Zvi Yaron

Endocrine control of gametogenesis and spawning induction in the carp

Abstract The gonadotropic regulation of spermatogenesis and spermiogenesis is apparently mediated by 11-ketotestosterone (11-KT) secreted by Leydig cells. However, the acquisition of sperm motility requires the stimulation of the sperm duct by gonadotropin, or by 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) which is produced by spermatozoa from 17α-hydroxyprogesterone (17-OH-P). The theca of the ovarian follicle performs most of the steroidogenic steps culminating in the formation of testosterone which diffuses into the granulosa layer to be aromatized to estradiol. A parallel two-cell type mechanism operates towards final oocyte maturation: theca cells produce 17-OH-P which diffuses into the granulosa cells to be converted to 17,20-P. Nevertheless, this model does not apply to all teleost fish. The synthesis of vitellogenin (VTG) by the liver is regulated by estradiol in synergism with growth hormone. The uptake of VTG, through low-affinity/high-capacity binding sites on the plasma membrane of the oocytes is stimulated by GTH-I. The proteins forming the chorion are produced by the liver and carried in the circulation to be incorporated at the periphery of the oocyte. The cascade of hormones involved in final oocyte maturation includes GTH-II which stimulates the formation of 17,20-P, or other maturation-inducing steroids, which bind to receptors on the oocyte membrane and activate the maturation-promoting factor (MPF). This factor is composed of cyclin B and a kinase of the CDC2 type. Ovulation involves the formation of prostaglandins and synthesis of proteases which degrade part of the follicular wall to form a rupture site through which the egg can emerge. Contractility of the follicular wall may take part in the process of ovulation. There is ample evidence for gonadotropin duality in salmonids. The two GTHs share a common α-subunit but differ considerably in their β-subunits. Also in the carp two GTHs have been isolated from pituitaries of adult females, and although shown to be chemically distinct, their function is quite similar. However, gonadotropin duality in cyprinid fish has not been confirmed by other laboratories and further research is required to elucidate this point. The release of GTH from the pituitary is regulated by various brain hormones, the most active being gonadotropin-releasing hormone (GnRH), which stimulates the release, and dopamine, which is inhibitory. Spawning induction in cyprinid fish is currently carried out by the hypophyseal approach (hypophysation or administration of chorionic gonadotropin) or by the hypothalamic approach (GnRH superactive analogues in combination with dopamine antagonists — the Linpe method). Comparison between the two approaches under the conditions of Israeli hatcheries has shown that both are suitable for spawning induction in carp, provided that the GTH content of the pituitary extract is calibrated. However, the Linpe method is advantageous because of the availability and lower cost of the compounds required for hypothalamic manipulations. In addition, the hazard of spreading pathogens along with pituitary material is a point to be considered. Adapting the Linpe method to Israeli cyprinid culture has required research int selecting the most suitable GnRH analogue and the dopamine antagonist, and the most suitable mode of their administration. In addition, the research examined the proper time of injection, the response latency and its temperature dependence. The results of the study indicate that the response to spawning induction by either approach cannot be assumed to be the same in every country due to differences in the environment, hatchery procedures and carp lines. In order to establish an optimal procedure for spawning induction at a given site, a study under the specific local conditions should be carried out using previous results only as guidelines.

Endocrine regulation of gonadotropin and growth hormone gene transcription in fish

