Expression profile of kisspeptin2 and gonadotropin-releasing hormone2 mRNA during photo-thermal and melatonin treatments in the female air-breathing catfish Heteropneustes fossilis.

In seasonally breeding vertebrates, extrinsic factors like photoperiod and temperature are major determinants, controlling the annual reproductive cycle. In teleosts, kisspeptin, which occurs in two molecular forms: kisspeptin1 (Kiss1) and kisspetin2 (Kiss2), has been reported to alter gonadotropin (Lh and Fsh) secretion but its effect on gonadotropin-releasing hormone (Gnrh) secretion is not unequivocally proved. In the catfish Heteropneustes fossilis, we isolated and characterized kiss2 and gnrh2 cDNAs and the present work reports effects of altered photo-thermal conditions and melatonin (MT, a pineal hormone) on their expressions in the brain. The exposure of the catfish to long photoperiod (LP, 16 h light) at normal temperature (NT) or high temperature (HT, 28 °C) at normal photoperiod (NP) for 14 or 28 days stimulated both kiss2 and gnrh2 expression in both gonad resting and preparatory phases with the combination of LP + HT eliciting maximal effects. Short photoperiod (SP, 8 h light) under NT or HT altered the gene expression according to the reproductive phase and temperature. MT that mediates photo-thermal signals to the brain inhibited brain kiss2 and gnrh2 gene expression in the NP + HT, LP + NT, and SP + NT groups. The altered photo-thermal conditions elicited changes in steroidogenic pathway as evident from changes in plasma E2, progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction.

Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation.

Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93-405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17ß (E2; 0.5 µg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions.

Molecular cloning and characterization of a gonadotropin-releasing hormone 2 precursor cDNA in the catfish Heteropneustes fossilis: Expression profile and regulation by ovarian steroids.

Gonadotropin-releasing hormone 2 (Gnrh2) is one of the three classes of Gnrh distributed in vertebrates and is highly conserved. In the present study, the cDNA encoding Gnrh2 was isolated and characterized in the ostariophysan catfish Heteropneustes fossilis (hf). The cDNA is 611 bp long with an open reading frame (ORF) of 261 bp that encodes a highly conserved protein of 86 amino acids. The deduced Gnrh2 precursor protein clustered with the vertebrate Gnrh2 type. The sequence identity of hfgnrh2 is 94% with African catfish (Clarias gariepinus) gnrh2 mRNA (accession no. X78047). The hfgnrh2 transcripts were expressed only in the brain and gonads with a higher expression in the female brain and ovary in both resting and prespawning phases. The expression was higher in the prespawning phase than the resting phase. The gnrh2 expression in the brain and ovary showed significant seasonal variations but with opposite patterns. In the brain, the expression was the highest in the preparatory phase, decreased progressively to low levels in the postspawning and resting phases. In the ovary, the transcript level was low in the resting and preparatory phases, increased sharply in the prespawning phase reaching the peak level in the spawning phase and declined sharply in the postspawning phase. The gnrh2 mRNA showed the highest expression in the hind brain-medulla oblongata and moderate to low expression in forebrain regions and pituitary. Ovariectomy resulted in a duration-dependent inhibition of hfgnrh2 mRNA levels in the resting and prespawning phases. Steroid (E2, testosterone and progesterone) replacement treatments (0.5 µg/g body weight) in the 3- week ovariectomized fish restored the inhibition due to ovariectomy, elevated the expression over and above the sham level in the resting phase (E2 group), and raised the levels almost to that of the sham group (testosterone and progesterone groups) in the prespawning phase. In the sham control groups, the steroid replacement resulted in a significant reduction in the mRNA levels. The expression of the gnrh2 mRNA in the brain-pituitary-gonadal axis and its regulation by gonadal steroids suggest that Gnrh2 may have a reproductive role in the catfish.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

Receptor, c-Kit is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analyzed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of c-kit was analyzed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression during the pre-spawning phase in male compared to females. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression levels of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes that are involved in germ cell development. Decrease in the levels of 11-ketotestosterone and testosterone in serum were also observed after esiRNA silencing. The findings suggests that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish.

