Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print June 28, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.051540
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BIOLOGY OF REPRODUCTION 75, 642–650 (2006)
DOI: 10.1095/biolreprod.106.051540
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary1

Berta Levavi-Sivan 2, Jakob Biran , and Einat Fireman 

Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University, Rehovot 76100, Israel

ABSTRACT

Althoughmolecular mechanisms underlying steroid effects on GnRH and dopamine receptors are well documented in mammals, little is known in fish. Herein, we describe the expression of pituitary GnRH and dopamine receptors relative to gonadotropin expression and release. We exposed female tilapia to graded doses of estradiol or 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) in vitro, and of estradiol in vivo, and determined mRNA levels ofgnrhr1,gnrhr3,drd2,lhb, andfshbby real-time PCR. We also determined gonadotropin levels using specific ELISAs. Exposure to low doses of estradiol caused increasedgnrhr3mRNA levels in vivo and in vitro, probably related to positive feedback on FSH release. Increasing concentrations of estradiol resulted in increaseddrd2mRNA levels in vivo and in vitro, inhibition of LH and FSH release, and inhibition oflhbmRNA levelsin vivo, possibly related to negative feedback. At high doses of estradiol, FSH release increased in preparation for a new generation of follicles. Exposure to nanomolar doses of DHP resulted in increaseddrd2mRNA levels, probably related to negative feedback on LH release. A decrease indrd2levels at the micromolar range of DHP (concomitant with increasedgnrhr3andfshbmRNA levels) may be related to the recruitment of a new generation of oocytes. Exposure to DHP also resulted in increasedlhbmRNA levels toward final oocyte maturation. Salmon GnRH analog (sGnRHa) increased mRNA levels ofgnrh1andgnrh3; when combined with DHP, sGnRHa synergistically increased expression ofgnrh3only. These results emphasize the role of sex steroids on positive and negative feedbacks controlling the reproductive cycle.

dopamine, estradiol, gonadotropin-releasing hormone receptor, neuroendocrinology, pituitary

INTRODUCTION

Regulation of the hypothalamic-pituitary-gonadal axis involves a complex interplay between peptides and steroid hormones. The mechanism of feedback regulation by sex steroids is complex and includes effects at the hypothalamic and pituitary levels. Gametogenesis is a critical and major process in vertebrate reproduction in which gonadotropins play a central role. Gonadotropins exert their action through gonadal biosynthesis of steroid hormones that, in turn, mediate various steps in gametogenesis (reviewed by Patino and Sullivan [1] and by Schulz and Miura [2]).

Oogenesis is launched with oocyte growth (vitellogenesis), during which estradiol secreted by the ovary regulates the synthesis of vitellogenins and choriogenins in the liver. When vitellogenesis is complete, there is a steroidogenic shift in which the production of testosterone and estradiol by the ovary is usually reduced, while the production of 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) is dramatically enhanced [3–5], leading to meiotic maturation [6]. The rise in DHP during this phase, rather than the decline in other steroids, is responsible for induction of the final maturation of oocytes [7].

LH and FSH secretion in fish is stimulated by several hypothalamic agents, with GnRH being the central stimulator and dopamine being the central inhibitor (reviewed by Yaron and Levavi-Sivan [5]). Three GnRH peptides and three cognate receptors with distinct distributions and functions have been identified in vertebrates. According to their sequences, the receptors can be grouped into distinct classes, types 1, 2, and 3 [8], which can be segregated into three branches. The first branch contains all type 1 GnRH receptors (GnRHr1) from mammals and fish; the second branch clusters mainly amphibian and human type 2 GnRH receptors; and the third branch includes type 3 GnRH receptors (GnRHr3) of evolved fish, mainly perciform species [9]. According to this classification, two types of GnRH receptors were found in the pituitary of tilapia, named according to phylogenetic analysis (the nomenclature used in this article is from Miller et al. [8] and from Levavi-Sivan and Avitan [9]). In tilapiines such as astatotilapia (Astatotilapia burtoni) or the Nile tilapia (Oreochromis niloticus), the distributions of GnRHr1 and GnRHr3 types are distinctly different. GnRHr3 is found mainly in brain areas related to reproductive function and is highly expressed in the posterior part of the pituitary that contains LH and FSH cells, suggesting that this receptor type may be important for regulating reproduction. Conversely, GnRHr1 is detected widely throughout the brain, from the olfactory bulb to the medulla, as well as in the dorsal anterior and posterior parts of the pituitary, suggesting that this receptor type is in a position to allow GnRH modulation of sensory input and growth [10–12].

