon

Olfactory bulbspre-exposurecontrol

nonovulatory

ovulatory

c d b ac d b ab c d a

b d c ab c d ab c d a

0.2

0.4

0.6

GnRH

concentration(ng/mgp

rotein)

dc

13:0006:00

Day 3Day 2

Time

Day 1

13:0013:00

ba

0.8

1.0

0

1

2

3

group and 1012 for the ovulatory group. Fish were kept on a 16 h photoperiod (darkened horizontal

bars represent daily dark period). Sampling times are indicated by lines and letters. ANOVA and

Duncans multiple range test were used to compare data from all experimental groups at each

sampling time ( 3 and { indicate significant difference from the respective control group and non-

spawning group, respectively, at the same sampling time; p , 0.05), and to compare changes over

time within each of the experimental groups (underlined letters represent means that are statistically

equivalent at p . 0.05). Modified from Yu et al. (1991).



10/25

regulation of terminal nerve and ventral telencephalic preoptic anterior hypothalamicGnRH neuronal activities during the periovulatory period, a recent study by Kobayashiand co-workers (1994) indicates that gonadal maturation and ovulation in femalegoldfish can occur in the absence of the terminal nerve-containing olfactory GnRHsystem, suggesting a relatively greater importance of the preoptic GnRH in the process.

Dopamine and other monamines

Male goldfish respond to pheromonal stimuli from ovulating females with a surge in

blood levels of GtH-II (Stacey and Sorensen, 1991; Stacey et al., 1994), causingincreased spermiation in preparation for spawning (Stacey et al., 1994). A decrease in

pituitary DA turnover has been demonstrated to occur during the initial phases of thesurge release of GtH-II in male goldfish responding to the pheromone 17,20 -P (Dulkaet al., 1992). Pretreatment of the males with the GnRH antagonist [Ac- 3-Pro1, 4FD-Phe2, D-Trp3,6]-mGnRH blocks the surge in GtH-II levels in male goldfish responding tothe pheromone 17,20 -P (Murthy et al., 1994b). Together these data strongly suggestthat the surge of GtH-II in male goldfish involves a decrease in the inhibitory actions ofDA, as well increased stimulation of GtH-II release by GnRH peptides. Although asimilar decrease in pituitary DA turnover has been predicted to occur during the

preovulatory period in female goldfish, the changes in pituitary DA turnover duringspontaneous ovulation have not been measured. The periovulatory changes in

hypothalamic 5-HT and pituitary DA in rainbow trout indicate the involvement of brainDA and other monoamines in the neuroendocrine regulation of ovulation in this species(Saligaut et al., 1992).

Pituitary gonadotrophins

Studies in chum salmon and coho salmon (Oncorhynchus kisutch, Salmonidae) haveshown that GtH-I is the predominant GtH in the blood and pituitary of vitellogenicfemales, and that plasma levels of GtH-I decline during final oocyte maturation. Incontrast, plasma levels of GtH-II remain low until the period of final oocyte maturationand ovulation (Swanson, 1991). Results of a recent study in rainbow trout demonstratedthat GtH-I mRNA is predominantly expressed during gonadal development in bothmales and females, whereas GtH-II mRNA is predominantly expressed during thespermiation and periovulatory periods (Weil et al., 1995). Unlike the differential changesof the two GtHs during gonadal maturation in salmonid species, both GtH-I and GtH-II mRNA levels in the pituitary of goldfish increased during gonadal development(Kobayashi, 1996). Furthermore, predominant expression of GtH-II versus GtH-I mRNA occurs in the pituitary of female goldfish from juvenile through maturing, matureand sexually regressed stages (Yoshiura et al., 1995). Because the ovarian steroidogenicactivities of common carp (Cyprinus carpio, Cyprinidae) GtH-I and GtH-II are similar(Van Der Kraak et al., 1992), and because substantial amounts of GtH-II can be measuredin the blood serum or plasma of goldfish throughout the reproductive cycle (Trudeau etal., 1991), GtH-II is interpreted to be of primary importance throughout the reproductivecycle.

In several teleost species, a surge of GtH-II associated with ovulation has beendemonstrated (Goetz, 1983; Peter et al., 1991). The periovulatory GtH-II surge in

goldfish is well described (e.g. Fig. 2). In rainbow trout, GtH-II begins to increase atabout 10 days prior to ovulation and reaches high levels of 10 20 ng ml 1 just prior to

182 Peter and Yu


11/25

or at ovulation (Goetz, 1983). In both rainbow trout and brown trout, postovulatoryserum GtH-II levels are higher than the preovulatory GtH-II levels. It is interesting thatthe postovulatory levels of serum GtH-II are higher in females that retain eggscompared with the levels in spent females (Goetz, 1983). In gilthead sea bream, parallelactivation of GtH-II gene expression and secretion during the periovulatory periodhave also been demonstrated (Meiri et al., 1995).

In salmonids, the preovulatory increase in blood levels of GtH-II is associated withthe appearance of type II GtH receptors, which bind GtH-II only. In contrast, serum

levels of GtH-I decrease and the number of type I GtH receptors, which bind both GtH-I and GtH-II, declines in the granulosa layers of preovulatory follicle (Yan et al., 1992;Miwa et al., 1994). The persistent presence of type II GtH receptors is considered to beresponsible for the unique stimulatory effects of GtH-II on production of 17,20 -P, thesteroid associated with the process of oocyte maturation, as well as inhibition of E2

production by granulosa layers during final oocyte maturation (Yan et al., 1991, 1992).

Ovarian steroids

In teleosts exhibiting synchronous ovarian development, serum levels of E2 and Tincrease during oogenesis to reach peak levels at, or near, the beginning of ovulation.During final oocyte maturation and ovulation, plasma E2 and T levels decrease, whereas

plasma progestogen levels increase (Kime, 1993; Linard et al., 1995a). In salmonid andnon-salmonid species, a sharp peak of 17,20 -P occurs prior to spontaneous or inducedovulation (Kime, 1993; Nagahama, 1994; Nagahama et al., 1995).

In group synchronous species such as orange roughy, a progressive decrease in plasma T levels also occurs during the preovulatory period, while in others, there is a prespawning decrease in plasma E2 levels (Pankhurst and Carragher, 1991). Never-theless, T and E2 remain elevated for most of the preovulatory period in some groupsynchronous species. For example, circulating E2 levels increase at the time of the

preovulatory surge of GtH-II in goldfish (Kobayashi et al., 1987).In many multiple spawning species, short-term fluctuations in plasma T and E2 levels

are associated with the maturation of different groups of oocytes at intervals of days orweeks during the spawning season (Pankhurst and Carragher, 1991; Kime, 1993). Forexample, a diurnal pattern of plasma E2 and 17,20 -P is found in the daily spawningJapanese whiting (Sillago japonica, Sillaginidae; Matsuyama et al., 1990).