Cloning and characterization of a riboflavin-binding hexamerin from the larval fat body of a lepidopteran stored grain pest, Corcyra cephalonica.

In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat body of Corcyra cephalonica. The complete cDNA (2121bp) encodes a 706-amino acid protein with a molecular mass ~82kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is prominently expressed during the last instar larval development. Homology modeling and docking studies predicted riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect. However, JH in the presence of 20HE maintains the RbHex 82 expression which indicates the JH's role as a status quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran pest. Further, the possibility of RbHex 82 as a pest control target is discussed.

Molecular cloning, expression analysis and transcript localization of testicular orphan nuclear receptor 2 in the male catfish, Clarias batrachus.

Testicular receptor 2 (TR2; also known as Nr2c1) is one of the first orphan nuclear receptors identified and known to regulate various physiological process with or without any ligand. In this study, we report the cloning of full length nr2c1 and its expression analysis during gonadal development, seasonal testicular cycle and after human chorionic gonadotropin (hCG) induction. In addition, in situ hybridization (ISH) was performed to localize nr2c1 transcripts in adult testis and whole catfish (1day post hatch). Tissue distribution and gonadal ontogeny studies revealed high expression of nr2c1 in developing and adult testis. Early embryonic stage-wise expression of nr2c1 seems to emphasize its importance in cellular differentiation and development. Substantial expression of nr2c1 during pre-spawning phase and localization of nr2c1 transcripts in sperm/spermatids were observed. Significant upregulation after hCG induction indicate that nr2c1 is under the regulation of gonadotropins. Whole mount ISH analysis displayed nr2c1 expression in notochord indicating its role in normal vertebrate development. Taken together, our findings suggest that nr2c1 may have a plausible role in the testicular and embryonic development of catfish.

Early exposure of 17α-ethynylestradiol and diethylstilbestrol induces morphological changes and alters ovarian steroidogenic pathway enzyme gene expression in catfish, Clarias gariepinus.

Environmental estrogens are major cause of endocrine disruption in vertebrates, including aquatic organisms. Teleosts are valuable and popular models for studying the effects of endocrine disrupting chemicals (EDCs) in the environment. In the present study, we investigated the changes caused by exposure to the synthetic estrogens 17α-ethynylestradiol (EE2 ) and diethylstilbesterol (DES) during early stages of growth and sex differentiation of air-breathing catfish, Clarias gariepinus, at the morphological, histological, and molecular levels. Catfish hatchlings, 0 day post hatch (dph) were exposed continuously to sublethal doses of EE2 (50 ng/L) and DES (10 ng/L) until 50 dph and subsequently monitored for ovarian structural changes and alteration in the gene expression of steroidogenic enzymes till adulthood. Treated fish exhibited morphological deformities such as spinal curvature, stunted growth, and yolk-sac fluid retention. In addition to ovarian atrophy, DES-treated fish showed either rudimentary or malformed ovaries. Detailed histological studies revealed precocious oocyte development as well as follicular atresia. Further, transcript levels of various steroidogenic enzyme and transcription factor genes were altered in response to EE2 and DES. Activity of the rate-limiting enzyme of estrogen biosynthesis, aromatase, in the ovary as well as the brain of treated fish was in accordance with transcript level changes. These developmental and molecular effects imparted by EE2 and DES during early life stages of catfish could demonstrate the deleterious effects of estrogen exposure and provide reliable markers for estrogenic EDCs exposure in the environment.

Two-dimensional proteomic analysis of gonads of air-breathing catfish, Clarias batrachus after the exposure of endosulfan and malathion.