It is well documented that fish with strong dopaminergic inhibition, such as cyprinid and tilapia, need cotreatment with GnRH and dopamine antagonists to achieve the LH surge that will induce final maturation and ovulation [13]. In vitro experiments have demonstrated the involvement of D2-like, but not D1-like, receptors in the dopaminergic inhibition of gonadotropin secretion directly at the pituitary level in goldfish [14], tilapia [15, 16], catfish [17], eel [18], and mullet [19]. Recently, the dopamine D2 receptor (Drd2) was cloned from the pituitary of tilapia, and its high similarity to the Drd2 of mullet and fugu was shown, as well as greater than 70% similarity to those of Xenopus, mouse, and turkey [20]. Phylogenetic analysis aligned the tilapia Drd2 with all vertebrate D2-like receptor sequences cloned to date. Pharmacological findings demonstrated that the response of tilapia Drd2 to the dopamine D2 agonist bromocriptine is higher than that to quinpirole [20].

Seasonal and annual changes in the plasma steroid levels of teleost fish have been extensively investigated [21–28]. However, these studies were carried out mostly on species that present synchronous oocyte development and spawn only once every 2 yr, once a year, or once during their lifetime (salmon, trout, carp, sturgeon, catfish, etc.). Fish with asynchronous or group-synchronous ovaries have received little attention, particularly due to the fact that (because their ovaries contain oocytes at all stages of development) occasional sampling cannot yield any significant results. Nevertheless, some fish possessing asynchronous ovaries are important fish models (zebra fish, tilapia, and medaka) or important aquaculture species (tilapia, striped bass [Morone saxatilis], gilthead sea bream [Sparus aurata], sea bass [Dicentrarchus labrax], etc.). Hence, the approach taken in the present study was to expose tilapia fish to the most important steroids dominating the vitellogenic period (estradiol) or the maturation phase (DHP) of the cycle.

Studies on the endocrine regulation of gonadotropin synthesis and secretion in fish have emphasized the modulatory role of GnRH and the rate of dopamine turnover in controlling seasonal changes in gonadotropin levels [29, 30]. Other studies have indicated that the responses to GnRH vary in conjunction with the season, sexual maturity, gender, and age [31–37]. This would indicate that the changes in GnRH and dopamine levels to which the gonadotrophs are exposed are not sufficient for modulating the gonadotropin response. An alternative candidate for such modulation would be the prevalence of GnRH and dopamine receptors in the pituitary. Indeed, in contrast to investigations on GnRH and dopamine in teleosts, the effort expended on studying their receptors has lagged behind significantly. It is possible that this informational gap has hindered, to some extent, our understanding of the physiological role of these key hormones in controlling the reproductive cycle. Therefore, the main goal of this research was to describe the pattern of pituitary GnRH and the expression of dopamine receptors (in relation to gonadotropin expression and release) in female tilapia during the vitellogenic and maturation stages of the reproductive cycle. The combined effect of the sex steroids and GnRH was also studied.

MATERIALS AND METHODS

Fish and Experimental Design

Nile tilapia (O. niloticus) were kept and bred in the fish facility unit at the Hebrew University in 500-L tanks at 26°C and 14L:10D. They were fed every morning ad libitum with commercial pellets and flakes containing 50% protein, 6% fat, 5.6% ash, and 2.6% cellulose (Zemach Feed Mills, Zemach, Israel).

Female tilapia can reach puberty at a very young age (3 mo) and small body weight [BW] (≥10 g) [38]. Consequently, we used young vitellogenic females (17.7 ± 1.5 g BW; gonadosomatic index [GSI, i.e., gonadal weight percentage of BW], 1.52% ± 0.029%; n = 30 and n = 50 for the in vivo and in vitro studies, respectively; and estradiol plasma levels, 11.79 ± 2.78 ng/ml) to study the effect of estradiol during vitellogenesis. Fish were anesthetized with 2-phenoxyethanol (Sigma, Ness Ziona, Israel) at a concentration of 1 ml/L before being weighed and injected intraperitoneally with the indicated doses of estradiol (Sigma) three times every 48 h at an injection volume of 1 µl/g BW. Blood samples were collected from the caudal vasculature and centrifuged at 3000 rpm for 20 min to obtain plasma samples, which were stored at –20°C until assayed. The fish were killed 48 h after the last injection. Controls were injected with fish saline. For the DHP studies, dispersed pituitary cells from mature females (65.21 ± 3.55 g BW; GSI, 2.55% ± 0.03%; n = 50; and estradiol plasma levels, 19.47 ± 3.49 ng/ml) were exposed to increasing doses of the steroid.

