Estrogen-regulated expression of P450arom genes in the brain and ovaries of adult female Indian climbing perch, Anabas testudineus.

Cytochrome P450arom (CYP19), a product of cyp19a1 gene, catalyzes the conversion of androgens to estrogens and is essential for regulation of reproductive function in vertebrates. In the present study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female perch, Anabas testudineus and investigated their regulation by estrogen in vivo. Results demonstrated that cyp19a1a and cyp19a1b predominate in ovary and brain respectively, with quantity of both attuned to reproductive cycle. To elucidate estrogen-regulated expression of cyp19a1b in brain and cyp19a1a in ovary, dose- and time-dependent studies were conducted with estrogen in vitellogenic-stage fish in the presence or absence of specific aromatase inhibitor fadrozole. Results demonstrated that treatment of fish with 17ß-estradiol (E2; 1.0 µM)) for 6 days caused significant upregulation of cyp19a1b transcripts, aromatase B protein, and aromatase activity in brain in a dose- and time-dependent manner. Ovarian cyp19a1a mRNA, aromatase protein, and aromatase activity, however, was less responsive to E2 than brain. Treatment of fish with an aromatase inhibitor fadrozole for 6 days attenuated both brain and ovarian cyp19a1 mRNAs expression and stimulatory effects of E2 was also significantly reduced. These results indicate that expression of cyp19a1b in brain and cyp19a1a in ovary of adult female A. testudineus was closely associated to plasma E2 levels and seasonal reproductive cycle. Results further show apparent differential regulation of cyp19a1a and cyp19a1b expression by E2/fadrozole manipulation.

Effects of bisphenol A (BPA) on brain-specific expression of cyp19a1b gene in swim-up fry of Labeo rohita.

Estrogen regulates numerous developmental and physiological processes and effects are mediated mainly by estrogenic receptors (ERs), which function as ligand-regulated transcription factor. ERs can be activated by many different types endocrine disrupting chemicals (EDCs) and interfere with behaviour and reproductive potential of living organism. Estrogenic regulation of membrane associated G protein-coupled estrogen receptor, GPER activity has also been reported. Bisphenol A (BPA), a ubiquitous endocrine disruptor is present in many household products, has been linked to many adverse effect on sexual development and reproductive potential of wild life species. The present work is aimed to elucidate how an environmentally pervasive chemical BPA affects in vivo expression of a known estrogen target gene, cyp19a1b in the brain, and a known estrogenic biomarker, vitellogenin (Vg) in the whole body homogenate of 30 days post fertilization (dpf) swim-up fry of Labeo rohita. We confirm that, like estrogen, the xenoestrogen BPA exposure for 5-15 days induces strong overexpression of cyp19a1b, but not cyp19a1a mRNA in the brain and increase concentration of vitellogenin in swim-up fry. BPA also induces strong overexpression of aromatase B protein and aromatase activity in brain. Experiments using selective modulators of classical ERs and GPER argue that this induction is largely through nuclear ERs, not through GPER. Thus, BPA has the potential to elevate the levels of aromatase and thereby, levels of endogenous estrogen in developing brain. These results indicate that L. rohita swim-up fry can be used to detect environmental endocrine disruptors either using cyp19a1b gene expression or vitellogenin induction.

Estrogen-regulated expression of cyp19a1a and cyp19a1b genes in swim-up fry of Labeo rohita.

P450 aromatase is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. The expression of cyp19a1 genes in brain and gonad of Indian major carp, Labeo rohita swim-up fry was measured by quantitative real-time polymerase chain-reaction. Results demonstrated that cyp19a1b and cyp19a1a predominate in brain and gonad respectively. Treatment of fry with an aromatase inhibitor fadrozole for 6days attenuated brain cyp19a1b expression, but not cyp19a1a of gonad. Fadrozole also attenuated brain aromatase activity. Treatment with 17ß-estradiol (E2) for 6days resulted in up-regulation of brain cyp19a1b transcripts in a dose- and time-dependent manner, but not cyp19a1a. Whole-body concentration of vitellogenin also increased in response to E2. Altogether, these results indicate L. rohita swim-up fry can be used to detect environmental estrogens either using vitellogenin induction or cyp19a1b gene expression.

