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Browsing Animal Biology - Publications by Subject "11-Ketotestosterone"
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    Cloning, expression and enzyme activity analysis of testicular 11β-hydroxysteroid dehydrogenase during seasonal cycle and after hCG induction in air-breathing catfish Clarias gariepinus
    ( 2010-05-01) Rasheeda, M. K. ; Kagawa, H. ; Kirubagaran, R. ; Dutta-Gupta, A. ; Senthilkumaran, B.
    A full-length cDNA encoding 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11β-HSD2 was then transfected to COS-7 cells, which converted 11β-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD+, 11β-HSD2 from testicular microsomes oxidized 11-OHT with apparent Km 56±4nM and Vmax 55±6pmol/h/mgprotein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11β-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11β-HSD2 transcript at 4h followed by gradual increase at 8h, 12h and peaking at 24h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11β-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11β-HSD2 during testicular differentiation and seasonal testicular cycle in catfish. © 2010 Elsevier Ltd.
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    Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus
    ( 2012-04-01) Rajakumar, A. ; Singh, R. ; Chakrabarty, S. ; Murugananthkumar, R. ; Laldinsangi, C. ; Prathibha, Y. ; Sudhakumari, C. C. ; Dutta-Gupta, A. ; Senthilkumaran, B.
    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 (2.5μg/L) and flutamide (33μg/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 (11β-hsd2, 17β-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. © 2012 Elsevier B.V..
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    Sox3 binds to 11β-hydroxysteroid dehydrogenase gene promoter suggesting transcriptional interaction in catfish
    ( 2016-04-01) Rajakumar, Anbazhagan ; Senthilkumaran, Balasubramanian
    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, 11β-hsd, enzyme involved in 11-KT production is important. Regulation of 11β-hsd gene was never studied in any fishes. At first 11β-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, 11β-hsd expression was higher during spawning phase of reproductive cycle and was found to be gonadotropin inducible both in vivo and in vitro. ∼2 kb of 5′ upstream region of 11β-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 11β-hsd. This is the first report to show that Sox3 binds to the 11β-hsd gene promoter and transactivates to regulate male reproduction in a teleost.