This study aimed to improve, using the zebrafish model, our understanding of the distinct roles of pituitary gonadotropins FSH and LH in regulating testis functions in teleost fish. We report, for the first time in a vertebrate species, that zebrafish Leydig cells as well as Sertoli cells express the mRNAs for both gonadotropin receptors (fshr and lhcgr). Although Leydig cell fshr expression has been reported in other piscine species and may be a common feature of teleost fish, Sertoli cell lhcgr expression has not been reported previously and might be related to the undifferentiated gonochoristic mode of gonadal sex differentiation in zebrafish. Both recombinant zebrafish (rzf) gonadotropins (i.e. rzfLH and rzfFSH) stimulated androgen release in vitro and in vivo, with rzfFSH being significantly more potent than rzfLH. Forskolin-induced adenylate cyclase activation mimicked, whereas the protein kinase A inhibitor H-89 significantly reduced, the gonadotropin-stimulated androgen release. Therefore, we conclude that both FSH receptor and LH/choriogonadotropin receptor signaling are predominantly mediated through the cAMP/protein kinase A pathway to promote steroid production. Despite this similarity, other downstream mechanisms seem to differ. For example, rzfFSH up-regulated the testicular mRNA levels of a number of steroidogenesis-related genes both in vitro and in vivo, whereas rzfLH or human chorionic gonadotropin did not. Although not fully understood at present, these differences could explain the capacity of FSH to support both steroidogenesis and spermatogenesis on a long-term basis, whereas LH-stimulated steroidogenesis might be a more acute process, possibly restricted to periods during which peak steroid levels are required.

Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11 beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11 beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially--via Sertoli cells--also to germ cell differentiation in zebrafish testis.

To develop new tools to study the regulation of testis physiology in teleost fish, a medium-term ex vivo organ culture system was adopted for zebrafish testis tissue. The addition of 100nM 11-ketotestosterone to the system supported complete spermatogenesis, as determined by morphological, molecular and immunohistochemical analyses. Under basal conditions, however, the development of differentiated spermatogonia, spermatocytes, and spermatids was seriously disturbed, probably related to the rapid (within 2 days) down-regulation of the steroidogenic system. Forskolin (0.5microM) stimulated acute androgen release from freshly removed tissue and partially prevented down-regulation of the steroidogenic system. The present ex vivo culture system can serve as a tool to evaluate effects of a wide range of substances on the two main functions of the testis, spermatogenesis and hormone production.

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This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

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Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17b-oestradiol (E 2 ) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E 2 ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.

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