Endocrine disrupting chemicals have raised public concern, since their effects have been found to interfere with the physiological systems of various organisms, especially during critical stage of development and reproduction. Endosulfan and malathion, pesticides widely used for agricultural purposes, have been known to disrupt physiological functions in aquatic organisms. The current work analyzes the effects of endosulfan (2.5 parts per billion [ppb]) and malathion (10 ppb) on the reproductive physiology of catfish (Clarias batrachus) by evaluating protein expression profiles after 21 days of exposure. The proteomic profile of testis and ovary after exposure to endosulfan showed downregulation of proteins such as ubiquitin and Esco2, and upregulation in melanocortin-receptor-2 respectively. Malathion exposed ovary showed upregulated prolactin levels. Identification of proteins differentially expressed in gonads due to the exposure to these pesticides may serve as crucial indications to denote their disruptive effects at the level of proteins.

Gene expression analysis in gonads and brain of catfish Clarias batrachus after the exposure of malathion.

Pesticides like malathion have the potential to disrupt development and reproduction of aquatic organisms including fishes. To investigate the likely consequences of malathion exposure at low doses in juvenile catfish, Clarias batrachus, we studied the expression pattern of genes encoding certain transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes which are known to be involved in gonadal development along with histological changes. To compare further, we also analyzed certain brain specific genes related to gonadal axis. Fifty days post hatch catfish fingerlings were exposed continuously to 1 and 10 µg/L of malathion for 21 days. Results from these experiments indicated that transcript levels of various genes were altered by the treatments, which may further affect the gonadal development either directly or indirectly through brain. Histological analysis revealed slow progression of spermatogenesis in testis, while in ovary, the oil droplet oocytes were found to be higher after treatment (10 µg/L). Our findings revealed that the exposure of malathion, even at low doses, hinder or modulate early gonadal development differentially by targeting gene expression pattern of transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes with an evidence on histological changes. Further, some of the genes showed differential expression at the level of brain in male and female sex after the exposure of malathion.

Effect of juvenile hormone analog, methoprene on H-fibroin regulation during the last instar larval development of Corcyra cephalonica.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E), co-ordinately orchestrate insect growth and development. The process of silk synthesis and secretion in lepidopteran insects is known to be under hormonal control. However, the role of JH in this process has not been demonstrated hitherto. The present study is aimed to elucidate the role of JH in H-fibroin regulation in Corcyra cephalonica, a serious lepidopteran pest. Reiterated amino acid stretches and the large molecular weight of H-fibroin render its cloning and characterization cumbersome. To address this, a commercially synthesized short amino acid peptide conjugated with a carrier protein was used to generate antibodies against the N-terminal region of H-fibroin. ELISA and immunoblot experiments demonstrated the sensitivity and specificity of antibody. Further, immunohistochemical analyses revealed the antibody's cross-reactivity with H-fibroins of C. cephalonica and Bombyx mori in the silk gland lumen. Quantitative RT-PCR and Western blot analysis demonstrated the tissue-specificity and developmental expression of H-fibroin. Hormonal studies revealed that JH alone does not alter the expression of H-fibroin. However, in the presence 20E, JH reverses the declined expression caused by 20E administration to normal levels. This study provides molecular evidence for the regulation of H-fibroin by the cumulative action of JH and 20E.

20ß-hydroxysteroid dehydrogenase gene promoter: potential role for cyclic AMP and xenobiotic responsive elements.