Membrane receptor cross talk in gonadotropin-, IGF-I-, and insulin-mediated steroidogenesis in fish ovary: An overview.

Gonadal steroidogenesis is critical for survival and reproduction of all animals. The pathways that regulate gonadal steroidogenesis are therefore conserved among animals from the steroidogenic enzymes to the intracellular signaling molecules and G protein-coupled receptors (GPCRs) that mediate the activity of these enzymes. Regulation of fish ovarian steroidogenesis in vitro by gonadotropin (GtH) and GPCRs revealed interaction between adenylate cyclase and calcium/calmodulin-dependent protein kinases (CaMKs) and also MAP kinase pathway. Recent studies revealed another important pathway in GtH-induced fish ovarian steroidogenesis: cross talk between GPCRs and membrane receptor tyrosine kinases. Gonadotropin binding to Gαs-coupled membrane receptor in fish ovary leads to production of cAMP which in turn trans-activate the membrane-bound epidermal growth factor receptor (EGFR). This is followed by activation of ERK1/2 signaling that promotes steroid production. Interestingly, GtH-induced trans-activation of EGFR in the fish ovary uniquely requires matrix-metalloproteinase-mediated release of EGF. Inhibition of these proteases blocks GtH-induced steroidogenesis. Increased cAMP production in fish ovarian follicle upregulate follicular cyp19a1a mRNA expression and aromatase activity leading to increased biosynthesis of 17ß-estradiol (E2). Evidence for involvement of SF-1 protein in inducing cyp19a1a mRNA and aromatase activity has also been demonstrated. In addition to GtH, insulin-like growth factor (IGF-I) and bovine insulin can alone induced steroidogenesis in fish ovary. In intact follicles and isolated theca cells, IGF-I and insulin had no effect on GtH-induced testosterone and 17a,hydroxysprogeaterone production. GtH-stimulated E2 and 17,20bdihydroxy-4-pregnane 3-one production in granulosa cells however, was significantly increased by IGF-I and insulin. Both IGF-I and insulin mediates their signaling via receptor tyrosine kinases leading to activation of PI3 kinase/Akt and MAP kinase. These kinase signals then activates steroidogenic enzymes which promotes steroid production. PI3 kinase, therefore considered to be an initial component of the signal transduction pathways which precedes MAP kinase in IGF-1 and insulininduced steroidogenesis in fish ovary. Thus, investigation on the mechanism of signal transduction regulating fish ovarian steroidogenesis have shown that multiple, apparently independent signal transduction pathways are needed to convey the message of single hormone or growth factor.

Gonadotropin and sf-1 regulation of cyp19a1a gene and aromatase activity during oocyte development in the rohu, L. rohita.

Cytochrome P450 aromatase (P450arom), a product of cyp19a1 gene, plays pivotal roles in vertebrate steroidogenesis and reproduction. In this study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female rohu, Labeo rohita and investigated the regulation of cyp19a1a by gonadotropin and SF-1. The cyp19a1a and cyp19a1b were expressed predominantly in the ovary and brain respectively, with quantity of the former attuned to reproductive cycle. To elucidate gonadotropin regulation of cyp19a1a mRNA expression and P450 aromatase activity for 17ß-estradiol (E2) biosynthesis in vitro by the vitellogenic ovarian follicles, time- and dose-dependent studies were conducted with HCG and porcine FSH. Results demonstrated that HCG stimulated significantly higher expression of cyp19a1a mRNA and aromatase activity leading to increased biosynthesis of E2 than FSH. To understand the involvement of SF-1 to in the regulation of cyp19a1a and aromatase activity, ovarian follicles were incubated with increasing concentrations of HCG and expression of sf1gene and activation of SF-1 protein were measured. Results demonstrated that HCG significantly induced expression of sf-1 gene and activation of SF-1 protein suggesting a link between SF-1 and P450 aromatase activation in this fish ovary during gonadotropin-induced steroidogenesis.

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17ß-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20ß-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes.