Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

BACKGROUND: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. RESULTS: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. CONCLUSIONS: This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.

Antagonistic regulation of spermatogonial differentiation in zebrafish (Danio rerio) by Igf3 and Amh.

Fsh-mediated regulation of zebrafish spermatogenesis includes modulating the expression of testicular growth factors. Here, we study if and how two Sertoli cell-derived Fsh-responsive growth factors, anti-Müllerian hormone (Amh; inhibiting steroidogenesis and germ cell differentiation) and insulin-like growth factor 3 (Igf3; stimulating germ cell differentiation), cooperate in regulating spermatogonial development. In dose response and time course experiments with primary testis tissue cultures, Fsh up-regulated igf3 transcript levels and down-regulated amh transcript levels; igf3 transcript levels were more rapidly up-regulated and responded to lower Fsh concentrations than were required to decrease amh mRNA levels. Quantification of immunoreactive Amh and Igf3 on testis sections showed that Fsh increased slightly Igf3 staining but decreased clearly Amh staining. Studying the direct interaction of the two growth factors showed that Amh compromised Igf3-stimulated proliferation of type A (both undifferentiated [Aund] and differentiating [Adiff]) spermatogonia. Also the proliferation of those Sertoli cells associated with Aund spermatogonia was reduced by Amh. To gain more insight into how Amh inhibits germ cell development, we examined Amh-induced changes in testicular gene expression by RNA sequencing. The majority (69%) of the differentially expressed genes was down-regulated by Amh, including several stimulators of spermatogenesis, such as igf3 and steroidogenesis-related genes. At the same time, Amh increased the expression of inhibitory signals, such as inha and id3, or facilitated prostaglandin E2 (PGE2) signaling. Evaluating one of the potentially inhibitory signals, we indeed found in tissue culture experiments that PGE2 promoted the accumulation of Aund at the expense of Adiff and B spermatogonia. Our data suggest that an important aspect of Fsh bioactivity in stimulating spermatogenesis is implemented by restricting the different inhibitory effects of Amh and by counterbalancing them with stimulatory signals, such as Igf3.

INSL3 stimulates spermatogonial differentiation in testis of adult zebrafish (Danio rerio).

INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (Aund) but not of type A differentiating (Adiff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating Aund. Since the area occupied by Aund decreases and that of Adiff increases, we conclude that hINSL3 recruits Aund into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single Aund spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fsh-stimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit Aund spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.

Thyroid hormone stimulates the proliferation of Sertoli cells and single type A spermatogonia in adult zebrafish (Danio rerio) testis.

Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor α (thrα) in SCs and the ß (thrß) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T3. However, in the presence of FSH, T3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17α-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T3 on the proliferation of A(und) and SCs. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.

Proteolytically activated, recombinant anti-mullerian hormone inhibits androgen secretion, proliferation, and differentiation of spermatogonia in adult zebrafish testis organ cultures.

Anti-Müllerian hormone (Amh) is in mammals known as a TGFß type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis.

Functional characterization and expression analysis of the androgen receptor in zebrafish (Danio rerio) testis.

The biological activity of androgens, important for male sexual differentiation and development, is mediated by the androgen receptor (AR) that binds to specific DNA recognition sites regulating the transcription of androgen target genes. We investigated androgen production by adult zebrafish testis tissue, and identified 11beta-hydroxyandrostenedione, 11-ketoandrostenedione (OA), and 11-ketotestosterone (11-KT) as main products, and hence potential ligands, for the zebrafish Ar. These androgens were then included in the pharmacological characterization of the zebrafish Ar. The zebrafish Ar responded well in terms of binding and transactivation to synthetic androgens as well as to testosterone and 11-KT, and reasonably well to OA and androstenedione. In situ hybridization analysis of zebrafish testis revealed that ar mRNA expression was detected in the subpopulation of Sertoli cells contacting early spermatogonia.

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor.

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.

Cloning and spatiotemporal expression of the follicle-stimulating hormone beta subunit complementary DNA in the African catfish (Clarias gariepinus).

The gene and cDNA encoding a putative follicle-stimulating hormone beta subunit (cfFSHbeta) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHbeta genes, the cfFSHbeta gene consisted of three exons interrupted by two introns. The cfFSHbeta cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone beta subunits were positionally conserved in cfFSHbeta. The cfFSHbeta mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHbeta transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSHbeta mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHbeta mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSHbeta transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

Gonadotropin-releasing hormone does not directly stimulate luteinizing hormone biosynthesis in male African catfish.

Besides gonadotropin release, GnRH stimulates gonadotropin subunit gene transcription and translation in gonadotrophs. In the African catfish, Clarias gariepinus, chicken GnRH-II (cGnRH-II: [His5,Trp7,Tyr8]-GnRH) and catfish GnRH (cfGnRH: [His5,Asn8]-GnRH) are two endogenous forms of GnRH. Studying their effects on LH subunit steady-state mRNA levels, LH de novo synthesis, and LH release in primary pituitary cell cultures of adult males, we found that cGnRH-II hardly influenced the steady-state levels of LH subunit mRNAs or LH de novo synthesis, although it stimulated LH release. Although cfGnRH stimulated LH secretion as well, high concentrations-although apparently still within the physiologic range-reduced LH transcript levels and de novo synthesis in primary pituitary cell cultures. In vivo experiments demonstrated a biphasic response of LH subunit transcript levels after a single GnRH injection: a decrease after 2 h was followed by an increase at 8 h. When the testes were removed before GnRH treatment, however, LH transcript levels remained depressed at 8 h after GnRH injection, indicating that the secondary increase in LH transcript levels depends on the presence of the testes. We conclude that the up-regulation of LH production subsequent to GnRH stimulation in adult male African catfish is mediated by factors originating from the testis. Previous work suggests that aromatizable androgens may play an important role in this context. Under the present experimental conditions, however, GnRHs had no, or an inhibitory, direct effect on LH production in catfish gonadotrophs.

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.

Gonadotropins, their receptors, and the regulation of testicular functions in fish.

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Steroid hormones stimulate gonadotrophs in juvenile male African catfish (Clarias gariepinus).

In juvenile African catfish (Clarias gariepinus), the pituitary LH content strongly increased after the beginning of spermatogonial proliferation. We hypothesized that a signal of testicular origin is involved in stimulating the gonadotrophs. We investigated the effects of castration and sex steroid treatment on gonadotrophs in juvenile males by quantifying LH production and release and LH subunit transcript levels and by examining gonadotroph morphology and proliferation. Castration reduced but did not abolish the maturation-associated elevation in pituitary LH content. Treatment with testosterone but not with 11-ketotestosterone, an otherwise potent androgen in fish, reversed the castration-induced decrease of pituitary LH levels. An increased pituitary LH content was accompanied by an increased number of cytologically mature gonadotrophs. However, no evidence was found for gonadotroph proliferation, so that quiescent gonadotrophs may have become activated. Although 11-ketotestosterone treatments had no effect in castrated males, this androgen attenuated gonadotroph activation in intact males. Because androgen production in juvenile catfish is downregulated by treatment with 11-ketotestosterone, its inhibitory effects on gonadotrophs in gonad-intact males may be due to suppression of Leydig cell testosterone production, which appears to be a limiting factor for the activation of catfish gonadotrophs. Aromatizable androgens may have opposite effects on fish (stimulatory) and mammalian (inhibitory) gonadotrophs.

Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic.

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD(65) and GAD(67), EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD(65) encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD(65). Goldfish GAD(67) encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD(67). Goldfish GAD(65) and GAD(67) are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD(65) mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD(67) mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD(65) and GAD(67) mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.

Gonadotropin-releasing hormone fibers innervate the pituitary of the male African catfish (Clarias gariepinus) during puberty.

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 microM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 microM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor.

The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish.

Both glutamate and gamma-aminobutyric acid (GABA) are involved in pituitary hormone release in fish. Glutamate serves 2 purposes, both as a neurotransmitter and as a precursor for GABA synthesis. Glutamate can be catabolized to GABA by the actions of 2 distinct but related enzymes, glutamate decarboxylase 65 (GAD65) and GAD67. They derive from 2 different genes that likely arose from an early gene duplication prior to the emergence of teleosts more than 400 million years ago. There is good evidence for the involvement of GABA in luteinizing hormone (LH) release in fish. The mechanism of GABA action to stimulate LH release appears to be a combination of effects on GnRH release, potentiation of gonadotropin hormone-releasing hormone (GnRH) action, and in some cases directly at the LH cell. These actions appear to be dependent on such factors as sex or sex steroid levels, and there may also be species differences. Nevertheless, the stimulatory effects of GABA on LH are present in at least 4 fish species. In contrast, convincing data for the inhibitory effects of GABA on LH release have only been observed in 1 fish species. The sites and mechanisms of action of amino acid neurotransmitters on LH release have yet to be fully characterized. Both 130N-methyl-D-aspartic acid (NMDA) and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors are likely to have important roles. We suggest that it is a receptor similar to the GABA(A) type which mediates the effects of GABA on LH release in fish, at least partially acting on the GnRH neuron, but likely directly acting at the gonadotroph as well. GABA may also be involved in regulating the release of other pituitary hormones in fish, namely follicle stimulating hormone (FSH = GTH-I), prolactin, and growth hormone. Based on the findings described in this review, a working model for the involvement of glutamate and GABA in the regulation of LH release in teleost fish is proposed.

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish.

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (¿Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ¿Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/¿Ca(2+)(i) to stimulate the release of LH.

Regulation of steady-state luteinizing hormone messenger ribonucleic acid levels, de novo synthesis, and release by sex steroids in primary pituitary cell cultures of male African catfish, Clarias gariepinus.

Primary pituitary cell cultures from sexually mature adult male African catfish, Clarias gariepinus, were used to study the regulation of LH biosynthesis by sex steroids. The cell cultures were exposed to testosterone (T), estradiol (E(2)), or 5alpha-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11beta-hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E(2) elevated glycoprotein alpha (GPalpha) and LHbeta steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E(2) on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GPalpha and LHbeta mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [(3)H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish.

Inhibitory and stimulatory interactions between endogenous gonadotropin-releasing hormones in the African catfish (Clarias gariepinus).

In the brain of all vertebrate classes, chicken (c) GnRH-II ([His(5), Trp(7),Tyr(8)]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary-cGnRH-II and catfish GnRH ([His(5),Asn(8)]GnRH, cfGnRH)-on the cytosolic free calcium concentration ([Ca(2+)](i)) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca(2+)](i) were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.

Reproductive development of male and female tilapia hybrids (Oreochromis niloticus x O. aureus) and changes in mRNA levels of gonadotropin (GtH) Ibeta and IIbeta subunits.

A study was carried out in tilapia in order to see whether the gonadotropin (GtH) beta subunits show distinct patterns of expression at different stages of their reproductive development. Male and female tilapia hybrids (Oreochromis niloticus x 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 (E(2)) 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 Ibeta and IIbeta mRNA levels. In maturing fish of both sexes, the circulating steroid levels were positively correlated with each other (r(2) = 0.66-0.91) and in males, also with the GSI values (r(2) = 0.68). A positive correlation was also seen in these fish between GSI values and the prevalence of spermatocytes and spermatids (r(2) = 0.54). In maturing females, the maximal oocyte diameter was positively correlated with circulating E(2) levels (r(2) = 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 Ibeta mRNA levels were highest in fish with steroids ranging 1-10 ng T or E(2)/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 IIbeta 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 E(2) at >1 nM) was followed by a decrease in the steady-state levels of the Ibeta transcript, while those of IIbeta 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 beta subunit in distinct cell types specific for each hormone.