Teleostean 20ß-hydroxysteroid dehydrogenase (20ß-HSD) is involved in final oocyte maturation and steroid hormone metabolism. It has structural and functional similarities to mammalian carbonyl reductases that are involved in the metabolism of endogenous carbonyl and xenobiotic compounds. To understand the transcriptional regulation of 20ß-HSD, here we report the cloning of 20ß-HSD promoter from two fish species, rainbow trout and air-breathing catfish. Analysis of the promoter motifs, in silico identified the presence of several sites for transcription factor binding including cAMP, xenobiotic and steroid hormone responsive elements. Luciferase reporter assays with progressive deletion constructs demonstrated that 20ß-HSD type B of trout has no promoter activity while 20ß-HSD type A of trout and catfish 20ß-HSD promoters showed basal promoter activity. A TATA box flanked by a CAAT box is important for basal transcription. Deletion of cAMP responsive element in the promoter decreased basal promoter activity significantly. Reporter assays with forskolin and IBMX, drugs that increase intracellular cAMP induced the promoter activity over the basal level. Intriguingly, ß-nafthoflavone, an arylhydrocarbon receptor ligand, induced the 20ß-HSD promoter activity and is further evidenced by the induction of 20ß-HSD expression in the livers of catfish, in vivo. These results demonstrate for the first time that 20ß-HSD expression is not only modulated by cAMP but also by xenobiotics and further studies may provide significance to the ubiquitous distribution and broad substrate specificity of this enzyme.

Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus.

Endosulfan and flutamide, a widely used pesticide and a prostate cancer/infertility drug, respectively, have an increased risk of causing endocrine disruption if they reach water bodies. Though many studies are available on neurotoxicity/bioaccumulation of endosulfan and receptor antagonism of flutamide, only little is known about their impact on testicular steroidogenesis at molecular level. Sex steroids play an important role in sex differentiation of lower vertebrates including fishes. Hence, a small change in their levels caused by endocrine disruptors affects the gonadal development of aquatic vertebrates significantly. The aim of this study was to evaluate the effects of endosulfan and flutamide on testis-related transcription factor and steroidogenic enzyme genes with a comparison on the levels of androgens during critical period of catfish testicular development. We also analyzed the correlation between the above-mentioned genes and catfish gonadotropin-releasing hormone (cfGnRH)-tryptophan hydroxylase2 (tph2). The Asian catfish, Clarias batrachus males at 50 days post hatch (dph) were exposed to very low dose of endosulfan (2.5 µg/L) and flutamide (33 µg/L), alone and in combination for 50 days. The doses used in this study were far less than those used in the previous studies of flutamide and reported levels of endosulfan in surface water and sediments. Sampling was done at end of the treatments (100 dph) to perform testicular germ cell count (histology), measurements of testosterone (T) and 11-ketotestosterone (11-KT) by enzyme immunoassay and transcript quantification by quantitative real-time PCR. In general, treatments decreased the expression of several genes including testis-related transcription factors (dmrt1, sox9a and wt1), steroidogenic enzymes (11ß-hsd2, 17ß-hsd12 and P450c17), steroidogenic acute regulatory protein and orphan nuclear receptors (nr2c1 and Ad4BP/SF-1). In contrast, the transcripts of cfGnRH and tph2 were elevated in the brain of all treated groups with maximum elevation in the endosulfan group. However, combination of endosulfan and flutamide (E+F) treatment showed minor antagonism in a few results of transcript quantification. Levels of T and 11-KT were elevated after flutamide and E+F treatments while no change was seen in the endosulfan group signifying the effect of flutamide as an androgen receptor antagonist. All the treatments modulated testis growth by decreasing the progression of differentiation of spermatogonia to spermatocytes. Based on these results, we suggest that the exposure to endosulfan and flutamide, even at low doses, impairs testicular development either directly or indirectly at the level of brain.

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-17ß 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-17ß 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.

Expression and immunolocalization of 20ß-hydroxysteroid dehydrogenase during testicular cycle and after hCG induction, in vivo in the catfish, Clarias gariepinus.

The maturation inducing hormone, 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DP) is required for the meiotic maturation and is produced from the precursor 17α-hydroxyprogesterone by the enzyme 20ß-hydroxysteroid dehydrogenase (20ß-HSD) in several teleosts. Central role of 20ß-HSD in ovarian cycle and final oocyte maturation is well studied when compared to spermatogenesis. In the present study, we investigated the localization and expression of 20ß-HSD in testicular cycle and gonadotropin induced sperm maturation. During testicular ontogeny, 20ß-HSD expression was detectable at 50 and 100 days post-hatch (dph), while the expression was high at 150 dph. In testicular cycle, highest levels of mRNA and protein of 20ß-HSD were observed during spawning phase. Intraperitoneal injection of human chorionic gonadotropin (hCG) to prespawning catfish elevated both 20ß-HSD transcripts and protein levels when compared to saline treated controls in a time-dependent manner. Serum 17α,20ß-DP levels, measured during different phases of testicular cycle as well as following the treatment of hCG, showed a positive correlation with the expression of 20ß-HSD. Immunolocalization revealed the presence of 20ß-HSD protein predominantly in interstitial cells and spermatogonia/spermatocytes while 20ß-HSD was undetectable in haploid cells (spermatids/sperm). These results together with high expression during spawning phase of testicular cycle and after hCG treatment in the prespawning catfish suggests a pivotal role for 20ß-HSD during testicular recrudescence leading to sperm maturation. Further studies using various fish models on testicular 20ß-HSD may provide interesting details to understand its importance in teleostean spermatogenesis.

FTZ-F1 and FOXL2 up-regulate catfish brain aromatase gene transcription by specific binding to the promoter motifs.

Cytochrome P450 aromatase (cyp19) catalyzes the conversion of androgens into estrogens. Teleosts have distinct, ovarian specific (cyp19a1a) and brain specific (cyp19a1b) cyp19 genes. Previous studies in teleosts demonstrated regulation of cyp19a1a expression by the NR5A nuclear receptor subfamily as well as a fork head transcription factor, FOXL2. In the present study, we investigated the involvement of fushi tarazu factor 1, FTZ-F1, a NR5A subfamily member, and FOXL2 in the regulation of cyp19a1b expression in brain of the air-breathing catfish, Clarias gariepinus. Based on the synchronous expression pattern of cyp19a1b, FTZ-F1 and FOXL2 in the brain, we isolated the 5' upstream region of cyp19a1b to analyse regulatory motifs. Promoter motif analysis revealed FTZ-F1/NR5A1 and FOXL2 binding nucleotide sequences. Transient transfection studies showed that FTZ-F1 and FOXL2 together enhanced the transcriptional activity of cyp19a1b gene in mammalian cell lines. Mutation in either of their putative binding sites within the cyp19a1b promoter abolished this effect. Electrophoretic gel mobility shift experiments indicated that FTZ-F1 and FOXL2 proteins bind to the synthesized radio-labelled oligomers used as probes and mobility shifted upon addition of their respective antibodies. Chromatin immunoprecipitation assay confirmed the binding of both these transcription factors to their corresponding cis-acting elements in the upstream region of cyp19a1b. To our knowledge, this study is the first report on the transcriptional regulation of cyp19a1b by FTZ-F1 and FOXL2 in a teleost fish.

Cloning and differential expression of FOXL2 during ovarian development and recrudescence of the catfish, Clarias gariepinus.

FOXL2 is a member of the forkhead/HNF-3-related family of transcription factors which provides tissue-specific gene regulation. It is known to regulate ovarian aromatase, (cyp19a1a) which plays a crucial role in ovarian differentiation. To understand the role of FOXL2 in gonads and brain during ovarian development and recrudescence, we cloned the full-length cDNA of FOXL2 and analyzed its spatio-temporal expression both at transcript and protein levels in the air-breathing catfish, Clarias gariepinus. Based on its deduced amino acid sequence, an antigenic peptide conjugated with a carrier protein was synthesized which was then used for raising antibody that reacted specifically with FOXL2. Tissue distribution pattern of FOXL2 revealed its presence prominently in ovary and female brain with sexual dimorphism. Highest expression of FOXL2 was observed in ovary and brain during prespawning phase indicating an important role for this correlate in ovarian recrudescence. Human chorionic gonadotropin (hCG) treatment, in vitro and in vivo, induced FOXL2 expression in the ovary during preparatory and prespawning phases. Similar type of enhanced expression was evident in brain after hCG-induction during the prespawning phase. The ontogeny of FOXL2 showed sexual dimorphic expression pattern both in gonads and brain. Based on our previous studies, the expression pattern of FOXL2 was found to be synchronous not only with that of ovarian cyp19a1a but also with brain cyp19a1b. Present study substantiates the role of FOXL2 in the regulation of aromatase in teleosts and also designates FOXL2 as a potential ovary and brain marker during female sex development in catfish.