Growth hormone

In goldfish, a periovulatory surge of growth hormone (GH) occurs in synchrony with theGtH-II surge (Yu et al., 1991; Fig. 2). The neuroendocrine regulation of GH secretion inteleosts is multifactorial, involving, at least in goldfish, the stimulation of GH release byGnRH, dopamine, growth hormone-releasing hormone, neuropeptide Y, thyrotrophin-releasing hormone, cholecystokinin, bombesin and activin (Peter and Marchant, 1995;Peng and Peter, 1997). Somatostatin is the primary inhibitor of basal and stimulated GHsecretion; however, norepinephrine and serotonin also have inhibitory actions on GHrelease (Peter and Marchant, 1995; Peng and Peter, 1997). Involvement of GH in the

regulation of fish ovarian functions have been demonstrated (Van Der Kraak et al., 1990;Singh and Thomas, 1991; Le Gac et al., 1993).



12/25

Other hormones

Insulin and insulin-like growth factor I (or IGF-I) have been shown to induce oocytematurational competence in red sea bream (Pagrus major, Sparidae; Kagawa et al., 1994)and spotted sea trout (Thomas and Ghosh, 1995). IGF-I is present in the blood of rainbowtrout (Niu et al., 1993) and IGF-I receptors are found in ovary of carp, brown trout andcoho salmon (Maestro et al., 1995). It has been suggested that insulin may have a role inreinitiation of meiosis in the goldfish oocyte (Lessman, 1985).

A sharp two-to-threefold increase in circulating cortisol levels occurs coincident withthe onset of the preovulatory GtH-II surge in goldfish (Cook et al., 1980). Plasmacortisol levels are also elevated during the spawning period in salmonids. Recent studiesin rainbow trout showed that 17,20 -P directly stimulates cortisol production byinterrenal tissue in vitro (Barry et al., 1995), suggesting that 17,20 -P may be a

physiological regulator of cortisol secretion during the periovulatory period.

Induction of ovulation

E N V I R O N M E N T A L S T I M U L I

Photoperiod

Sexual development and spawning in many fishes is modulated by photoperiod (Bromage

et al., 1984; Hontela and Stacey, 1990). In salmonids, the predominant environmental cuefor timing of the reproductive cycle and spawning is photoperiod (Bromage et al., 1984),whereas in many cyprinids, the predominant environmental cues are temperature and

photoperiod combined (Hontela and Stacey, 1990). Generally speaking, it was found thatlong days or continuous light during the earlier part of the reproductive cycle advance thetime of oocyte maturation and spawning, whereas the same photoperiod at or after thesummer solstice delays these processes (Bromage et al., 1984; Duston and Bromage,1986). Temperature and nutritional status are also known to affect the timing of spawning

by photoperiod(Bromage and Cumaraanatunga, 1988).Photoperiodic manipulation has been used to control the timing of ovulation in

salmonids since 1937 (Hoover and Hubbard). Results of recent studies have allowed thedevelopment of simple procedures for photoperiod control of ovulation (Duston andBromage, 1986). For example, abrupt changes to long photoperiod during winter and

spring accelerate ovulation in rainbow trout (Bromage et al., 1984). Continuousadditional light can also be used to either advance or delay ovulation in Atlantic salmon(Salmo salar, Salmonidae) depending on the duration of the light period and its positionin relation to the phase of the reproductive cycle (Taranger, 1993). Daily rhythms of

photosensitivity for gonadal recrudescence and spawning have been described for the barbel ( Barbus barbus, Cyprinidae; Poncin, 1989) and threespine stickleback(Gasterosteus aculeatus, Gasterosteidae; Baggerman, 1990).

Temperature

In cyprinids, ovarian development and ovulation is modulated both by temperature and by photoperiod, although temperature is the predominant influence on the overallreproductive cycle in most species (Lam, 1983; Hontela and Stacey, 1990). For example,

final oocyte maturation and ovulation in goldfish can be induced by an elevation intemperature from 12 8C to 20 8C without the presence of plant (or artificial spawning

184 Peter and Yu


13/25

substrate) and males (Stacey, 1983; Hontela and Stacey, 1990). Photoperiod, on the otherhand, influences the timing of the periovulatory GtH-II surge induced by temperatureelevation (Stacey, 1983; Hontela and Stacey, 1990). Similarly, in the red drum (Sciaenopsocellatus, Sciaenidae), ovulation can usually be induced by slowly raising the temperature(1 8C day 1) over the range 2228 8C (Roberts, 1987). On the contrary, autumn or winterspawners ovulate at relatively low temperatures and the timing of ovulation is againinfluenced by temperature. For example, the sea bass (Dicentrarchus labrax, Moronidae)spawns when temperature lowers to 1012 8C, and it was suggested that low temperature

can advance spawning whereas high temperatures cause a delay (Zanuy et al., 1986).

Spawning substrate

Ovulatory response of goldfish to elevation of water temperature can be enhanced byaquatic vegetation (Stacey et al., 1979; Stacey, 1984; Hontela and Stacey, 1990). Othercyprinids and African catfish are known to spawn in relation to rainfall and flooding,which presumably alters the availability of spawning substrate and= or water quality(Lam, 1983; Stacey, 1984; Hontela and Stacey, 1990).

Social and pheromonal interactions

Evidence that male fishes release a primer pheromone to trigger ovulation in the females

has been obtained for a number of teleost species including African catfish, yellowfinBaikal sculpin (Cottus sp., Cottidae) and zebrafish ( Brachydanio rerio, Cyprinidae)(Hontela and Stacey, 1990; Stacey and Sorensen, 1991). In zebrafish, females held withmales will normally ovulate every 45 days. Female zebrafish isolated from males willovulate upon re-exposure to males or male-holding water. Although testis extractcontaining steroid glucuronides has been demonstrated to induce ovulation in isolatedfemale zebrafish, the active component of the pheromone has not been fully characterized(Hontela and Stacey, 1990; Stacey and Sorensen, 1991). Because ovulation in zebrafish isstimulated by water from males and inhibited by water from crowded aquaria, ovulationin zebrafish responds both to stimulatory pheromones and to inhibitory social factors(Stacey, 1984).

In goldfish, exposure to spawning pair(s) enhances GtH-II secretion and ovulation.Results of recent studies demonstrated that the rate of ovulation in groups of goldfish

increased during exposure to the pheromone 17,20 -P (Stacey et al., 1994; Fig. 3).The results suggest that synchronous ovulation in groups of goldfish may be induced bythe primer pheromone 17,20 -P, released by ovulatory females. This intrasexual

pheromonal communication has also been suggested by the observed synchronization ofovulation in other cyprinid species (Lin, 1982).