To examine the combined effect of GnRH and estradiol or DHP, early vitellogenic female tilapia (38.91 ± 2.07 g BW; GSI, 0.23% ± 0.02%; n = 28; and estradiol plasma levels, 3.58 ± 0.23 ng/ml) were used. These estradiol plasma levels are consistent with those determined in tilapia from immature to spawning fish [33, 39]. Fish were anesthetized, weighed, and divided into six groups of 7–10 fish each and treated as follows in the respective groups: 1) three injections of estradiol (500 µg/kg BW) every 48 h; 2) 25 µg/kg BW of salmon GnRH analog [D-Ala6,Pro9-NEt]-mammalian GnRH (sGnRHa) (Bachem Inc., Torrance, CA) dissolved in 0.85% saline, according to the method by Levavi-Sivan et al. [40]; 3) estradiol plus sGnRHa; 4) 500 µg/kg BW of DHP; or 5) DHP plus sGnRHa; while 6) control fish were injected with fish saline. All fish receiving sGnRHa were injected 36 h before being killed at an injection volume of 1 µl/g BW. Blood was collected from the caudal vasculature into heparinized syringes from anesthetized fish. After centrifugation, the plasma was stored at –20°C until further analysis. estradiol levels in the plasma were determined by ELISA according to the method by Aizen et al. [19]. All experimental procedures were in compliance with the Animal Care and Use Guidelines at the Hebrew University as approved by the local Administrative Panel on Laboratory Animal Care Committee.

Primary Culture of Pituitaries

The primary culture of tilapia pituitary cells was generally according to the method by Levavi-Sivan and Yaron [41]. Briefly, pituitaries were collected aseptically from female tilapia into the culture medium (M199, penicillin [100 IU/ml], streptomycin [0.1 mg/ml], and nystatin [1.25 IU/ml]) to which 0.3% BSA was added and then buffered to pH 7.4 with 10 mM Hepes. The glands were cut and trypsinized in trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA in PBS containing 1% glucose). After counting and determining cell viability by trypan blue exclusion, the cells were plated on a 96-well plate (120 000 cells/well per 0.2 ml). The cells were cultured for 4 days at 28°C under an atmosphere of 5% CO2 and then challenged for 48 h with various doses of estradiol or DHP (Sigma) in the presence of steroid-free fetal calf serum.

ELISAs for Tilapia FSH and LH

FSH and LH are heterodimeric glycoprotein hormones, composed of a common {alpha} subunit noncovalently associated with a hormone-specific ß subunit. Recently, using the methylotrophic yeast Pichia pastoris, a recombinant tilapia LH was produced as a biologically active, single-chain polypeptide [42]. The tilapia LH beta subunit (tLHb; GenBank accession No. AY541609) or tilapia FSH beta subunit (tFSHb; GenBank accession No. AF289174) and {alpha} (GenBank accession No. AF303087) mature protein coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which one of the ß chains forms the N-terminal part and the {alpha} chain forms the C-terminal part. A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the ß and {alpha} chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the ß subunit to enable purification of the recombinant protein. Recombinant tilapia LH-ß{alpha} and FSH-ß{alpha} stimulated the release of 11-ketotestosterone from mature testes, confirming their biological activity (Kasuto and Levavi-Sivan [42] and our unpublished data).

The recombinant FSHb was used to develop specific antibodies, and both recombinant glycoproteins were used to develop specific and homologous competitive ELISAs, generally according to the method by Mananos et al. [43], using primary antibodies against LHb and FSHb, respectively, and recombinant LH-ß{alpha} or tFSH-ß{alpha} for the standard curves. The wells were coated with recombinant LHb or FSHb (0.5 ng/well), and the antibodies were diluted 1:5000 (for LH) or 1:50 000 (for FSH). The intra-assay and inter-assay coefficients of variation, respectively, were 7.2% and 14.8% for LH and 8.0% and 12.5% for FSH. The sensitivities of the assay were 0.65 ng/ml for LH and 0.55 ng/ml for FSH. More details about the establishment of the ELISAs for tilapia LH and FSH will be forthcoming in another article.

