Sudipta Paul

Regulation of ovarian steroidogenesis in vitro by IGF-I and insulin in common carp, Cyprinus carpio: stimulation of aromatase activity and P450arom gene expression.

Regulation of ovarian steroidogenesis in vitro by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) was investigated in intact follicles and isolated follicular cells of carp, Cyprinus carpio at vitellogenic stage of oocyte maturation. In intact follicles, IGF-I and b-insulin stimulated testosterone and 17beta-estradiol production in vitro. In isolated theca cells, IGF-I and b-insulin stimulated testosterone production, whereas in granulosa cells, they stimulated 17beta-estradiol production when testosterone was added in the incubation medium as precursor substrate. In intact follicles and in theca cells, IGF-I and b-insulin had no effect on HCG-stimulated testosterone production. HCG-stimulated 17beta-estradiol production, however, was significantly increased by IGF-I and b-insulin. To clarify the mechanism of 17beta-estradiol production by the ovarian follicles during vitellogenic stage of carp, effects of IGF-I and b-insulin either alone or in combination with HCG on aromatase activity (conversion of testosterone to 17beta-estradiol) and cytochrome P450 aromatase (P450arom) gene expression were investigated in vitro. IGF-I and b-insulin alone stimulated aromatase activity and P450arom gene expression and significantly enhanced HCG-induced enzyme activity and P450arom gene expression. Our results thus indicate that IGF-I and b-insulin alone can stimulate testosterone and 17beta-estradiol production in vitellogenic follicles of C. carpio by stimulating aromatase activity and P450arom gene expression. Evidence also provided for the modulation of HCG-induced aromatase activity and P450arom gene expression by IGF-I and b-insulin in such follicles.

Regulation of ovarian steroidogenesis in vitro by gonadotropin in common carp Cyprinus carpio: interaction between calcium- and adenylate cyclase-dependent pathways and involvement of ERK signaling cascade

Multiple signal transduction pathways mediating gonadotropin-induced testosterone and 17β-estradiol (E(2)) production were identified in carp ovarian theca and granulosa cells in short-term co-incubation. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin attenuated human chorionic gonadotropin (HCG)-induced steroid production, whereas modulators of adenylate cyclase and protein kinase A (PKA) increased their production, indicating that both calcium- and PKA-dependent pathways are involved in the regulation of gonadotropin-induced steroidogenesis in carp ovary. Interactions between these two pathways are evident from the positive effect of elevated intracellular calcium on HCG-induced steroid production and the reduction of forskolin (FK)- and dibutyryl cAMP (dbcAMP)-induced steroidogenesis by inhibitors of VSCCs and calmodulin. In this study, we found the involvement of a third signaling pathway, a mitogen-activated protein kinase (MAP kinase), in the regulation of gonadal steroidogenesis in this fish. An antagonist of mitogen-activated protein kinase kinases 1/2 (MEK1/2; also known as MAP2K1/MAP2K2) markedly attenuated HCG-induced steroid production. Cells treated with HCG stimulated MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERKs1/2) in a concentration and time-dependent manner. Moreover, ERK1/2 activation in cells was mimicked by FK and dbcAMP suggesting that ERK1/2 transduce signal downstream of PKA in HCG-induced ovarian steroidogenesis. Evidence for presence of cross talk between calcium-dependent pathways and this MAP kinase cascade has been shown by demonstrating the inhibitory effects of verapamil and calmodulin on ERK1/2 activation after HCG stimulation. Our results suggest that activation of ERK1/2 by HCG as well as other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis in carp ovary.

Involvement of PI3 kinase and MAP kinase in IGF-I- and insulin-induced oocyte maturation in Cyprinus carpio

Previously, we observed that in vitro germinal vesicle breakdown (GVBD) in Cyprinus carpio oocytes was induced by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) and this induction was steroid-independent. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during oocyte maturation was examined. IGF-I- and b-insulin-induced oocyte maturation was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase after 90 min of their treatment. Both IGF-I and b-insulin were found to activate cdc2 kinase at 21h of treatment. We examined the relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in IGF-I- and b-insulin-induced oocyte maturation in C. carpio. MAP kinase was rapidly phosphorylated and activated (30-150 min) in response to exposure of the oocytes with IGF-I and b-insulin. This response preceded the phosphorylation and activation of cdc2 by several hours (almost 19h). A potent and selective inhibitor of MEK, PD98059, the protein kinase that phosphorylates and activate MAP kinase, blocked the phosphorylation and activation of MAP kinase and cdc2 kinase and GVBD induction. Likewise, PI3 kinase inhibitors strongly inhibited phosphorylation and activation of MAP kinase, which was increased during oocyte maturation. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes MAP kinase, and MPF activation during IGF-I- and b-insulin-induced oocyte maturation in C. carpio.

