Steroidogenesis and its regulation in teleost-a review.

Steroid hormones modulate several important biological processes like metabolism, stress response, and reproduction. Steroidogenesis drives reproductive function wherein development and differentiation of undifferentiated gonads into testis or ovary, and their growth and maturation, are regulated. Steroidogenesis occurs in gonadal and non-gonadal tissues like head kidney, liver, intestine, and adipose tissue in teleosts. This process is regulated differently through multi-level modulation of promoter motif transcription factor regulation of steroidogenic enzyme genes to ultimately control enzyme activity and turnover. In view of this, understanding teleostean steroidogenesis provides major inputs for technological innovation of pisciculture. Unlike higher vertebrates, steroidal intermediates and shift in steroidogenesis is critical for gamete maturation in teleosts, more essentially oogenesis. Considering these characteristics, this review highlights the promoter regulation of steroidogenic enzyme genes by several transcription factors that are involved in teleostean steroidogenesis. It also addresses different methodologies involved in promoter regulation studies together with glucocorticoids and androgen relationship with reference to teleosts.

In vivo induction of human chorionic gonadotropin by osmotic pump advances sexual maturation during pre-spawning phase in adult catfish.

Gonadal maturation is a critical event wherein gonads, under the influence of several hormones and factors, undergo cyclic morphological and physiological changes to produce functional gametes during the spawning phase. However, artificial induction can be effectively used to advance the maturation of gonad vis-à-vis spawning like behavior in seasonal breeders during the off-breeding season. In the present study, osmotic pumps loaded with 5000IU of human chorionic gonadotropin (hCG) or saline as control were implanted intraperitoneally for 21days during the pre-spawning phase (May-June) in catfish Clarias batrachus and C. gariepinus. Significant increase in gonado-somatic index and sperm motility, and in the levels of certain sex steroids were observed in the hCG treated catfish when compared to control while estradiol-17ß (E2) was low. Histological analysis in hCG treated testis revealed densely packed sperm and/or spermatids inside the lumen wherein the control testis displayed normal characteristics of the pre-spawning phase. In females, histological analysis showed a significant increase in post-vitellogenic full-grown immature follicles as seen in the spawning phase. In accordance with this, the steroid hormone profile correlated well with steroidogenic shift from E2 to 17α,20ß-DP indicating oocyte maturation. However, in the control ovaries of C. batrachus, perinucleolar and pre-vitellogenic oocytes were seen to be predominant. In addition, when compared with the control, the hCG treated group displayed a significant increase in the transcripts of several genes associated with gonadal growth. Taken together, artificial induction by slow release of hCG is an effective strategy to advance sexual maturation in catfish in a programmed manner.

Sox3 binds to 11ß-hydroxysteroid dehydrogenase gene promoter suggesting transcriptional interaction in catfish.

In fishes, the expression of steroidogenic enzyme genes and their related transcription factors (TFs) are critical for the regulation of steroidogenesis and gonadal development. 11-KT is the potent androgen and hence, 11ß-hsd, enzyme involved in 11-KT production is important. Regulation of 11ß-hsd gene was never studied in any fishes. At first 11ß-hsd was cloned and recombinant protein was tested for enzyme activity prior to expression and promoter motif analysis. Expression changes revealed stage- and sex-dependent increase in the ontogenic studies. Further, 11ß-hsd expression was higher during spawning phase of reproductive cycle and was found to be gonadotropin inducible both in vivo and in vitro. ∼2kb of 5' upstream region of 11ß-hsd, was cloned from catfish genomic DNA library and in silico promoter analysis revealed putative TF binding sites such as Sox3, Wt1, Pax2, Dmrt1 and Ad4BP/SF-1. Luciferase reporter assay using the sequential deletion constructs in human embryonic kidney and Chinese hamster ovary cells revealed considerable promoter activity of the constructs containing Sox3, but not with other motifs largely. Site-directed mutagenesis, Sox3 over expression, electrophoretic mobility shift and chromatin immunoprecipitation assays further substantiated the binding of Sox3 to its corresponding cis-acting element in the upstream promoter motif of 11ß-hsd. This is the first report to show that Sox3 binds to the 11ß-hsd gene promoter and transactivates to regulate male reproduction in a teleost.

Dynamic expression of 11ß-hydroxylase during testicular development, recrudescence and after hCG induction, in vivo and in vitro in catfish, Clarias batrachus.

Cytochrome P450 11ß-hydroxylase (11ß-h), is involved in the production of 11-hydroxytestosterone, an immediate precursor for 11-ketotestosterone (11-KT), a potent androgen in teleosts. To understand the role of 11ß-h in gonadal development, maturation, function and recrudescence in an annually reproducing teleost, the present study was conducted using Clarias batrachus. Four forms of 11ß-h cDNA, regular type (2.253 kb), variant 1 (1.290 kb), variant 2 (1.223 kb) and variant 3 (1.978 kb) were identified from the testis of catfish which expressed ubiquitously with high levels in testis. 11ß-h transcripts were detected as early as 0 days post hatch further, stage- and sex-dependent increase in the 11ß-h transcripts were seen during gonadal differentiation/development. In addition, high expression of 11ß-h (regular type) in pre-spawning phase was detected. Corroboratively, levels of 11-KT in serum and testicular tissue was high during pre-spawning and spawning phases, which might facilitate initiation and normal progression of spermatogenesis. The expression of 11ß-h was high after human chorionic gonadotropin induction in vivo (all forms), and in vitro (regular type). Immunohistochemical and immunofluorescence localization showed the presence of 11ß-h in Sertoli and interstitial/Leydig cells of the testis. These results suggest that 11ß-h is involved in late stages of testicular development, together with the regulation of seasonal reproductive cycle in catfish.

Molecular cloning and expression analysis of 17b-hydroxysteroid dehydrogenase 1 and 12 during gonadal development, recrudescence and after in vivo hCG induction in catfish, Clarias batrachus.

In teleosts, the levels of steroids during critical period of sex differentiation are critical for gonadogenesis. Hence, steroidogenesis and expression of steroidogenic enzyme genes are very critical for gonadal development and function. In this regard, 17b-HSDs are important as they are involved in both 17b-estradiol (E2) and testosterone (T) biosynthesis. Full length cDNAs of 17b-HSD 1 (1791 bp) and 12 (1073 bp) were cloned from catfish gonads which encodes a protein of 295 and 317 amino acids, respectively. To understand the importance of these enzymes in teleost reproduction, mRNA expression was analyzed during gonadal development, seasonal reproductive cycle and after human chorionic gonadotropin (hCG) induction. Phylogenetic analysis revealed that the 17b-HSD 1 and 12 share high homology with their respective 17b-HSD forms from other teleosts and both the 17b-HSD forms belong to short chain dehydrogenase/ reductase family. Tissue distribution analysis showed that the 17b-HSD 1 expression was higher in ovary and gills, while 17b-HSD 12 was higher expressed in testis, ovary, brain, intestine and head kidney compared to other tissues analyzed. Developing and mature ovary showed higher expression of 17b-HSD 1, while 17b-HSD 12 was higher in testis than the ovary of corresponding stages. Further, 17b-HSD 1 and 12 transcripts together with E2 and T levels were found to be modulated during different phases of the seasonal reproductive cycle. Expression of 17b-HSD 1 and 12 was upregulated after hCG induction which shows possible regulation by gonadotropin. Our findings suggest that 17b-HSD 1 and 12 might play important role in regulating gonadal development and gametogenesis through modulation of sex steroid levels.

Expression analysis of sox3 during testicular development, recrudescence, and after hCG induction in catfish, Clarias batrachus.