Real-Time PCR

To test the effect of steroids on the relative abundance of tilapia drd2, gnrhr1, gnrhr3, lhb, and fshb mRNA, the genes were normalized to the amount of an endogenous reference, the 18S subunit of rRNA, by the comparative threshold cycle method. A detailed description of the real-time quantitative PCR procedure can be found elsewhere [20, 40]. The 18S rRNA was used as an internal standard for the measurements because it is an abundant RNA and its expression is considered stable. The 18S rRNA levels were observed to have a greater uniformity than those of other commonly used internal standards, such as ß-actin. The 18S levels proved to be stable for other teleost fish and did not vary during different reproductive stages reflecting various estradiol levels [20]. Serial dilutions were prepared from a pituitary cDNA sample (0.5, 0.1, 0.05, 0.02, 0.01, and 0.005), and the efficiencies of the specific hormones or receptors and 18S amplifications were compared by plotting {Delta}CT versus log(template) according to the method described by PE Applied Biosystems (Perkin-Elmer, Foster City, CA). R2 values and slopes, calculated by linear regression for all the genes tested, are given in Table 1. Total RNA and cDNA were prepared as described by Levavi-Sivan et al. [20]. Gene-specific primers used for the real-time PCR were designed using Primer Express 2.0 software (Table 1). The PCR mixture consisted of 2 µl of diluted cDNA sample, 300 nM of each primer, and 10 µl of Mastermix for Syber Green I (Eurogentec, Seraing, Belgium) in a final volume of 20 µl. Amplification was carried out in an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) under the following conditions: initial denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing-extension at 60°C for 1 min, and then a final extension at 60°C for 20 min. The genes and 18S rRNA cDNAs were amplified simultaneously in separate tubes in duplicate, and results were analyzed with the ABI Prism 5700 Sequence Detection System using version 1.6 software (PE Applied Biosystems). A dissociation curve analysis was run after each real-time experiment to ensure the presence of only one product. To control for false positives, a reverse-transcriptase negative control was run for each template and primer pair. To verify amplification of the correct sequences, the real-time PCR products amplified with the relative gene primers were sequenced, and their sequence was confirmed.


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  		TABLE 1. Primers used for quantitative real-time PCR

Statistical Analysis

One-way ANOVA was used to compare mean values, followed by an a posteriori Student Newman Keuls-test using PRISM 3.02 software (GraphPad, San Diego, CA). Three independent experiments were carried out for the in vitro studies; four experiments were carried out for the estradiol in vivo studies, as well as three experiments for the combined effect of GnRH and steroids. The results are presented as means ± SEM.

RESULTS

Effect of Estradiol

Estradiol is the dominant steroid during vitellogenesis; hence, to elucidate the role of estradiol on the modulation of GnRH receptors, Drd2, and gonadotropins, we used females at this specific stage. Females were injected with increasing doses of estradiol (250–500 µg/kg BW), and gnrhr mRNA levels were measured. The levels of tilapia gnrhr1 and gnrhr3 mRNA exhibited a dose-dependent increase (Fig. 1A), peaking at 2-fold to 3-fold their initial levels at 500 µg/kg BW of estradiol. Tilapia drd2 mRNA levels also increased (≤2-fold) at the lower estradiol dose tested but stayed at that level even at the higher dose tested (Fig. 1B).


Figure 1
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  		FIG. 1. Effect of estradiol administered in vivo on the expression patterns of GnRH receptors, dopamine receptor, and gonadotropin mRNA levels in the pituitary of female tilapia, relative to plasma gonadotropin levels. A) Expression patterns of gnrhr1 and gnrhr3. B) Expression pattern of drd2. C) Expression patterns of fshb and lhb. D) Plasma levels of LH and FSH. Estradiol was injected every 48 h for 6 days, and the fish were killed 48 h after the last injection. Values are means ± SEM (5–10 fish per group). Different letters indicate statistically significant differences between groups (P < 0.05)

Estradiol injections significantly decreased mRNA levels of tilapia lhb and fshb, as well as the release of LH from vitellogenic females (Fig. 1, C and D). However, FSH release increased up to 25.21 ± 4.10 ng/ml (n = 8) after injection of 500 µg/kg BW of estradiol (Fig. 1D).