Stimulation of salmon calcitonin on secretion of 17β-estradiol by the ovarian follicles of common carp, Cyprinus carpio

The effects of salmon calcitonin (sCT) on the secretion of 17beta-estradiol (E(2)) were examined in female common carp, Cyprinus carpio. Vitellogenic stage fish adapted to high-Ca water were i.p. injected with vehicle, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. To determine whether ovarian follicles are equipped with CT receptors, a CT binding assay was conducted. In the in vitro experiments, vitellogenic follicles were incubated with stimulators and inhibitors. Administration of sCT increased the basal and hCG-stimulated E(2) release in vivo and in vitro. Binding characteristics of [(125)I]sCT to plasma membrane preparation of carp ovarian follicles showed saturability with high-affinity (K(d)=48.48 pmol/l and B(max)=1.2 pmol/mg protein). To clarify the mechanism of E(2) production by sCT, in vitro effect of sCT and hCG on aromatase activity (conversion of testosterone to E(2)) and cytochrome P450 aromatase (P450arom) gene expression in carp ovarian follicles were investigated. Salmon CT-stimulated both aromatase activity and P450arom gene expression in ovarian follicles of carp. sCT-stimulated E(2) release by the ovarian follicles in vitro was augmented in the presence of dibutyryl cAMP. Inhibitor of protein kinase A (PKA), SQ 22536 inhibited sCT-stimulated steroid production in a dose-dependent manner. Specific inhibitor of protein kinase C (PKC), NPC-15437 dihydrochloride had no inhibitory effects on sCT-induced E(2) release. The present study indicates that sCT binds specifically to carp ovary and stimulates E(2) production by increasing the activity of cytochrome P450 aromatase and P450arom gene expression. The results further suggest that stimulatory action of sCT on E(2) production is mediated through cAMP pathway.

Steroid-induced oocyte maturation in Indian shad Tenualosa ilisha (Hamilton, 1822) is dependent on phosphatidylinositol 3 kinase but not MAP kinase activation

Fully grown fish and amphibian oocytes exposed to a maturation-inducing steroid (MIS) activates multiple signal transduction pathways, leading to formation and activation of maturation-promoting factor (MPF) and induction of germinal vesicle breakdown (GVBD). The present study was to investigate if phosphatidylinositol 3 kinase (PI3 kinase) and mitogen-activated protein kinase (MAP kinase) activation are required for naturally occurring MIS, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that 17,20β-P-induced OM was significantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20 β-P was shown to activate PI3 kinase maximally at 90 min and cdc2 kinase at 16 h of treatment. Relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in 17,20β-P-induced OM was examined. MAP kinase was rapidly phosphorylated and activated (60-120 min) after MIS treatment and this response preceded the activation of cdc2 kinase by several hours. A selective inhibitor of MAP kinase (MEK), PD98059, sufficiently blocked the phosphorylation and activation of MAP kinase. Inhibition of MAP kinase activity using PD98059 however, had no effect on MIS-induced cdc2 kinase activation and GVBD. These results demonstrate that activation of the PI3 kinase is required for 17,20β-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20β-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species.

Involvement of PI3 kinase and MAP kinase in IGF-I and insulin-induced ovarian steroidogenesis in common carp Cyprinus carpio

Previously, we observed that in vitro steroidogenesis in intact ovarian follicles of common carp Cyprinus carpio can alone be induced by recombinant human insulin-like growth factor (IGF-I) and bovine insulin (b-insulin) and this induction was gonadotropin-independent. To investigate early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during ovarian steroidogenesis was examined. IGF-I and b-insulin induced testosterone and 17β-estradiol production in carp ovarian theca and granulosa cells in short-term coincubation and this induction was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase from 30 min onwards with a maximum at 90 min. In this study, we found the involvement of mitogen-activated protein kinase (MAP kinase) in the regulation of IGF-I- and b-insulin-induced steroidogenesis in carp ovary. An antagonist of mitogen-activated protein kinase kinase1/2 (MEK1/2) markedly attenuated IGF-I- and b-insulin-induced steroid production. Cells treated with IGF-I and b-insulin stimulated ERK1/2-dependent phosphorylation of extracellular signal regulated protein kinase1/2 (ERKs1/2) in a time-dependent manner, which was significantly attenuated in presence of MEK1/2 inhibitor. PI3 kinase inhibitors strongly attenuated phosphorylation and activation of MAP kinase, which was increased during IGF-I and b-insulin-induced steroidogenesis. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes the MAP kinase during IGF-I- and b-insulin-induced steroidogenesis in C. carpio ovarian follicles.

Changes in plasma steroid levels during oocyte development in Indian shad, Tenualosa ilisha (Hamilton, 1822): role of gonadotropins on in vitro steroid production and development of oocyte maturational competence.

