K. Raghuveer

Dimorphic Expression of Various Transcription Factor and Steroidogenic Enzyme Genes during Gonadal Ontogeny in the Air-Breathing Catfish, Clarias gariepinus

In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17–20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30–40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30–40 dph when sex-specific genes showed differential expression.

Endosulfan and flutamide, alone and in combination, target ovarian growth in juvenile catfish, Clarias batrachus

Juvenile Catfish(es), Clarias batrachus of 50 days post hatch (dph) were exposed to endosulfan (2.5 parts per billion [ppb]) and flutamide (33 ppb), alone and in combination for 50 days to access their impact on ovarian development. The doses used in this study were nominal considering pervious reports. Sampling was done at 100 dph to perform histology and measurement of various transcripts, estradiol-17β and aromatase activity. In general, treatments enhanced expression of ovary-specific transcription factors, steroidogenic enzymes steroidogenic acute regulatory protein and aromatases while transcripts of tryptophan hydroxylase2 (tph2) and catfish gonadotropin-releasing hormone declined in the brain of all treated groups with maximum reduction in the endosulfan group. Significant reduction of tph2 immunoreactivity in the forebrain/telencephalon-preoptic area endorsed our results. Increased number of pre-vitellogenic and less immature oocytes in the treated groups indicated hastened ovarian growth. Elevated ovarian aromatase activity and plasma estradiol-17β levels were noticed in the treated groups with maximum being in the endosulfan group. These data together demonstrate that the exposure of endosulfan causes synchronous precocious ovarian development better than flutamide, alone or in combination. Our results suggest that both endosulfan and flutamide alter ovarian growth by triggering precocious development in catfish.

Cloning and differential expression pattern of vasa in the developing and recrudescing gonads of catfish, Clarias gariepinus.

Vasa gene codes for a DEAD box family protein, which plays a crucial role in primordial germ cell proliferation. In this study, we report cloning of vasa from gonads of air-breathing catfish, Clarias gariepinus, a seasonally reproducing teleost fish. We studied the expression pattern of vasa during gametogenesis using real-time PCR. We also examined the hormonal regulation on vasa in gonads of catfish. RT-PCR analysis revealed that vasa was detectable only in the gonads. Further, real-time PCR results showed that expression of vasa was seen throughout the development from embryonic stage to adult. However, the expression was more in ovary than in testis during gonadal development. In adult testis, the vasa transcripts were significantly high during spermatogenesis and it declined during spermiation. On the other hand, during ovarian recrudescence, vasa transcripts were high in immature oocytes (stages I and II oocytes) when compared to mature oocytes (stages III and IV oocytes). Human chorionic gonadotropin treatment in recrudescing ovary (in vivo) as well as in testicular slices (in vitro) resulted in up regulation of vasa mRNA in a time-dependent manner. These results together suggest that vasa gene has got an important role to play in spermatogenesis and oogenesis during recrudescence in addition to development.

Isolation of sox9 duplicates in catfish: localization, differential expression pattern during gonadal development and recrudescence, and hCG-induced up regulation of sox9 in testicular slices

In vertebrates, sox9 is a transcription factor that plays a crucial role in testicular development and chondrogenesis. Here, we report cloning of isoforms of sox9 (sox9a and sox9b) from air-breathing catfish Clarias gariepinus, which undergoes an annual reproductive cycle. Tissue distribution pattern showed differential expression of sox9 duplicates, wherein both forms were highly expressed in brain and gonads. Furthermore, we observed a dimorphic expression pattern of sox9a and sox9b in both adult and developing gonads using RT-PCR, indicating that sox9a retained its function in testis while sox9b might have a new role to play in ovary. Changes in sox9 mRNA levels using real-time quantitative PCR (qRT-PCR) during the seasonal reproductive cycle revealed that sox9a transcript in testis was abundant during testicular recrudescence (during spermatogenesis), and its expression significantly decreased during spawning and post-spawning phases. Furthermore, treatments of human chorionic gonadotropin and 11-ketotestosterone in vitro up-regulated sox9a mRNA levels in the testicular slices at 12 and 24 h time points, suggesting that gonadotropins might stimulate sox9 expression. These results suggest that sox9 might have a plausible role in the entrainment of the testicular cycle. In contrast, during the ovarian cycle, sox9b mRNA levels gradually declined from preparatory to post-spawning phases. Immunohistochemical (IHC) data showed that, in testis, sox9 is detectable in Sertoli and spermatogonial cell types except spermatid/spermatozoa. In the ovary, it is localized in the ooplasm of primary and pre-vitellogenic oocytes. These results were further confirmed by whole-mount IHC and qRT-PCR.