The pituitary of a number of teleosts contains two gonadotropins (GtHs) which are produced in distinct populations of cells; the beta subunit of the GtH I being found in close proximity to the somatotrophs, while the II beta cells are more peripheral. In several species the GtH beta subunits are expressed at varying levels throughout the reproductive cycle, the I beta dominating in early maturing fish, after which the II beta becomes predominant. This suggests differential control of the beta subunit synthesis which may be regulated by both hypothalamic hormones and gonadal steroids. At ovulation and spawning, changes also occur in the somatotrophs, which become markedly more active, while plasma growth hormone (GH) levels increase. In a number of species, GnRH elevates either the I beta or the II beta mRNA levels, depending on the reproductive state of the fish. In tilapia, the GnRH effect on the II beta appears to be mediated through both cAMP-PKA and PKC pathways. GnRH also stimulates GH release in both goldfish and tilapia, but it increases the GH transcript levels only in goldfish; both GnRH and direct activation of PKC are ineffective in altering GH mRNA in tilapia pituitary cells. Dopamine (DA) does not alter II beta transcript levels in cultured tilapia pituitary cells, but increases GH mRNA levels in both rainbow trout and tilapia, in a PKA-dependent manner. This effect appears to be through interactions with Pit-1 and also by stabilizing the mRNA. Somatostatin (SRIF) does not alter GH transcript levels in either tilapia or rainbow trout, although it may alter GH synthesis by modulation of translation. Gonadal steroids appear to have differential effects on the transcription of the beta subunits. In tilapia, testosterone (T) elevates I beta mRNA levels in cells from immature or early maturing fish (in low doses), but depresses them in cells from late maturing fish and is ineffective in cells from regressed fish. Similar results were seen in early recrudescing male coho salmon injected with T or E2. T or E2 administered in vivo has dramatic stimulatory effects on the II beta transcript levels in immature fish of a number of species, while less powerful effects are seen in vitro. A response is also seen in cells from early maturing rainbow trout or tilapia, or regressed tilapia, but not in cells from late maturing or spawning fish. These results are substantiated by the finding that the promoter of the salmon II beta gene contains several estrogen responsive elements (EREs) which react and interact differently when exposed to varying levels of E2. In addition, activator protein-1 (AP-1) and steroidogenic factor-1 (SF-1) response elements are also found in the salmon II beta promoter; the AP-1 site is located close to a half ERE, while the SF-1 acts synergistically with the E2 receptor. The mRNA levels of both AP-1 and SP-1 are elevated, at least in mammals, by GnRH, suggesting possible sites for cross-talk between GnRH and steroid activated pathways. Reports of the effects of T or E2 on GH transcription differ. No effect is seen in vitro in pituitaries of tilapia, juvenile rainbow trout or common carp, but T does increase the transcript levels in pituitaries of both immature and mature goldfish. Reasons for these discrepancies are unclear, but other systemic hormones may be more instrumental than the gonadal steroids in regulating GH transcription. These include T3 which increases both GH mRNA levels and de novo synthesis (in tilapia and common carp) and insulin-like growth factor-I (IGF-I) which reduces GH transcript levels as well as inhibiting GH release.

Spawning induction in common carp (Cyprinus carpio) using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocyte maturation and dependence on temperature

Spawning experiments under routine hatchery conditions on Israeli farms showed that successful ovulation in the common carp (Dor 70 x Yugoslavian lines) can be achieved by a single administration of 10 pg/kg sGnRHa ( [D-Arg6,Prop-NEt I-sGnRH) combined with 20 mg/kg of the water-soluble dopamine receptor antagonist, metoclopramide (GnRH+MET). An initial rise in the maturational gonadotropin (cGtH) level occurred 3 h after treatment, gradually increasing thereafter, and reaching a peak of 227f 41.8 ng/ml (mean? s.e.m., n= 10) 14 h after treatment, when full ovulation took place, as reflected by the presence of a few expelled eggs on the bottom of the tank. This rise was associated with increased levels of oestradiol-17/I (Ez; 19.5 f 3.4 ng/ml) and 17a,20/?-dihydroxy-4pregnen-3-one ( 17,20-P; 23.9 f 19.7 ng/ml). Ovarian biopsies showed a gradual progress in oocyte maturation with germinal vesicle breakdown (GVBD) occurring during the rise in both steroids. At the time of initial egg release, GtH remained at a high level while the steroids started to decline. In a parallel group, fish were primed with a dose of carp pituitary extract (CPE), calibrated to contain 0.07 mg/kg cGtH, and 11 h later with a resolving dose of CPE (0.34 mg/kg cGtH). Following priming, circulating GtH and E, increased moderately, and the germinal vesicle migrated towards the oocytes’ periphery. A further increase in cGtH and E2, and a sharp peak in 17,20-P, occurred concomitantly with GVBD, 6 h after the resolving dose was given. Data compiled from several spawning experiments showed that the interval between hormone administration and initial egg release (latency) was negatively correlated with water temperature over the range of 20-26°C. While latency was always longer in GnRH + MET than in CPE treatment, in each treatment it was nonetheless constant between 22.5 ’ C and 25 ” C. GnRH + MET given at various hours of the day or evening resulted in identical spawning with the same latency. This fact, together with the predicted latency at a given temperature, may provide the aquaculturist with a protocol to accurately schedule spawning induction in the common carp.