To test the effect of estradiol on vitellogenic tilapia females, dispersed pituitary cells were exposed to increasing doses of estradiol (10 nM–150 µM). The mRNA levels of tilapia gnrhr1 did not change significantly in response to increasing doses of estradiol, whereas those of gnrhr3 increased gradually, reaching a 3-fold increase at 100 µM and declining at the highest dose (Fig. 2A). The mRNA levels of tilapia drd2 increased dose-dependently, reaching 2-fold induction at 1 µM, and decreased gradually at higher doses (Fig. 2B). No significant differences were found in mRNA levels of tilapia fshb in response to increasing doses of estradiol (Fig. 2C). However, mRNA levels of lhb increased up to 2-fold at 10 nM to 10 µM estradiol and reached a peak at 100 µM, declining thereafter.


Figure 2
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  		FIG. 2. Effect of estradiol on the expression patterns of GnRH receptors, dopamine receptor, and gonadotropin mRNA levels in primary cultures of tilapia pituitary cells. A) Expression patterns of gnrhr1 and gnrhr3. B) Expression pattern of drd2. C) Expression patterns of fshb and lhb. Cells were treated with various doses of estradiol for 48 h. The mRNA levels of the different genes are expressed as percentage of the levels in untreated cells at 48 h. Values are means ± SEM (n = 4 per dose). Means marked by an asterisk are significantly different from time 0

Effect of DHP

DHP is the maturation-inducing steroid in tilapia, as in many other teleosts. It increases during the final stages of oocyte maturation, usually in parallel to an increase in estradiol levels [39]. Pituitaries of mature female tilapia were exposed to increasing doses of DHP in vitro. The mRNA levels of tilapia gnrhr1 did not change in response to increasing doses of DHP, except at very high doses of 100–150 µM. However, gnrhr3 mRNA levels increased dose-dependently at all DHP doses tested (Fig. 3A). Tilapia drd2 mRNA levels increased 2.7-fold, even at the lower dose tested (10 nM), reached a peak at 100 nM, and decreased gradually at higher DHP doses (Fig. 3B). Tilapia lhb and fshb increased at lower doses, reached a peak at 100 nM DHP, and decreased gradually at higher doses (Fig. 3C).


Figure 3
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  		FIG. 3. Effect of DHP on the expression patterns of GnRH receptors, dopamine receptor, and gonadotropin mRNA levels in primary cultures of tilapia pituitary cells. A) Expression patterns of gnrhr1 and gnrhr3. B) Expression pattern of drd2. C) Expression patterns of fshb and lhb. Cells were treated with various doses of DHP for 48 h. The mRNA levels of the different genes are expressed as percentage of the levels in untreated cells at 48 h. Values are means ± SEM (n = 4 per dose). Means marked by an asterisk are significantly different from time 0

Combined Effects of Estradiol or DHP and sGnRHa

The combined effect of sGnRHa with estradiol or DHP was examined in vivo using early vitellogenic females. In time-course and dose-response experiments, it was previously shown that the optimal dose of sGnRHa is 25 µg/kg BW and that the optimal time is 36 h after injection [40]. Therefore, this concentration and time were used in this study. Salmon GnRH analog increased mRNA levels of gnrhr1 and gnrhr3, an increase that was not significantly raised with the addition of estradiol (Fig. 4A). In early vitellogenic females, DHP increased mRNA levels of gnrhr1 but not those of gnrhr3. The combined treatment of sGnRHa and DHP synergistically increased mRNA levels of gnrhr3 but did not change those of gnrhr1.


Figure 4
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  		FIG. 4. Combined effect of sGnRHa with estradiol or DHP administered in vivo on the expression patterns of GnRH receptors, dopamine receptor, and gonadotropin mRNA levels in the pituitary of female tilapia, relative to plasma gonadotropins levels. A) Expression patterns of gnrhr1 and gnrhr3. B) Expression pattern of drd2. C) Expression patterns of fshb and lhb. D) Plasma levels of LH and FSH. Estradiol or DHP was injected every 48 h for 6 days; sGnRHa was injected 36 h before the fish were sacrificed (see Materials and Methods for details). Values are means ± SEM (n = 7 fish per group). Different letters indicate statistically significant differences between groups (P < 0.05)

The most effective inducer of drd2 mRNA levels was estradiol (Fig. 4B). However, the addition of sGnRHa, together with estradiol, did not significantly change mRNA levels of drd2. Remarkably, sGnRHa increased mRNA levels of both gonadotropins, while estradiol decreased their levels (Fig. 4C). The combination of estradiol and sGnRHa did not change mRNA levels of lhb but slightly increased those of fshb (Fig. 4C); DHP alone reduced mRNA levels of fshb, and sGnRHa synergistically increased its levels. Salmon GnRH analog increased FSH and LH plasma levels (Fig. 4D). Administration of estradiol decreased LH plasma levels. Administration of sGnRHa with either of the steroids did not change the plasma levels of the gonadotropins compared with the levels obtained by administration of the steroid alone.

DISCUSSION

Gonadal steroids have been shown to exert negative or positive effects on gonadotropin secretion in various vertebrates [44–48], with the type of influence varying with the gonadal stage of development. Studies have been conducted to elucidate the mechanisms of steroid feedback regulation of gonadotropin secretion in mammals and birds [46–48]. However, these mechanisms are poorly understood in lower vertebrates, including fish. In particular, little is known about the feedback control of gonadotropin secretion by gonadal steroids in perciform fishes, which possess asynchronous or group-synchronous ovaries.

The present study provides evidence that sex steroids modulate mRNA levels of the receptors for the key hypothalamic hormones (GnRH and dopamine), in addition to their effect on the release and synthesis of gonadotropins. The reproductive stage of the fish may affect its responsiveness to the various steroids. Because estradiol is the main steroid secreted by tilapia ovary during all stages [33, 39], we chose to determine its levels as a parameter of the reproductive stage of the female, in parallel to its GSI values. Accordingly, estradiol levels were low in the plasma of early vitellogenic females, increased gradually in vitellogenic females, and reached its maximum in mature females. Gonadotropin levels were also higher in vitellogenic females than in early vitellogenic females. This is in agreement with previous findings in tilapia [33, 39, 49]. In this study, we used early vitellogenic or vitellogenic females for the analysis of estradiol alone or in combination with GnRH. However, the effect of DHP (the maturation-inducing hormone) was studied on mature females to identify the dominant role of the steroid at this specific stage.

Our results show that estradiol is an important regulator of synthesis of GnRHr3, the reproductive type of GnRH receptors. This is in agreement with previous findings that synthesis of this receptor type is higher in females than in males [40]. In mammals, the hormone that has been examined in the greatest detail with respect to regulation of pituitary sensitivity to GnRH is estradiol, and this steroid was found to increase pituitary responsiveness to GnRH [50]. Moreover, it has been shown to increase mRNA levels of GnRH receptors and their numbers in many species, including sheep [51], cows [52], and several species of laboratory animals [53, 54]. However, although this action occurs directly on gonadotrophs, its mechanism of action has yet to be defined [55].

Neuroendocrine regulation of gonadotropins involves a complex interplay of neurotransmitters. Among the main monoamines with important inhibitory effects on LH release in fish is dopamine (reviewed by Yaron et al. [49]). The present study shows a positive effect of estradiol on the synthesis of drd2 in vivo and in vitro. In goldfish and rainbow trout, dopaminergic neurons of the anterior part of the ventral preoptic region project to the proximal pars distalis of the pituitary [56]. Furthermore, these dopaminergic neurons of the preoptic area express the estrogen receptor [57], providing a neuroanatomical substrate for dopamine-estradiol interactions. Our results show a significant increase in mRNA levels of tilapia drd2 with increasing doses of estradiol in vivo and in vitro. These results corroborate experiments conducted on trout in which estradiol was found to be essential for maintenance of the dopaminergic inhibition of LH release in immature or vitellogenic trout [58]. The dopaminergic inhibition appears to positively correlate with circulating levels of estradiol during the preovulatory period [59], as well as to modulate the release of gonadotropins [60], in female trout. Also, a remarkable increase in mRNA levels of drd2 in the pituitary of male tilapia on stimulation with estradiol has previously been shown [20]. Taken together, our results suggest that the effect of estradiol on drd2 is part of the negative steroid feedback mechanism at vitellogenic stages. The recent finding that the promoter of mullet drd2 possesses a functional estradiol-responsive element [61] further corroborates our hypothesis.