Gender differences in tryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan levels in the brain of catfish, Clarias gariepinus, during sex differentiation

Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.

Thiourea-induced alteration in the expression patterns of some steroidogenic enzymes in the air-breathing catfish Clarias gariepinus.

Previous study from our laboratory on thiourea-induced thyroid hormone depletion in mature male catfish demonstrated that thyroid hormones play a significant role in testicular function. In the present study, we aimed to analyze the changes in the expression pattern of several steroidogenic enzyme genes after thyroid hormone depletion using semi quantitative RT-PCR in both adult male and female catfish. There was a marked decrease in the 11β-hydroxylase expression in the testis and liver while no change was observed in case of kidney. A significant decrease in 11β-hydroxysteroid dehydrogenase transcript level in testis, liver and kidney were observed in the thiourea treated males. The results obtained corroborated with our earlier findings of testicular regression after thyroid hormone depletion. In females, expression of aromatase transcript increased in experimental group compared to control. There was no considerable change observed in the transcript level of 3β-hydroxysteroid dehydrogenase, P450 17α-hydroxylase/C17-20-lyase, and 20β-hydroxysteroid dehydrogenase in both males and females. Thus, thyroid hormones might exert modulating effect on steroidogenic enzyme genes at the transcription level.

Dimorphic expression of tryptophan hydroxylase in the brain of XX and XY Nile tilapia during early development

Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.

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.

Cloning and expression of 3β-hydroxysteroid dehydrogenase during gonadal recrudescence and after hCG induction in the air-breathing catfish, Clarias gariepinus.

3β-hydroxysteroid dehydrogenase (3β-hsd) plays an important role in biosynthesis of both androgens and estrogens during steroidogenesis. In this study, we report the cloning of a full-length cDNA of 3β-hsd from gonads of the air-breathing catfish, Clarias gariepinus a seasonally reproducing teleost fish. We studied the expression pattern of 3β-hsd during gonadal ontogeny and recrudescence (flanking two years of reproductive cycle) using real-time PCR. We also examined the influence of gonadotropin on 3β-hsd expression in gonads of catfish by human chorionic gonadotropin (hCG) induction. The real-time PCR results revealed that 3β-hsd transcript was detectable much earlier in undifferentiated gonads i.e. before the sex differentiation and later on its expression was seen in both male and female gonads throughout the development. The expression analysis during subsequent seasonal reproductive cycle in catfish (older than one year) showed that in adult males, the transcripts were significantly high during prespawning phase (spermatogenesis) and declined during spermiation. In adult females, the transcripts were abundantly expressed in the ovarian follicles both at prespawning and spawning phases. Furthermore, the 3β-hsd mRNA levels in different follicular stages were markedly high in vitellogenic follicles (maturing oocytes; stage III) compared to other stages. Treatment of hCG in recrudescing female fish, in vivo as well as in testicular slices, in vitro resulted in the up-regulation of gonadal 3β-hsd mRNA indicating that it is under the regulation of gonadotropins. These results together suggest that 3β-hsd gene plays an important role during spermatogenesis and oogenesis as well as in the gonadal recrudescence of catfish.

Dimorphic expression of GnRH, gonadotropin and tryptophan hydroxylase provides new markers to follow brain sex development in teleosts

resence of multiple forms of gonadotropin-releasing hormone (GnRH) is comon in teleosts. Among these, species-specific form of GnRH plays an important ole in regulating gonadotropin (GTH) in turn reproduction. Interestingly, serotonin 5-HT) plays an important role to regulate GnRH and GTH release. Nevertheless, ole of GnRH, GTH and 5-HT has not been implicated in sex differentiation of eleosts. Studies from our laboratory addressed this issue to some extent. Genetic Y males have differential seabream GnRH immunoreactivity (-ir) while in females teady -ir was evident during early development. It is true with reference to GTH. ur studies on tryptophan hydroxylase, rate limiting enzyme for 5-HT biosynthesis lso showed male-specific expression in teleosts. Based on these, we presume that uitable markers can be developed to identify male brain sex differentiation.