In teleosts, the expression of steroidogenic enzymes and related transcription factor genes occurs in a stage- and tissue-specific manner causing sexual development. The role of sox3, an evolutionary ancestor of SRY, has not been studied in detail. Therefore, the full-length cDNA of sox3 (1,197 kb) was cloned from catfish testis, and mRNA expression was analyzed during gonadal development, during the seasonal reproductive cycle, and after human chorionic gonadotropin (hCG) induction. Tissue distribution analysis showed that sox3 expression was higher in testis, ovary, and brain compared to other tissues analyzed. Developing and mature testis showed higher sox3 expression than ovary of corresponding stages, and more sox3 transcripts were found during the spawning phase of the seasonal reproductive cycle. Expression of sox3 was upregulated by hCG after in vivo and in vitro induction, suggesting that gonadotropins might stimulate it. In situ hybridization and immunohistochemistry showed the presence of sox3 mRNA and protein in somatic and interstitial cell layers of the testis. Sox3 could also be found in the zona radiata of developing and mature oocytes. Exposure of methyltestosterone (1 µg/l) and ethinylestradiol (1 µg/l) for 21 days during testicular development showed lower sox3 expression levels in the testis and brain, indicating a certain feedback intervention. These results suggest a possible role for Sox3 in the regulation of testicular development and function.

Expression analysis of cyp11a1 during gonadal development, recrudescence and after hCG induction and sex steroid analog treatment in the catfish, Clarias batrachus.

In teleosts, the levels of steroids are critical for sexual development and hence, expression of steroidogenic enzyme genes and specific substrate availability are indispensable for gonadal steroidogenesis. Early stages of steroidogenesis specifically cholesterol to pregnenolone conversion by Cyp11a1 is crucial for estradiol and testosterone biosynthesis. Based on this, in this study, full length cDNA of cyp11a1 (2581bp) was cloned from catfish testis to investigate the importance of Cyp11a1 by analyzing the expression of cyp11a1 during gonadal development, seasonal reproductive cycle, after human chorionic gonadotropin (hCG) induction and sex steroid analog treatment. Phylogenetic analysis revealed that the Cyp11a1 is more conserved across teleosts. Tissue distribution analysis showed that the cyp11a1 expression was higher in the testis followed by the brain, head kidney, muscle and ovary compared to other tissues analyzed. High expression of cyp11a1 in the head kidney and muscle revealed that Cyp11a1 could potentially regulate the extra-gonadal and/or circulating steroid levels in teleosts. Developing and mature testes showed higher expression of cyp11a1 than the ovary of corresponding age group. Further, cyp11a1 expression was found to be higher during pre-spawning and spawning phases of testicular cycle and was upregulated by hCG, in vivo and in vitro, which indicates the possible regulation by gonadotropin. Exposure of methyltestosterone (1µg/L) and ethinylestradiol (1µg/L) for 21days during catfish testicular development showed lower cyp11a1 expression levels in the testis and brain indicating a certain feedback intervention. These results suggest possible role for Cyp11a1 in the testis development and recrudescence.

Cloning and expression analysis of tyrosine hydroxylase and changes in catecholamine levels in brain during ontogeny and after sex steroid analogues exposure in the catfish, Clarias batrachus.

Tyrosine hydroxylase (Th) is the rate-limiting enzyme for catecholamine (CA) biosynthesis and is considered to be a marker for CA-ergic neurons, which regulate the levels of gonadotropin-releasing hormone in brain and gonadotropins in the pituitary. In the present study, we cloned full-length cDNA of Th from the catfish brain and evaluated its expression pattern in the male and female brain during early development and after sex-steroid analogues treatment using quantitative real-time PCR. We measured the CA levels to compare our results on Th. Cloned Th from catfish brain is 1.591 kb, which encodes a putative protein of 458 amino acid residues and showed high homology with other teleosts. The tissue distribution of Th revealed ubiquitous expression in all the tissues analyzed with maximum expression in male and female brain. Copy number analysis showed two-fold more transcript abundance in the female brain when compared with the male brain. A differential expression pattern of Th was observed in which the mRNA levels were significantly higher in females compared with males, during early brain development. CAs, l-3,4-dihydroxyphenylalanine, dopamine, and norepinephrine levels measured using high-performance liquid chromatography with electrochemical detection in the developing male and female brain confirmed the prominence of the CA-ergic system in the female brain. Sex-steroid analogue treatment using methyltestosterone and ethinylestradiol confirmed our findings of the differential expression of Th related to CA levels.