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 3ß-hydroxysteroid dehydrogenase (3ß-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-17ß biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30-40 days post hatch (dph). The steroidogenic enzyme, 11ß-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17ß 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.

20-Hydroxyecdysone regulation of H-fibroin gene in the stored grain pest Corcyra cephalonica, during the last instar larval development.

20-Hydroxyecdysone (20E) controls molting, metamorphosis and reproduction of insects. It binds to a heterodimeric complex of ecdysone receptor (EcR) and ultraspiracle (USP), and regulates the transcription of genes containing ecdysone response elements (EcREs). However, the 20E regulation of silk fibroin genes is largely unexplored. In most lepidopteran larvae, the silk fibroin primarily consists of a large protein, heavy chain fibroin (H-fibroin) that is associated with two small proteins, L-chain fibroin and P25. In the present study, we demonstrate that 20E regulates the expression of H-fibroin gene in Corcyra cephalonica, in a dose-dependent manner during the last instar larval development. Semi-quantitative and real-time PCR studies reveal that physiological doses of 20E do not alter the normal expression, whereas higher doses cause a significant decline in the expression. Luciferase activity assays and gel shift experiments further confirm the presence of a functional EcRE in the upstream region of H-fibroin which regulates the ecdysteroid dependent transcriptional activity of fibroin gene through EcR. In vitro treatment with 20E mimicking insecticides, RH-5849 and RH-5992 decreases the expression of H-fibroin in isolated salivary glands. Insects fed with similar concentrations of these insecticides, metamorphose abnormally. Differences are also observed in the ultrastructure of the silk fibers of control and insecticide fed insects providing additional insight into the disruptive effects of these non-steroidal ecdysteroid agonists.

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.

Cytochrome P450 aromatases: Impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus.

Present study analyzed the importance of two forms of aromatases during ovarian development and recrudescence of north African/air-breathing catfish. We cloned both CYP19A1 (1941bp; ovarian form) and CYP19A2 (1786bp; brain form), which showed 47% homology between the two forms. Characterization of encoded proteins in non-steroidogenic COS-7 cells illustrated that both isoforms efficiently catalyzed the aromatization reaction by producing estradiol-17beta (E(2)) from testosterone. Tissue distribution pattern revealed preferential expression of CYP19A2 in brain while CYP19A1 predominated in ovary with trace amounts detected in other tissues including brain. Relative real-time PCR analysis revealed high transcript levels of both isoforms in the prespawning phase of ovarian cycle, which is in accordance with serum E(2) level. Aromatase activity in brain was comparatively lower than ovary, indicating the predominant requirement of aromatase in ovary. Ontogeny studies displayed sexual dimorphism, with early expression of CYP19A1 and CYP19A2 in ovary and brain, respectively. Phase-dependent rise of expression and enzyme activity of aromatase after hCG treatment revealed the stimulatory role of gonadotropin during preparatory and prespawning phases, preferentially to promote vitellogenesis. Lack of influence of hCG treatment during spawning phase endorses it further. A good correlation of expression, enzyme activity and serum E(2) levels suggests a crucial role of CYP19A1 during ovarian differentiation and ovarian cycle of catfish. Likewise, CYP19A2 might also be involved in these processes either indirectly or directly.