GnRH, which increases the synthesis and release of gonadotropins, also increases the secretion of growth hormone in fish [49]. Moreover, gnrhr1 was recently found to be expressed on the somatotrophs of the tilapia pituitary [10–12]. In our study, estradiol increased gnrhr1 mRNA levels, an increase that can be correlated with vitellogenesis, as growth hormone levels have been found to be high at this reproductive stage in trout [62] and in tilapia [63]. DHP also increased gnrhr1 mRNA levels, indicating that growth hormone might play a role during final oocyte maturation. In salmon, it was also found that GnRH may indirectly regulate growth hormone family genes through the sex steroids, particularly in the late stage of gametogenesis [64]. Taken together, these data may lead to the postulation that the presence of an endocrine control mechanism—that is able to regulate the interplay between body growth (as reflected by growth hormone secretion) and gonadal function (as reflected by gonadotropin secretion)—may regulate the processes toward body mass increase or toward reproductive function and, in turn, may regulate the cyclical reproduction of tilapia.

Exposing vitellogenic female tilapia to increasing doses of estradiol in vivo led to a dose-dependent decrease in mRNA levels of lhb and fshb, probably due to the negative feedback of estradiol on the expression of both subunit genes. However, exposing pituitary cells to estradiol in vitro produced a different result, namely, mRNA levels of the lhb subunit increased, reaching a peak at 0.1 µM estradiol, whereas no significant change was evident in mRNA levels of fshb.

Negative effects of gonadal steroids on gonadotropin secretion have been demonstrated in a variety of teleost species by using gonadectomy and steroid replacement protocols. Gonad removal increases LH secretion in goldfish (Carassius auratus) [65], African catfish (Clarias gariepinus) [66], Atlantic croaker [37], Indian catfish (Heteropneustes fossilis) [67], hybrid striped bass [68], Mediterranean sea bass (Dicentrarchus labrax) [69], and some salmonids [70, 71] during later stages of gonadal recrudescence. This effect can be reversed by treatment with testosterone or estradiol [37, 65, 66, 68, 70]. However, in trout, which possess synchronous ovaries, an estradiol implant increased the circulating levels of LH and decreased those of FSH in previtellogenic fish [60].

The discrepancy between the in vivo and in vitro results could be explained in two ways. First, the effect of estradiol in vivo is not only at the pituitary level but also at the brain level. In trout, estradiol decreases the levels of sGnRHa mRNA in the telencephalon of previtellogenic fish, suggesting that brain GnRH neurons are involved, in part, in a negative feedback of estradiol on the release of gonadotropins, especially FSH, in early vitellogenic fish [60]. Second, the inhibition of gonadotropin synthesis (at least that of LH) could be associated with the effect of gonadal peptides such as activin [49]. These results are also in agreement with the finding of estradiol-responsive elements on the promoter and 5' flanking regions of lhb and fshb subunits of tilapia [72] and salmon [73].

In tilapia, we did not find any synergism between the effects of GnRH and estradiol. Because estradiol acts via intracellular receptors, while GnRH exerts its effects through a membrane receptor, it is likely that these hormones exert their effects via different mechanisms. This is in agreement with the case of the ewe [74]. However, in salmon, GnRH and estradiol synergistically modulated synthesis and release of gonadotropins by pituitary cells [75, 76].

We found that the level of reproductive-type gnrhr (gnrhr3) increases in response to DHP in vivo and in vitro. The levels of all five gnrhr types cloned from the masu salmon are low during the spawning period, when DHP levels are high relative to vitellogenesis [77]. Using real-time PCR, significantly higher mRNA levels of gnrhr (similar to gnrhr3) have been shown in the pituitaries of striped bass at advanced stages of ovarian development, compared with the pituitaries of fish with less developed ovaries [78].

In contrast to estradiol, progesterone appears to have a negative effect on the number of GnRH receptors in the mammalian pituitary gland. The lowest gnrhr mRNA levels and receptor numbers have been observed during the luteal phase, when the corpus luteum is actively secreting progesterone [79]. Furthermore, progesterone decreases the number of receptors for GnRH [80] and the amount of mRNA encoding gnrhr in cultured ovine anterior pituitary cells [81]. Finally, administering estradiol to ewes during their luteal phase fails to increase the number of GnRH receptors [55], implying that progesterone can prevent the estradiol-induced increase in gnrhr gene expression. Based on these data, Turzillo et al. [82] inferred that progesterone might directly affect the ability of the pituitary gland to respond to GnRH.