Differential Effects of Gonadotropin-Releasing Hormone, Dopamine and Somatostatin and Their Second Messengers on the mRNA Levels of Gonadotropin IIβ Subunit and Growth Hormone in the Teleost Fish, Tilapia

In cultured pituitary cells of tilapia, gonadotropin-releasing hormone (GnRH; 10 nM 4-24 h), elevation of cyclic AMP (by 10 microM forskolin or 0.2 mM 3-isobutyl-1-methylxanthine: IBMX 0.5-36 h) or activation of protein kinase C (PKC; by 12.5 nM tetradecanoyl phorbol-13-acetate: TPA, 0.5-24 h) all increased gonadotropin (GtH) II beta steady state mRNA levels by three to four-fold. The involvement of PKA and PKC in the GnRH stimulatory effect on both GtH release and GtH II beta mRNA levels was corroborated by use of the PKA and PKC inhibitors, H89 and GF109203X, respectively (100 nM) which attenuated the GnRH effect. Incubation with actinomycin D (8 microM, 4-21 h) after preexposure for 24 h to either forskolin (10 microM) or TPA (12.5 nM), revealed that rates of transcript degradation were slower in forskolin-treated cells (T 1/2 = 14.1 h) than in control or TPA-treated cells (T 1/2 = 8.47 or 8.38 h), suggesting a stabilizing effect on the mRNA. Dopamine (DA; 10 microM, 4-36 h) had no apparent effect on steady state mRNA levels of GtH II beta, but reduced GtH release by as much as 75%. Steady state levels of growth hormone (GH) mRNA were not affected by exposure to GnRH (10 nM, 4-24 h), although GH release was more than doubled. Similarly, activation of PKC (by TPA 12.5 nM, 1.5-36 h), which was shown to be essential for the GnRH-stimulatory effect on GH release, did not alter levels of the GH transcript, but increased GH release by more than fivefold. DA (10 microM, 4-24 h) moderately increased GH transcript levels (160%) with similar kinetics but lower potency than direct elevation of cAMP (by 10 microM forskolin or 0.2 mM IBMX, 0.5-36 h) which increased transcript levels by more than fourfold. The involvement of PKA in the DA effect was confirmed when the PKA inhibitor H89 (100 nM, 15 min prior to DA exposure) attenuated the DA effect on GH mRNA levels. Exposure of cells to actinomycin D (8 microM, 2-16 h) after treatment with forskolin (10 microM, 24 h) led to a slower rate of transcript degradation than in control cells (T 1/2 = 6.5 h vs. T 1/2 = 4.36 h), suggesting that cAMP also elicits a stabilizing effect on GH mRNA. Somatostatin (100 nM, 0.5-36 h) had no clear effect on GH transcript levels, but reduced GH release by as much as 90%. These results suggest that activation of either cAMP-PKA or PKC pathways can, possibly by different mechanisms, stimulate mRNA levels of the GtH II beta gene, but that only the cAMP-PKA pathway stimulates GH mRNA levels. It would appear therefore that GnRH, although stimulating GH release, does not regulate GH transcription in this fish.

Changes in gonadotropin and ovarian steroids associated with oocytes maturation during spawning induction in the carp

The present study correlates the profiles of certain ovarian steroid hormones with stages of oocyte maturation in common carp induced to spawn by classic hypophysation. Sixteen females were injected at midday with a calibrated homologous pituitary extract (cPE) containing 0.07 mg immunoreactive cGTH/kg body wt, as a priming dose. Ten of those females were injected 11 hr later with cPE containing 0.35 mg IR cGTH/kg body wt as an induction dose (Group P + IN). The other females were left without further treatment (Group P). An additional 6 fish were injected around midnight with the induction dose only (Group IN). A control group was injected with 0.7% saline (0.2 ml/kg) at midday and at midnight (Group S). Ovarian biopsies were taken at intervals and the maturational stages of individual females were recorded. Blood was sampled simultaneously with the biopsies. GTH, estradiol (E2) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were determined by specific RIAs. Germinal vesicle breakdown (GVBD) and ovulation were observed only in Group P + IN 2-4 hr after the second injection. The change in circulating ovarian steroids, in response to hypophysation, was found to depend on the maturational stage of the oocytes. Fish with oocytes showing central and eccentric GV responded to the increased GTH level by elevating E2, but not 17,20-P in the circulation. However, fish with ovaries containing peripheral GV responded by an increase in circulating 17,20-P (111 +/- 14.6 ng/ml) concomitantly with a transient decrease in E2. Only fish that showed such a shift in the steroidogenic profile exhibited GVBD in their oocytes and consequently ovulated. This finding is consonant with the hypothesis that 17,20-P is the maturational-inducing steroid in the carp.

Possible sites of dopaminergic inhibition of gonadotropin release from the pituitary of a teleost fish, tilapia.

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 microM) totally abolished this stimulation. Dopamine (DA; 1 microM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 microM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D2 type mode of action. The inhibition of GtH release in culture, caused by 1 microM APO, the specific DA D2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 microM) in perifusion was not reduced by DA (1 microM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca2+ ionophore A23187, however, their inhibitory effect was reversed by 10 microM ionomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia☆

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence and biological activity of estradiol-17β in the intact and ovariectomized Tilapia aurea (Cichlidae, Teleostei)

Recent reports on ovarian steroids of teleosts have, in large part, emphasized the finding of substances other than the C18 steroids. Furthermore, most of these reports do not include findings concerning the physiologic role of the steroids measured. In the present work we studied both radioimmunoassay measurements of circulating estrogen and its functional significance in a female teleost. Plasma levels of estradiol-17β(E2) were measured in Tilapia aurea by radioimmunoassay, using a rabbit anti-17β-estradiol-6-bovine serum albumin serum. The cross-reactivity of the following steroids with E2 standards was: estrone, 1.4%; estriol, 0.3%; 11-deoxycorticosterone, 0.3%; testosterone, <0.1%; 16α-OH-estrone, 11-ketotestosterone, 11β-OH-testosterone, cortisol, deoxycortisol, and progesterone did not show any affinity to the antiserum. Estradiol, chromatographically purified from plasma, accounted for its total immunoreactivity. The E2 level in the plasma of adult females kept at 25° for 1 month or more was 3.1 ± 0.7 ng/ml (mean ± SEM; N = 26; range, 0.2–10 ng/ml). A positive correlation was found between plasma E2 concentrations and ovarian weight. Nine days following bilateral ovariectomy the plasma E2 level dropped to 0.08 ± 0.04 ng/ml (N = 26). The level remained low for the subsequent 55 days. Concentration of plasma calcium and proteins, indirect parameters of the plasma vitelloprotein level, decreased following ovariectomy, and increased in estradiol-injected ovariectomized fish. It is concluded that: (a) Immunoreactive estradiol-17β occurs in the blood of female Tilapia aurea; (b) the ovary is the main source of E2 in this fish; (c) the circulating level of this steroid depends on the maturity of the ovary; and (d) E2 stimulates the synthesis and release of vitelloproteins in this fish. All this evidence is consonant with the conclusion that estradiol-17β may be the estrogenic hormone of Tilapia aurea.