Environmental stress

Exposure to pollutants or various elements of deteriorating water quality will induce astress response in fish. Acute and chronic stress have been shown to have deleteriouseffects on reproductive functions in fishes (Sumpteret al., 1987). Specifically, it has beenshown that stress causes a delay in ovulation and decreased egg quality in brown andrainbow trout (Campbell et al., 1994). Although stress reaction is mainly mediated by the

increase in serum cortisol level, and deleterious effects of cortisol im plantation onreproductive functions have been demonstrated in salmonids (Carragher et al., 1989), the



14/25


15/25

by 10 9 M [D-Arg6, Pro9NEt]-sGnRH (sGnRH-A) and from two sites by 10 6 M sGnRH-A, confirming the presence of high- and low-affinity binding sites (Habibi and Peter,1991).

Seasonal variations in the responsiveness of pituitary GtH-II secretion to superactiveGnRH analogues have been demonstrated in goldfish (Peter et al., 1991). The greatestincreases in serum GtH-II to mGnRH-A, given either alone or in combination with

pimozide, were found in prespawning goldfish, whereas sexually regressed fish were theleast responsive. This seasonal variation in the responsiveness coincides with the

seasonal variation in the capacity of both high- and low-affinity GnRH receptors in thegoldfish pituitary (Habibi and Peter, 1991). These results suggest that pituitary GnRHreceptors may be regulated by gonadal steroids.

Two injections of sGnRH-A 12 h apart in goldfish cause an increase in GnRHreceptor capacity but no effects on affinity of the high-affinity= low-capacity bindingsites (Omeljaniuk et al ., 1989; Habibi and Peter, 1991), suggesting receptorautoregulation. Treatment with the DA antagonist domperidone increased, whereasthe agonist apomorphine decreased, the capacities of the high- and low-affinityGnRH binding sites both in vitro and in vivo in goldfish (De Leeuw et al., 1989;Habibi and Peter, 1991). This demonstrates that inhibition of the inhibitory DA systemon GtH-II cells in the goldfish pituitary causes an up-regulation in the GnRHreceptor system. Similarly, injection of pimozide and a superactive GnRH analogue both

caused an increase in GnRH receptor capacity in the African catfish (Habibi and Peter,1991).

Dopamine receptors

Although results of studies in goldfish suggest the periovulatory GtH-II surge isassociated with increased GnRH and decreased DA neuronal activities (Habibi and Peter,1991; Yu et al., 1997), the precise regulation of the pituitary GnRH and DA receptorsduring the periovulatory period awaits further investigation. In this regard, the isolation ofcomplementary cDNAs encoding receptors for GnRH (Goos et al., 1996) and DA (Tse etal., 1996) will help to reveal the structure and the regulation of these two important

pituitary receptors involved in ovulation in teleosts.

H O R M O N A L I N D U C T I O N : N E U R O P E P T I D E S V E R S U S P I T U I T A R Y H O R M O N E S

Early attempts to induce ovulation were carried out using homologous or heterologousgonadotrophic preparations. Despite the lack of standardized GtH preparations andineffectiveness of some heterologous GtH preparations, pituitary extracts have been usedfor inducing ovulation in many cultured fish species (Weil et al., 1986; Peter et al.,1988). Protocols generally required two injections of the GtH preparation. In commoncarp, the first or priming injection of GtH stimulates the onset of final oocyte maturationand primes the ovary for production of maturation-inducing steroids, while the secondinjection is the resolving dosage, that triggers the production of maturation-inducingsteroids and ovulation (Levavi-Zermonsky and Yaron, 1986). Disadvantages of thissystem are the need to handle broodstock fish twice to administer injections, lack ofstandardization of GtH preparations (note: Levavi-Zermonsky and Yaron used a

calibrated common carp pituitary extract for their experiments), and immune responsesto injection of heterologous GtH preparations.



16/25

Development of superactive fish GnRH analogues

Similar to native mammalian GnRH, native fish GnRH, such as sGnRH, is rapidlydegraded by enzymes in the pituitary, kidney and liver of gilthead sea bream (Zohar etal., 1990). The substitution of selected D-amino acids in position 6 of GnRH providesresistance to cleavage between positions 5 and 6 in mammals (Karten and Rivier, 1986).There is also a cleavage site between Pro9-Gly10-NH2 in GnRH, and substitution ofethylamide for amino acid 10 stabilizes the C-terminal of mGnRH in mammals (Karten

and Rivier, 1986). Using the goldfish as a model, structureactivity relationships of theGnRH peptides have been extensively studied. Results of a series of studies using peptides based on mGnRH and sGnRH revealed that substitution of D-amino acids in position 6 is important for developing superactive analogues for stimulation of GtH-IIrelease in goldfish. sGnRH-A has been found to be superactive in all teleosts studied(Peter et al., 1991). Modifications at position 6 enhance the potency of GnRH analogues

by increasing resistance to peptidase degradation (Zohar et al., 1990) and receptor binding affinity (Habibi and Peter, 1991).

The amino acid structures of the five fully characterized teleost GnRH decapeptides(mGnRH, sGnRH, cGnRH-II, cfGnRH and sbGnRH) differ from each other only in

positions 5, 7 and 8. Results of studies on the activity of GnRH analogues with variantamino acid residues in positions 5, 7 and 8 in goldfish revealed that the presence oftryptophan in position 7 of the native sGnRH and cGnRH-II peptides is essential for the

high potency of these peptides in release of GtH-II from perifused goldfish pituitaryfragments in vitro (Habibi et al., 1992). Compared with cGnRH-II, [His5, Tyr8]-GnRHhad significantly lower GtH-II releasing potency. Similarly, [Leu8]-GnRH showedsignificantly lower GtH-II releasing potency compared with sGnRH. Further studiesshowed that [Trp7]- of the native sGnRH and cGnRH-II peptides in goldfish alters therequirement of the D-amino acid substitution in position 6 to produce superactiveanalogues when compared with the mGnRH peptide having leucine in position 7 (Peteret al., 1995). It was found that [Trp7]- of sGnRH and cGnRH-II require a positivelycharged [D-Arg6]- or [D-Lys6]- to confer increased GtH-II releasing activity. Consistentwith this analysis, results of recent studies showed that [D-hArg(Et2)6, Pro9-NHEt]-sGnRH has the highest potency in stimulating GtH-II release among the GnRH peptidestested in goldfish (Murthy et al., 1994a). Given that cGnRH-II is most potent among the

native teleost GnRHs in stimulating GtH-II release in goldfish and other teleosts, itwould be of interest to design superactive analogues based on the cGnRH-II structure.Results of a recent study showed that goldfish pituitaries in vitro were easilydesensitized by [D-Arg6, Pro9-NEt]-cGnRH-II com pared with responses to sGnRH,suggesting that it may have superagonistic activity (Lovejoy et al., 1995).