Dopamine inhibits the release of LH in cyprinids (reviewed by Yaron [13]), silurids, European eel, salmonids [58], and tilapia [15, 16]. Dopamine also inhibits the release of FSH from the gonadotrophs of mature trout via Drd2 [83]. We show that with increasing doses of DHP there is an increase in the synthesis of tilapia drd2, probably as part of the negative feedback that occurs toward the stages of final oocyte maturation. However, at the very high doses of DHP (micromolar range in the in vitro study and in the in vivo study), mRNA levels of drd2 decreased significantly concomitant with the increase in gnrhr3 mRNA levels. Moreover, we found a synergistic effect of GnRH and DHP on gnrhr3 and fshb gene expression concomitant with a reduction in FSH release. These events are probably part of the recruitment of a new generation of follicles in the asynchronous ovary of the tilapia.

Oocyte maturation in fish is triggered by maturation-inducing hormone, which acts on receptors located on the oocyte membrane and induces the activation of maturation-promoting factor in the oocyte cytoplasm (reviewed by Yaron and Levavi-Sivan [5]). During the course of maturation, oocytes undergo drastic morphological changes associated with progression of the meiotic cell cycle. The breakdown of the oocyte nuclear envelope (germinal vesicle breakdown) occurring at the prophase-metaphase transition is usually regarded as a hallmark of the progress of oocyte maturation. The most common maturation-inducing hormone, DHP, has been identified in several fish species [84], including tilapia [39].

The present study shows that lhb synthesis increases significantly with lower doses of DHP. At higher doses, no change was found in LH levels; however, a synergistic effect of GnRH and DHP on lhb gene expression was found. This is in agreement with the hypothesis that LH is the determining factor regulating the production of the maturation-inducing hormone DHP. Moreover, mRNA levels of fshb increased with DHP dose, probably due to the recruitment of a new generation of oocytes for a new reproductive cycle. In salmon, DHP also increases the content of cellular LH [85].

GnRH is the principal mediator for secretion of gonadotropins (as described in the Introduction). GnRH has been shown to increase the amounts of mRNA encoding gonadotropin subunits in the pituitaries and to further elevate plasma levels of sex steroid hormones in many teleost species (reviewed by Yaron et al. [49]).

Our results show a significant increase in the gene expression of fshb and lhb in response to GnRH in vivo. This is in agreement with previous results that showed an increase in ß subunit mRNA levels in both gonadotropins in response to GnRH in tilapia in vivo and in vitro [49]. In addition, the effect of GnRH on LH release is well documented [49]. However, we demonstrate herein that GnRH increases FSH levels in tilapia plasma, showing that GnRH can be the secretagogue of LH and FSH in tilapia.

In summary, the following steps describe the reproductive stages in female tilapia at vitellogenesis and at maturation: At vitellogenesis, the steps include 1) an increase in gnrhr3 and gnrhr1 mRNA levels as part of the positive feedback on FSH release, 2) an increase in drd2 synthesis with increasing concentrations of estradiol, and 3) the inhibition of LH and FSH release and lhb synthesis as part of the negative feedback. At the end of vitellogenesis (high levels of estradiol), there is an increase in FSH synthesis as part of the preparation for a new generation of follicles. At maturation, the steps include 1) an increase in gnrhr3 mRNA levels; 2) an increase in drd2 levels (at the nanomolar range of DHP), probably as part of the negative feedback on LH release; 3) a decrease in drd2 mRNA levels (at the micromolar range of DHP) concomitant with the increase in gnrhr3, probably as part of the recruitment of a new generation of oocytes for a new cycle; 4) an increase in the synthesis of lhb at lower doses of DHP (toward final oocyte maturation) and its decrease at higher doses of DHP, probably as part of the negative feedback; and 5) an increase in the synthesis of fshb at lower doses of DHP as part of the recruitment of a new generation of oocytes for a new cycle.

ACKNOWLEDGMENTS

We are very grateful to Mr. Joseph Aizen and Mr. Matan Golan for their help in sampling fish.

FOOTNOTES

1 Supported by grant 775/01 from the Israel Science Foundation. Back

2 Correspondence: Berta Levavi-Sivan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University, P.O. Box 12, Rehovot 76100, Israel. FAX: 972 8 946 5763; sivan{at}agri.huji.ac.il Back

Received: 9 February 2006.

First decision: 3 March 2006.

Accepted: 26 June 2006.

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