GnRH signaling pathways regulate differentially the tilapia gonadotropin subunit genes

Exposure of tilapia pituitary cells in culture to salmon gonadotropin-releasing hormone (sGnRH; 0.01-100 nM) elevated the phosphorylated extracellular signal-regulated kinase (pERK) levels. sGnRH also elevated the alpha, FSHbeta and LHbeta subunit mRNA levels. The phorbol ester, 1-O-tetradecanoyl phorbol-13-acetate (TPA; 12.5 nM) increased pERK levels, whereas protein kinase C (PKC) depletion or inhibition by GF109203X (GF; 0.01-10 microM) suppressed GnRH-activated ERKs. GF too abated the GnRH-induced alpha and LHbeta mRNA levels, but had no effect on those of FSHbeta. Forskolin (0.001-100 microM) activated ERK, while inhibition of protein kinase A (PKA) by H89 (0.01-10 microM) suppressed pERK levels and all GnRH-stimulated gonadotropin subunit transcripts. Exposure of cells to the mitogen-activated protein kinase kinase (MAPK kinase; MEK) inhibitor (PD98059; PD 10, 25 and 50 microM) completely blocked GnRH-induced increase in ERKs activation. Furthermore, PD suppressed the alpha and LHbeta mRNA responses to GnRH, but had no effect on FSHbeta mRNA levels. It is suggested that in tilapia the differential regulation of gonadotropin subunit gene expression by GnRH results from a divergent recruitment of signal transduction pathways, activated upon GnRH binding; PKC-ERK cascade is involved in elevating alpha and LHbeta mRNAs, whereas induction of FSHbeta transcript is ERK-independent and is under direct cAMP-PKA regulation or through other MAPK cascades.

Reproductive development of male and female tilapia hybrids (Oreochromis niloticus × O. aureus) and changes in mRNA levels of gonadotropin (GtH) Iβ and IIβ subunits.

A study was carried out in tilapia in order to see whether the gonadotropin (GtH) β subunits show distinct patterns of expression at different stages of their reproductive development. Male and female tilapia hybrids (Oreochromis niloticus × O. aureus) were collected at various times of the year, and a number of parameters were measured in order to establish the reproductive state of the fish. Circulating testosterone (T), estradiol (E2) and 11 ketotestosterone (11KT) levels were assayed, gonads were removed for calculation of gonadosomatic index (GSI) values and histological studies, and RNA was extracted from the pituitaries for measurement of GtH Iβ and IIβ mRNA levels. In maturing fish of both sexes, the circulating steroid levels were positively correlated with each other (r2 = 0.66–0.91) and in males, also with the GSI values (r2 = 0.68). A positive correlation was also seen in these fish between GSI values and the prevalence of spermatocytes and spermatids (r2 = 0.54). In maturing females, the maximal oocyte diameter was positively correlated with circulating E2 levels (r2 = 0.63), while GSI values showed no correlation; this presumably relates to the cycling nature of this asynchronous spawner. In regressing fish of both sexes, no clear correlation between these reproductive parameters was seen. In all fish, the GtH Iβ mRNA levels were highest in fish with steroids ranging 1–10 ng T or E2/ml for males or females, respectively, and were lower in fish with steroids at higher or lower levels. In fish with high steroid levels, the IIβ mRNA levels were also high, and in regressed males the increases were positively correlated. Exposure of cultured pituitary cells to either steroid (T at >10 nM, or E2 at >1 nM) was followed by a decrease in the steady-state levels of the Iβ transcript, while those of IIβ were left unaltered. In situ hybridization studies revealed that in pituitaries of both sexes, the cells producing each of these mRNAs are located in a distinct location. These results suggest that gonadal steroids may exert differential feedback mechanisms at the level of the pituitary to control transcription of each GtH β subunit in distinct cell types specific for each hormone. J. Exp. Zool. 286:64–75, 2000.