GnRH agonists and dopamine antagonists

The dual neurohormonal regulation of secretion of GtH-II by GnRH and DA acting as arelease-inhibitory factor has provided the basis for a highly effective technique forinduced ovulation of many cultured fishes. This involves intraperitoneal or intramuscularinjection of a superactive analogue of GnRH, specifically sGnRH-A or [D-Ala6,Pro9NEt]-mGnRH (mGnRH-A), with a DA receptor antagonist, specifically pimozide or

domperidone, to remove the inhibitory influence of DA on pituitary GtH-II secretion(Peter et al., 1988, 1991). This combination well simulates the normal ovulatory surge of

188 Peter and Yu


17/25

GtH-II in goldfish (Sokolowska et al., 1985) and common carp (Lin et al., 1987; Fig. 4).

This method for induced ovulation of cultured fish has been termed the Linpe technique(Peter et al., 1988). A formulation of sGnRH-A and domperidone has been marketed as aspawning kit OvaprimTM.

Sustained release of GnRH

When using GnRH peptides or analogues to induce a sustained increase in circulatingGtH-II levels to induce multiple ovulations and spawnings over a prolonged period,multiple injections are often required because of the rapid clearance of the injected GnRHanalogues from the circulation. Sustained delivery systems for GnRH analogues provide auseful alternative to frequent handling and injections. Sustained delivery systems aremade in the form of a cholesterol pellet or polyanhydride microspheres (Mylonas et al.,1992; Clearwater and Crim, 1995). For exam ple, multiple spawners such as sea bass

( Lates calcarifer, Centropomidae; Almendras et al., 1988), gilthead sea bream (Zohar etal., 1990) and American shad ( Alosa sapidissima, Clupeidae; Mylonas and Zohar, 1995)

0 20 40 60 80

100

200

300

SerumG

tH

(ng/ml)

d

dd

cba

cba

bba

cba

c TEMPERATURE 21-26c

PS 0/20a

DOM 3/19a

sGnRH-A 2/18a

DOM 1 sGnRH-A 18/20b

OVULATION

HOURS POST-INJECTION

Fig. 4. The time course of the serum GtH-II release-response to injection of physiological saline (PS),

domperidone (DOM; 1 g g 1 body weight), [D-Arg6, Pro9-NEt]-salmon gonadotrophin-releasing

hormone (sGnRH-A; 0.01 g g 1 body weight), or the combination of DOM plus sGnRH-A in

sexually mature female common carp. At each sampling time, groups with statistically similar GtH- II

levels are identified by the same superscript (ANOVA and Duncans multiple range test, p , 0.05).

Groups with statistically similar rates of ovulation (inset) are identified by the same superscript

(Fishers exact probability test, p , 0.05). Reproduced from Lin et al. (1987).



18/25

may be induced to spawn several times using a single sustained-release preparation ofGnRH-A. Similar to Atlantic croaker (Copeland and Thomas, 1989), combination with aDA receptor antagonist, such as domperidone, is not necessary in these species as DAinhibition of GtH-II release is either absent or very weak. A commercially availablecontrolled-release GnRH-A implant has been found to advance and synchronize ovulationin several salmonid species (Goren et al., 1995; Solar et al., 1995).

Sustained-release preparations of GnRH may suffer from some disadvantages,however. In some fishes, a diurnal variation in efficacy of GnRH in inducing GtH-II

secretion and= or ovulation has been suggested (common carp: Billard et al., 1987;goldfish: Peter, 1980; Peter et al., 1982; sea bass: Alvarino et al., 1992). Furthermore,results of studies have shown that excessive dosages of GtH or GnRH preparations mayhave deleterious effects (Mylonas et al., 1992). Prolonged exposure of the pituitary toGnRH peptides may also lead to desensitization or down-regulation of GnRH high-affinity receptor binding sites. It is interesting to note that cGnRH-II has a higheraffinity for the high-affinity binding sites than sGnRH, and it causes a greater down-regulation of the high-affinity binding sites than sGnRH (Peter et al., 1991). It is notknown whether this receptor down-regulation and desensitization of the hormone releaseresponse occurs when fishes are exposed to GnRH analogues over extended periods.However, in goldfish, two injections 12 h apart of sGnRH-A were found to cause anincrease in capacity of the high-affinity GnRH binding sites (Omeljaniuk et al., 1989),

suggesting that over the longer term, receptor down-regulation may not be a factor.

Routes of GnRH delivery

Apart from intraperitoneal or intramuscular injection or implantation of GnRH preparations, other routes of GnRH administration have also been studied in teleosts.Oral and rectal administration of GnRH-A and DA antagonist (pimozide or domperidone)were both effective in stimulation of GtH-II secretion in the agastric common carp(Breton et al., 1995). However, oral delivery of GnRH-A in a gastric species, the Africancatfish, was ineffective in stimulation of GtH-II secretion. It was found that inclusion ofEDTA and a trypsin inhibitor in the GnRH-A preparation for oral delivery significantlyenhanced the GnRH-A-induced GtH-II secretion in African catfish (Breton et al., 1995).

Future directions in research

The role of GnRH peptides in stimulating the secretion of GtH-II is well accepted. Whilethe functions of the multiple forms of GnRH in the brain presents one set of questions,the regulation of secretion and the secretion patterns of GnRH peptides during ovulationremain poorly understood. We also have limited understanding of the dynamics of theDA inhibitory system and feedback actions of sex steroids during ovulation. Possibledifferences in the neuroendocrine mechanisms in fishes that ovulate large masses ofoocytes once or several times in any one spawning season, compared with fishes havingmultiple ovulations of relatively small numbers of oocytes over an extended period oftime, largely remain to be explored.

Regulation of reproduction of broodstock fish for production of fry is essential for theintroduction of new species to aquaculture. Study of the natural reproductive cycle to

develop an understanding of appropriate environmental conditions for folliculardevelopment, ovulation and spawning under culture conditions is one possible approach.