Circanual variations in plasma testosterone (T), 17-estradiol (E2), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels and ovarian steroid synthetic potential of Tenualosa ilisha of river Hooghly, West Bengal, India were examined. This fish exhibited bi-annual spawning; one during April-May and another during August-September. Coinciding with the GSI values, present study recorded a decline in plasma T and E2 levels from October, reaching their lowest values in January followed by a rapid rise in March when the ovary contained mostly vitellogenic follicles and remained high up to April (postvitellogenic stage). Plasma 17,20β-P level was detected in March and reached peak value in April during oocyte maturation. After spawning, all the steroid levels declined to reach lowest values in June. From June onwards, T and E2 levels again increased for the next cycle and peaked at the end of vitellogenesis. Plasma 17,20β-P was reappeared in August and reached maximum in September during oocyte maturation and spawning. Of the two gonadotropins tested, in vitro production of both T and E2 by the vitellogenic and postvitellogenic follicles was regulated by FSH and LH respectively. Production of 17,20-P by the post-vitellogenic follicles was regulated by LH only. Acquisition of in vitro oocyte maturational competence (OMC) was developed by the addition of HCG in culture medium. Treatment of a 3β-HSD inhibitor blocked LH-induced steroid production, but not development of OMC. Both Cycloheximide and actinomycin D inhibited LH-induced development of OMC, indicating the requirement of de novo protein synthesis for this process.

Expression of LH receptor in nonpregnant mouse endometrium: LH induction of 3β-HSD and de novo synthesis of progesterone

In mouse uterus, at the late diestrus stage LH binding sites have previously been described. The aim of our study was to confirm the existence of LH receptor (Lhr (Lhcgr)) mRNA and its protein in mouse endometrium. Endometrium at all stages of the estrous cycle contained Lhr mRNA, essentially identical to that found in mouse ovary. Endometrium also contained a 72  kDa immunoreactive receptor protein that bound to mouse anti-LHR antibody in western blot. Both receptor mRNA and protein were maximally expressed in the endometrium at metestrus and LH caused a significant increase in their expression levels. Endometrium also contained 3β-hydroxy steroid dehydrogenase (3β-hsd) mRNA and 3β-HSD protein. LH addition elevated their expression and activity as evident from increased conversion of labeled pregnenolone to progesterone (P(4)) and de novo P(4) synthesis. LH-induced endometrial P(4) synthesis is mediated through expression of steroidogenic acute regulatory (Star) gene. Results demonstrated that LH-induced P(4) synthesis in endometrium is possibly mediated through the cAMP pathway. Involvement of a MAPK pathway was also evident. Gonadotropin-stimulated endometrial P(4) synthesis was markedly attenuated by an antagonist of MEK1/2, PD98059. LH-stimulated MEK1/2-dependent phosphorylation of ERK1/2 in a concentration- and time-dependant manner in cultured endometrial tissues. Moreover, involvement of cAMP in LH-stimulated activation of ERK1/2 was also evident. It is therefore possible that the major signaling pathways regulating endometrial steroidogenesis in mouse, including the adenylate cyclase and MAP kinase pathways, converge at a point distal to activation of protein kinase A and ERK1/2.

Calcitonin Functions Both as a Hypocalcemic Hormone and Stimulator of Steroid Production and Oocyte Maturation in Ovarian Follicles of Common Carp, Cyprinus carpio

Calcitonin (CT) is a calcium regulating hormone produced mainly by the parafollicular C cells of the thyroid gland in mammals. In lower vertebrates, these cells are concentrated primarily in a specialized gland, the ultimobranchial gland. The common structure of CT is 32-amino acid residues with a seven-residue cyclic loop formed by disulfide bond between cysteines at position 1 and 7 and prolinamide at carboxyl terminal. Calcitonin in mammals and birds is acknowledged to be the principal hypocalcemic agent, but the situation in fish is less clear. Several laboratories although showed a hypocalcemic action of CT in fish, conflicting results are often reported. Moreover, fish have other hypocalcemic factor like Staniocalcin, a product of Corpuscles of Stannius, secretion of which is positively regulated by extracellular calcium levels. Control of calcium homeostasis in fish may likely to be different from that in terrestrial vertebrates because of the aquatic environment. In addition to the involvement of CT in calcium homeostasis in mammals, an endocrine role of endogenous CT at brain, pituitary and gonad has also been suggested. Similarly, a role for CT in calcium ion regulation in fish may be subordinate to other functions which include acting as neurotransmitter or inducer of ovarian steroid hormone synthesis. A major question thus ariseswhat is the exact role of CT in fresh water teleost? Does it really has any role in calcium homeostasis or involved in many other functions including reproduction? The evolutionary history and functions of CT in aquatic vertebrate therefore require further investigation and as part of ongoing studies in to endocrine/paracrine factors involved in calcium homeostasis and the role of CT, if any, on reproduction in fish, we tried to answer these questions.