190 Peter and Yu


19/25

Frequently, however, appropriate environmental conditions cannot be provided underculture conditions, making artificial regulation of reproduction necessary. In this situationit is still necessary to study the natural reproductive cycle to know what strategies to usefor regulation of follicular development, and the optimal approach to induce ovulationand spawning. Radioimmunoassay or enzyme-linked immunosorbent assay (ELISA)measurement of GtH-II levels in blood becomes an essential tool to investigate both basicand applied aspects of the reproductive physiology of the species in question. A verygood example of success when this holistic approach is taken to investigate a new species

is the gilthead sea bream, which has recently been successfully introduced to aquacultureusing knowledge developed through basic research. Whether in a new species or in onethat has already been cultured for some time, knowledge based on sound research is theessential basis for strategies for regulation of reproduction.

Acknowledgements

The research described in this review was supported by grants from the Natural Sciencesand Engineering Research Council of Canada (to R.E.P.), the Croucher Foundation andthe Research Grant Council of Hong Kong (to K.L.Y.).

ReferencesAida, K. (1988) A review of plasma hormone changes during ovulation in cyprinid fishes. Aquaculture

74, 1121.

Almendras, J.M., Duenas, C., Nacario, J., Sherwood, N.M. and Crim, L.W. (1988) Sustained hormone

release. III. Use of gonadotropin releasing hormone analogues to induce multiple spawning in sea

bass, Lates calcarifer. Aquaculture 74, 97111.

Alvarino, J.M.R., Zanuy, S., Prt, F., Carrillo, M. and Mananos, E. (1992) Stimulation of ovulation and

steroid secretion by LHRHa injection in the sea bass Dicentrarchus labrax: effect of time of day.

Aquaculture 102, 177186.

Amano, M., Ikuta, K., Kitamura, S. and Aida, K. (1996) Salmon type GnRH neurons in the ventral

telencephalon and the preoptic area receive different feedback control signals in Masu salmon. In:

Abstracts of 3rd International Symposium on Fish Endocrinology, 1996, Hakodate, Japan, p. 62

(Abstract O-43).

Baggerman, B. (1990) Sticklebacks. In Munro, A.D., Scott, A.P. and Lam, T.J., eds. ReproductiveSeasonality in Teleosts: Environmental Influences. Boca Raton, FL: CRC Press, pp. 79107.

Barry, T., Riebe, J., Parrish, J. and Malison, J. (1995) 17,20 -dihydroxy-4-pregnen-3-one stimulates

cortisol production by rainbow trout interrenal tissue in vitro: mechanism of action. In Goetz,

F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium

95, p. 325.

Billard, R., Bieniarz, K., Popek, W., Epler, P., Breton, B. and Alagarswami, K. (1987) Stimulation of

gonadotropin secretion and spermiation in carp by pimozide-LHRHa treatment: effects of dose

and time of day. Aquaculture 62: 161170.

Breton, B., Roelands, I., Mikolajczyk, T., Epler, P. and Ollevier, F. (1995) Induced spawning in teleost

fish after oral administration of GnRH-A. In Goetz, F.W. and Thomas, P., eds. Reproductive

Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, pp. 102104.

Bromage, N.R. and Cumaraanatunga, R. (1988) Egg production in the rainbow trout. Rec. Adv.

Aquaculture 3, 63138.

Bromage, N.R., Elliot, J.A.K., Springate, J.R.C. and Whitehead, C. (1984) The effects of constantphotoperiods on the timing of spawning in the rainbow trout. Aquaculture 43, 213223.



20/25

Brooks, S., Pottinger, T.G., Tyler, C.R. and Sumpter, J.P. (1995) Does cortisol influence egg quality in

the rainbow trout, Oncorhynchus mykiss? In Goetz, F.W. and Thomas, P., eds. Reproductive

Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, p. 180.

Campbell, P., Pottinger, T. and Sumpter, J. (1994) Preliminary evidence that chronic confinement stress

reduces the quality of gametes produced by brown and rainbow trout. Aquaculture 120, 151169.

Canario, A.V.M., Couto, E., Vilia, P., Kime, D.E., Hassin, S. and Zohar, Y. (1995) Sex steroids during

the ovulatory cycle of the gilthead seabream (Sparus aurata). In Goetz, F.W. and Thomas, P., eds.

Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, p. 290

Carragher, J., Sumpter, J., Pottinger, T. and Pickering, A. (1989) The deleterious effects of cortisolimplantation on reproductive function in two species of trout, Salmo trutta L. and Salmo

gairdneri Richardson. Gen. comp. Endocrinol. 76, 310321.

Clearwater, S.J., and Crim, L.W. (1995) Milt quality and quantity produced by yellowtail founder

( Pleuronectes ferrugineus) following GnRH-analogue treatment by microspheres or pellet. In

Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX: Fish

Symposium 95, p. 113.

Cook, A.F., Stacey, N.E. and Peter, R.E. (1980) Periovulatory changes in serum cortisol levels in the

goldfish, Carassius auratus. Gen. comp. Endocrinol. 40, 507510.

Copeland, P.A. and Thomas, P. (1989) Control of gonadotropin release in Atlantic croaker: evidence

for a lack of dopaminergic inhibition. Gen. comp. Endocrinol. 74, 474483.

De Leeuw, R., Habibi, H.R., Nahorniak, C.S. and Peter, R.E. (1989) Dopaminergic regulation of

pituitary gonadotrophin-releasing hormone receptor activity in the goldfish (Carassius auratus).

J. Endocrinol. 121, 239247.

Donaldson, E.D. and Hunter, G.A. (1983) Induced final maturation, ovulation, and spermiation incultured fish. In Hoar, W.S., Randall, D.J. and Donaldson, E.M., eds. Fish Physiology, Vol. IXB.

New York: Academic Press, pp. 351 404.

Dulka, J.G., Sloley, B.D., Stacey, N.E. and Peter, R.E. (1992) A reduction in pituitary dopamine

turnover is associated with sex pheromone-induced gonadotropin secretion in male goldfish. Gen.

comp. Endocrinol. 86, 496505.

Duston, J. and Bromage, N. (1986) Photoperiodic mechanisms and rhythms of reproduction in the

female rainbow trout. Fish Physiol. Biochem. 2, 3551.

Ge, W. and Peter, R.E. (1994) Evidence for non-steroidal gonadal regulator(s) of gonadotropin release

in the goldfish, Carassius auratus. Zool. Sci. 11, 717724.

Ge, W., Chang, J.P., Peter, R.E., Vaughan, J., Rivier, J. and Vale, W. (1992) Effects of porcine

follicular fluid (pFF), inhibin A and activin A on goldfish gonadotropin release in vitro.

Endocrinology 131, 19221929.

Goetz, F.W. (1983) Hormonal control of oocyte final maturation and ovulation in fishes. In Hoar, W.S.,

Randall, D.J. and Donaldson, E.M., eds. Fish Physiology, Vol. IXB. New York: Academic Press,

pp. 117170.

Goetz, F.W., Ranjan, M., Berndtson, A.K. and Duman, P. (1987) The mechanism and hormonal

regulation of ovulation: the role of prostaglandins in teleost ovulation. In Idler, D.R., Crim, L.W.

and Walsh, J.M., eds. Proceedings of International Symposium of Reproductive Physiology of

Fish. St Johns, Newfoundland: Memorial University Press, pp. 1620.

Goetz, F.W., Berndtson, A.K. and Ranjan, M. (1991) Ovulation: mediators at the ovarian levels. In

Pang, P.K.T. and Schreibman, M.P., eds. Vertebrate Endocrinology: Fundamentals and Biomedical

Implications, Vol. 4A. New York: Academic Press, pp. 127 203.

Goetz, F.W., Garczynski, M.A. and Hsu, S.Y. (1995) Expression of kallikrein gene family mRNAs in

the trout ovary. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995.

Austin, TX: Fish Symposium 95, p. 310.

Goos, H.J.Th. (1987) Steroid feedback on pituitary gonadotropin secretion. In Idler, D.R., Crim, L.W.

and Walsh, J.M., eds. Proceedings of International Symposium of Reproductive Physiology of Fish. St Johns, Newfoundland: Memorial University Press, pp. 1620.

192 Peter and Yu


21/25

Goos, H.J.Th., Bosma, P.T., Tensen, C.P., Bogerd, J., Zandbergen, M.A. and Schulz, R.W. (1996)

Gonadotropin-releasing hormones in the African catfish: molecular forms, localization, potency

and receptors. In Abstracts of 3rd International Symposium on Fish Endocrinology, 1996,

Hakodate, Japan, p. 60 (Abstract O-41).

Goren, A., Gustafson, H. and Doering, D. (1995) Field trials demonstrate the efficacy and commercial

benefit of a GnRHa implant to control ovulation and spermiation in salmonids. In Goetz, F.W.

and Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95,

pp. 99101.

Gothilf, Y., Elizur, A. and Zohar, Y. (1995) Three forms of gonadotropin-releasing hormone ingilthead seabream and striped bass: physiological and molecular studies. In Goetz, F.W. and

Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95,

pp. 5254.

Habibi, H.R. and Peter, R.E. (1991) Gonadotropin-releasing hormone (GnRH) receptors in teleosts. In

Scott, A.P., Sumpter, J.P., Kime, D.E. and Rolfe, M.S., eds. Reproductive Physiology of Fish,

1991. Sheffield, England: FishSymp 91, pp. 109113.

Habibi, H.R., Peter, R.E., Nahorniak, C.S., Milton, R.C. de L. and Millar, R.P. (1992) Activity of

vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in

positions 5, 7 and 8 in the goldfish pituitary. Regulatory Peptides 37, 271284.

Hontela, A. and Stacey, N.E. (1990) Cyprinidae. In Munro, A.D., Scott, A.P. and Lam, T.J., eds.

Reproductive Seasonality in Teleosts: Environmental Influences. Boca Raton, FL: CRC Press,

pp. 5377.

Hoover, E.E. and Hubbard, H.E. (1937) Modification of the sexual cycle in trout by control of light.

Copeia 1937, 206210.Kagawa, H., Kobayashi, M., Hasegawa, Y. and Aida, K. (1994) Insulin and insulin-like growth factors

I and II induce final oocyte maturation of oocytes of red seabream, Pagrus major, in vitro. Gen.

comp. Endocrinol. 95, 293300.

Karten, M.J. and Rivier, J.E. (1986) Gonadotropin-releasing hormone analog design. Structure

function studies toward the development of agonists and antagonists: rationale and perspective.

Endocr. Rev. 7, 4466.

Kime, D.E. (1993) Classical and non-classical reproductive steroids in fish. Rev. Fish Biol. Fish. 3,

160180.

King, J.A. and Millar, R.P. (1995) Evolutionary aspects of gonadotropin-releasing hormone and its

receptor. Cell. mol. Neurobiol. 15, 523.

King, V.W., Ghosh, S., Thomas, P. and Sullivan, C.V. (1995) Ovarian receptors for 17,20 ,21-

trihydroxy-4-pregnen-3-one (20 -S) in striped bass. In Goetz, F.W. and Thomas, P., eds.

Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, p. 314.

Kobayashi, M. (1996) Gonadotropin-releasing hormone and gonadotropin in fish reproduction. In

Abstracts of 3rd International Symposium on Fish Endocrinology, 1996, Hakodate, Japan, p. 16

(Abstract L-1).

Kobayashi, M., Aida, K. and Hanyu, I. (1987) Hormone changes during ovulation and effects of

steroid hormones on plasma gonadotropin levels and ovulation in goldfish. Gen. comp.

Endocrinol. 67, 2432.

Kobayashi, M., Aida, K. and Hanyu, I. (1989) Involvement of steroid hormones in the preovulatory

gonadotropin surge in female goldfish. Fish. Physiol. Biochem. 7, 141146.

Kobayashi, M., Amano, M., Kim, M.H., Furukawa, K., Hasegawa, Y. and Aida, K. (1994)

Gonadotropin-releasing hormones of terminal nerve origin are not essential to ovarian

development and ovulation in goldfish. Gen. comp. Endocrinol. 95, 192200.

Lam, T.J. (1983) Environmental influences on gonadal activity in fish. In Hoar, W.S., Randall, D.J.,

and Donaldson, E.M., eds. Fish Physiology, Vol. IXB. New York: Academic Press, pp. 65 116.

Le Gac, F., Blaise, O., Fostier, A., Le Bail, P.Y., Loir, M., Mourot, B. and Weil, C. (1993) Growthhormone (GH) and reproduction: a review. Fish Physiol. Biochem. 11, 219232.



22/25

Lessman, C.A. (1985) Effect of insulin on meiosis reinitiation induced in vitro by progestogens in

oocytes of the goldfish (Carassius auratus). Dev. Biol. 107, 259263.

Levavi-Zermonsky, B. and Yaron, Z. (1986) Changes in gonadotropin and ovarian steroids associated

with oocyte maturation during spawning induction in the carp. Gen. comp. Endocrinol. 62,

8998.

Lin, H.R. (1982) Polycultural system of freshwater fish in China. Can. J. Fish. aquat. Sci. 39,

143150.

Lin, H.R., Liang, J.Y., Van Der Kraak, G. and Peter, R.E. (1987) Stimulation of gonadotropin secretion

and ovulation in common carp by an analogue of salmon GnRH and domperidone. In Ohnishi,E., Nagahama, Y. and Ishizaki, H., eds. Proceedings of the First Congress of the Asia

and Oceania Society for Comparative Endocrinology. Japan: Nagoya University Corporation,

pp. 155156.

Linard, B., Anglade, I., Bennani, S., Salbert, G., Navas, J.M., Bailhache, T., Pakdel, F., Jego, P.,

Valotaire, Y., Saligaut, C. and Kah, O. (1995a) Some insights into sex steroid feedback

mechanisms in the trout. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish,

1995. Austin, TX: Fish Symposium 95, pp. 4951.

Linard, B., Bennani, S. and Saligaut, C. (1995b) Involvement of estradiol in a catecholamine

inhibitory tone of gonadotropin release in the rainbow trout ( Oncorhynchus mykiss). Gen. comp.


Lovejoy, D.A., Corrigan, A.Z., Nahorniak, C.S., Perrin, M.H., Porter, J., Kaiser, R., Miller, C., Pantoja,

D., Craig, A.G., Peter, R.E., Vale, W.W., Rivier, J.E. and Sherwood, N.M. (1995) Structural

modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of

novel GnRH analogues. Regul. Peptides 60, 99115.Maestro, M.A., Planas, J.V., Swanson, P. and Gutierrez, J. (1995) Insulin-like growth factor I (IGF-I) in

the fish ovary. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995.

Austin, TX: Fish Symposium 95, pp. 279283.

Matsuyama, M., Adachi, S., Nagahama, Y., Maruyama, K. and Matsura, S. (1990) Diurnal rhythm of

serum hormone levels in the Japanese whiting, Sillago japonica, a daily spawning teleost. Fish.

Physiol. Biochem. 8, 32938.

Meiri, I., Gothilf, Y., Knibb, W.R., Zohar, Y. and Elizur, A. (1995) Preovulatory changes in

gonadotropin gene expression and secretion in the gilthead seabream, Sparus aurata. In Goetz,

F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium

95, p. 37.

Miwa, S., Yan, L. and Swanson, P. (1994) Localization of two gonadotropin receptors in the salmon

gonad by in vitro ligand autoradiography. Biol. Reprod. 50, 629642.

Modesto, T. and Canario, A.V.M. (1995) Steroid levels during ovulation and spermiation in toadfish

Halobatrachus didactylus. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish,

1995. Austin, TX: Fish Symposium 95, p. 322.

Montero, M., Le Belle, N., King, J.A., Millar, R.P. and Dufour, S. (1995) Differential regulation of the

two forms of gonadotropin-releasing hormone (mGnRH and cGnRH-II) by sex steroids in the

European female silver eel (Anguilla anguilla). Neuroendocrinology 61, 525535.

Muginier, C., Gaignon, J.L., Legegue, E., Fauvel, C. and Fostier, A. (1995) Maturation inducing

steroid in turbot, Scophthalmus maximus L. In Goetz, F.W. and Thomas, P., eds. Reproductive

Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, p. 323.

Murthy, C.K., Turner, R.J., Nestor, J.J., jun., Rivier, J.E. and Peter, R.E. (1994a) A new gonadotropin-

releasing hormone (GnRH) superagonist in goldfish: influence of dialkyl-D-homoarginine at

position 6 on gonadotropin-II and growth hormone release. Regul. Peptides 53, 115.

Murthy, C.K., Zheng, W., Trudeau, V.L., Nahorniak, C.S., Rivier, J.E. and Peter, R.E. (1994b) In vivo

actions of a gonadotropin-releasing hormone (GnRH) antagonist on gonadotropin-II and growth

hormone secretion in goldfish, Carassius auratus. Gen. comp. Endocrinol. 96, 427437.Mylonas, C.C. and Zohar, Y. (1995) Preparation and evaluation of GnRHa-loaded, polymeric delivery

194 Peter and Yu


23/25


24/25

Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, pp. 26.

Scott, A.P., Canario, A.V.M. and Prat, F. (1990) Radioimmunoassay of ovarian steroids in plasma of

ovulating female sea bass (Dicentrarchus labrax). Gen. comp. Endocrinol. 78, 299302.

Sherwood, N.M., Parker, D.B., McRory, J.E. and Lescheid, D.W. (1994) Molecular evolution of growth

hormone-releasing hormone and gonadotropin-releasing hormone. In Sherwood, N.M. and Hew,

C.L., eds. Fish Physiology, Vol. XIII. New York: Academic Press, pp. 3 66.

Singh, H. and Thomas, P. (1991) Mechanism of stimulatory action of growth hormone on ovarian

steroidogensis in spotted seatrout. In Scott, A.P., Sumpter, J.P., Kime, D.E. and Rolfe, M.S., eds.

Reproductive Physiology of Fish, 1991. Sheffield, England: FishSymp 91, p. 104.Sokolowska, M., Peter, R.E. and Nahorniak, C.S. (1985) The effects of different doses of pimozide

and [D-Ala6, Pro9-N ethylamide]-LHRH (LHRH-A) on gonadotropin release and ovulation in

female goldfish. Can. J. Zool. 63, 12521256.

Solar, I.I., Smith, J., Dye, H.M., MacKinlay, D.D., Zohar, Y. and Donaldson, E.M. (1995) Induced

ovulation of chinook salmon using a GnRHa implant: effect on spawning, egg variability and

hormone levels. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995.

Austin, TX: Fish Symposium 95, pp. 144145.

Sorensen, P.W., Hara, T.J., Stacey, N.E. and Goetz, F.W. (1988) F prostaglandins function as potent

olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish. Biol

Reprod. 39, 10391050.

Stacey, N.E. (1983) Hormones and reproductive behavior in teleosts. In Rankin, J.C., Pitcher, T.J. and

Duggan, R.T., eds. Control Processes in Fish Physiology. London: Croom Helm, pp. 117129.

Stacey, N.E. (1984) Control of the timing of ovulation by exogenous and endogenous factors. In Potts,

G.W. and Wootton, R.J., eds. Fish Reproduction: Strategies and Tactics. New York: AcademicPress, pp. 207222.

Stacey, N.E. and Peter, R.E. (1979) Central action of prostaglandins in spawning behaviour of female

goldfish. Physiol. Behav. 22, 11911196.

Stacey, N.E. and Sorensen, P. (1991) Function and evolution of fish hormonal pheromones. In

Hochachka, P.W. and Mommsen, T.P., eds. Biochemistry and Molecular Biology of Fishes, Vol. 1.

Amsterdam: Elsevier, pp. 109 135.

Stacey, N.E., Cook, A.F. and Peter, R.E. (1979) Ovulatory surge of gonadotropin in the goldfish,

Carassius auratus. Gen. comp. Endocrinol. 37, 246249.

Stacey, N.E., Cardwell, J.R., Liley, N.R., Scott, A.P. and Sorensen, P.W. (1994) Hormonal sex

pheromones in fish. In Davey, K.G., Peter, R.E. and Tobe, S.S., eds. Perspectives in Comparative

Endocrinology. Ottawa: National Research Council of Canada, pp. 438448.

Sumpter, J.P., Cararagher, J.F., Pottinger, T.G. and Pickering, A.D. (1987) The interaction of stress and

reproduction in trout. In Idler, D.R., Crim, L.W. and Walsh, J.M., eds. Proceedings of

International Symposium of Reproductive Physiology of Fish. St Johns Newfoundland: Memorial

University Press, pp. 299302.

Swanson, P. (1991) Salmon gonadotropins: reconciling old and new ideas. In Scott, A.P., Sumpter, J.P.,

Kime, D.E. and Rolfe, M.S., eds. Reproductive Physiology of Fish, 1991. Sheffield, England:

FishSymp 91, pp. 27.

Taranger, G.L. (1993) Sexual maturation in Atlantic salmon, Salmo salar L.; aspects of environmental

and hormonal control. Dr scient. thesis, Univ. Bergen, Norway. ISBN 82-7744-006-5. 41 pp.

Thomas, P. and Ghosh, S. (1995) Regulation of the maturation-inducing receptor in spotted seatrout

ovaries. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish, 1995. Austin, TX:

Fish Symposium 95, p. 376.

Thomas, P. and Trant, J.K. (1989) Evidence that 17,20 ,21-trihydroxy-4-pregnen-3-one is a

maturation-inducing steroid in spotted seatrout. Fish Physiol Biochem. 7, 185191.

Trudeau, V.L. and Peter, R.E. (1995) Functional interactions between neuroendocrine systems

regulating GtH-II release. In Goetz, F.W. and Thomas, P., eds. Reproductive Physiology of Fish,1995. Austin, TX: Fish Symposium 95, pp. 4448.

196 Peter and Yu


25/25

Trudeau, V.L., Sloley, B.D. and Peter, R.E. (1991) Testosterone and estradiol potentiate the serum

gonadotropin response to gonadotropin releasing hormone in goldfish. Biol. Reprod. 44, 951960.

Trudeau, V.L., Sloley, B.D. and Peter, R.E. (1993a) Norepinephrine turnover in the goldfish brain is

modulated by sex steroids and GABA. Brain Res., Amsterdam 624, 2934.

Trudeau, V.L., Sloley, B.D., Wong, A.O.L. and Peter, R.E. (1993b) Interactions of gonadal steroids

with brain dopamine and gonadotropin-releasing hormone in the control of gonadotropin-II

secretion in the goldfish. Gen. Comp. Endocrinol. 89, 3950.

Tsafriri, A., Daphna-Iken, D., Abisogun, A.O. and Reich, R. (1991) Follicular rupture during

ovulation: activation of collagenolysis. In Mashiach, S., Ben-Rafael, Z., Laufer, N. andSchenker, J.G., eds. Advances in Assisted Reproductive Technologies. New York: Plenum Press,

pp. 103112.

Tse, C.H., Dong, K.W. and Yu, K.L. (1996) Molecular cloning of the goldfish dopamine D2 receptor.

Am. Zool. 36 29A (abstract 113).

Van Der Kraak, G., Rosenblum, P.M. and Peter, R.E. (1990) Growth hormone dependent potentiation

of gonadotropin stimulated steroid production by ovarian follicles of the goldfish. Gen. comp.


Van Der Kraak, G., Suzuki, K., Peter, R.E., Itoh, H. and Kawauchi, H. (1992) Properties of common

carp gonadotropin I and gonadotropin II. Gen. comp. Endocrinol. 85, 217229.

Wallace, R.A. and Selman, K. (1981) Cellular and dynamic aspects of oocyte growth in teleosts. Am.

Zool. 21, 325343.

Weil, C., Fostier, A. and Billard, R. (1986) Induced spawning (ovulation and spermiation) in carp and

related species. In Billard, R. and Marcel, J., eds. Aquaculture of Cyprinids. Paris: INRA,

pp. 119137.Weil, C., Bougoussa-Houadec, M., Gallais, C., Itoh, S., Sekine, S. and Valotaire, Y. (1995) Preliminary

evidence suggesting variations GtH 1 and GtH 2 mRNA levels at different stages of gonadal

development in rainbow trout, Oncorhynchus mykiss. Gen. comp. Endocrinol. 100, 327333.

Yan, L., Swanson, P. and Dickhoff, W.W. (1991) Binding of gonadotropins (GtH I and GtH II) to coho

salmon gonadal membrane preparations. J. exp. Zool. 258, 221230.

Yan, L., Swanson, P. and Dickhoff, W.W. (1992) A two-receptor model for salmon gonadotropins

(GTH I and GTH II). Biol. Reprod. 47, 418427.

Yoshiura, Y., Kobayashi, M., Kato, Y. and Aida, K. (1995) Molecular cloning of cDNA encoding two

gonadotropin subunits (GTH I and II ) from the goldfish. In Goetz, F.W. and Thomas, P., eds.

Reproductive Physiology of Fish, 1995. Austin, TX: Fish Symposium 95, p. 41.

Yu, K.L., Nahorniak, C.S., Peter, R.E., Corrigan, A., Rivier, J.E. and Vale, W.W. (1987) Brain

distribution of radioimmunoassayable gonadotropin-releasing hormone in female goldfish:

seasonal variation and periovulatory changes. Gen. comp. Endocrinol. 67, 234246.

Yu, K.L., Peng, C. and Peter, R.E. (1991) Changes in brain levels of gonadotropin-releasing hormone

and serum levels of gonadotropin and growth hormone in goldfish during spawning. Can. J. Zool.

69, 182188.

Yu, K.-L., Lin, X.-W., Bastos, J.C. and Peter, R.E. (1997) Neural regulation of gonadotropin-releasing

hormones in teleost fishes. In Parhar, I.S. and Sakuma, Y., eds. GnRH Neurons: Gene to

Behavior. Tokyo: Brain Shuppan Publishers (in press).

Zanuy, S., Carrillo, M. and Ruiz, F. (1986) Delayed gametogenesis and spawning of sea bass

( Dicentrarchus labrax L.) kept under different photoperiod and temperature regimes. Fish

Physiol. Biochem. 2, 5363.

Zohar, Y., Goren, A., Fridkin, M., Elhanati, E. and Koch, Y. (1990) Degradation of gonadotropin-

releasing hormones in the gilthead seabream, Sparus aurata. II. Cleavage of native salmon

GnRH, mammalian LHRH, and their analogs in the pituitary, kidney, and liver. Gen. comp.


Accepted 6 February 1997


reviews in fish biology and fisheries 7,

Documents