Estrogen receptors as potential therapeutic target in endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecological carcinomas in both developed and developing countries. Majority of the gynecological malignancies are hormonally driven where estrogen signaling acts as an oncogenic signal. Estrogen's effects are mediated via classical nuclear estrogen receptors; estrogen receptor alpha and beta (ERα and ERß) and a trans-membrane G protein-coupled estrogen receptor (GPR30 and GPER). ERs and GPER through ligand binding triggers multiple downstream signaling pathways causing cell cycle regulation, cell differentiation, migration, and apoptosis in various tissues including endometrium. Although the molecular aspect of estrogen function in ER-mediated signaling is now partly understood, the same is not true for GPER-mediated signaling in endometrial malignancies. Understanding the physiological roles of ERα and GPER in EC biology therefore leads to the identification of some novel therapeutic targets. Here we review the effect of estrogen signaling through ERα-and GPER in EC, major types, and some affordable treatment approaches for endometrial tumor patients which has interesting implications in understanding uterine cancer progression.

Functions of interleukin-6 in ovulation of female climbing perch, Anabas testudineus.

In mammals, interleukin 6 (IL-6) has an important function during ovulation, however, the functions of IL-6 in fish have not been elucidated. In the present study, there was quantification of de novo synthesis of ovarian IL-6 and tumor necrosis factor-alpha (TNFα) in control and hCG-treated fish and results were compared with those from an in vitro study where there was evaluation of the regulatory functions of gonadotropins and TNFα of IL-6 secretions. Relatively greater concentrations of ovarian IL-6 at the post-GVBD (post-germinal vesicle breakdown) stage indicates IL-6 modulates ovulatory processes. The hCG-induced increase in relative abundance of IL-6 (in vitro) mRNA transcript and secretion from the ovary were attenuated when there was administration of the inhibitor of TNFα secreting enzyme, TAPI-I, which indicates TNFα modulates IL-6 secretion. Treatments with IL-6 induced a marked increase in ovulation rate in vitro when there was induction of activating matrix metalloproteinase (MMP). Furthermore, treatment with IL-6 resulted in production of prostaglandin as indicated by the IL-6 induced increase in the abundance of ptgs2 mRNA transcript in the ovary of Anabas testudineus. Furthermore, results indicate the source of IL-6 in the ovary is the granulosa cells with secretion of IL-6 being induced by the additions of hCG and TNFα in the medium. There was also an IL-6-induced increase in abundance of receptors (IL-6 Rα and gp130) to which it binds indicating IL-6 autoregulates this population of receptors. Results from this study, for the first time, elucidate the reproductive functions of IL-6 in a teleost fish.

Seasonal ovarian development in relation to the gonadotropins, steroids, aromatase and steroidogenic factor 1 (SF-1) in the banded gourami, Trichogaster fasciata.

The endocrine regulation of gonadal development and annual variation of key sex steroids is the basic knowledge to understand the reproductive cycle of teleost fish. Present study was aimed to investigate the levels of gonadotropins in relation to the follicular development and plasma steroids during the reproductive cycle of female Trichogaster fasciata. Female fish were sampled and ovarian development is described histologically throughout the year in relation to the seasonal variations of gonadosomatic index (GSI); follicle stimulating hormone (FSH) and luteinizing hormone (LH); three key steroids for folliculogenesis and maturation i.e. testosterone (T), 17ß-estradiol (E2) and 17α20ßdihydroxy4pregnen3one (17,20ß-P). Relatively higher level of FSH was observed till the ovary reaches in late vitellogenic stage confirms that FSH regulates the early folliculogenesis of the ovary, whereas LH peak was observed in the postvitellogenic stage, which indicates that maturation and ovulation were controlled by LH. Seasonal steroid profiles show that both T and E2 reach its maximum level prior to the 17,20ß-P which attain its peak value in the month of August. Thus, single peak values of LH and 17,20ß-P coinciding with GSI peak, clearly indicates that T. fasciata breeds only once in a year. Furthermore, to elucidate the molecular basis of the reproductive cycle, this study analyzes the other key factors of ovarian function such as cyp19a1a gene expression, aromatase activity and SF-1 localization throughout the year. cyp19a1a gene expression and the aromatase activity were highest in vitellogenic stages indicate that relatively higher E2 production in this stage is regulated by FSH. Immunohistochemical localizations of aromatase and SF-1 in the cellular layer of oocytes demonstrated that aromatase is FSH-dependent and SF-1 could be regulated by both FSH and LH as relatively higher amount of aromatase was localized in the vitellogenic stage oocytes than the postvitellogenic and post germinal vesicle breakdown (post-GVBD) stages; whereas, high amount of SF-1 was observed in vitellogenic, postvitellogenic and post-GVBD stages. These data regarding the reproductive endocrinology of T. fasciata may be useful to understand the interaction between gonadotropins, steroids, aromatase and SF-1 in teleost fishes and may contribute to restoration of the ecologically important fish through artificial reproduction.

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.

Molecular mechanism of mercury-induced reproductive impairments in banded gourami, Trichogaster fasciata.

Mercury is one of the key pollutants responsible for the degradation of natural aquatic ecosystems. Among the different forms of mercury that exist in the environment, mercuric chloride (HgCl2) is the dominant pollutant for freshwater environments as it is used as an ingredient in antiseptics, disinfectants and preservatives, insecticides, batteries and in metallurgical and photographic operations. Pollutant may exert their action on organisms or populations by affecting their normal endocrine function as well as reproduction. Thus, the present study tried to understand the effect of mercuric chloride (HgCl2) on reproductive function and to decipher the molecular mechanism of Hg-induced reproductive impairments of female Trichogaster fasciata. Both in vivo and in vitro experiments were performed by using ecologically relevant doses of HgCl2 and the resulting effects on follicular development, steroidogenic potentiality, aromatase activity, aromatase gene expression and steroidogenic factor-1 (SF-1) expression pattern were analysed. In vivo exposure to HgCl2 caused reproductive impairments as shown by the inhibitory role of HgCl2 on follicular development, steroid biosynthesis and SF-1 activity. In vitro experiments revealed that aromatase activity, steroidogenesis, aromatase and SF-1 expression were blocked by HgCl2. The results obtained from this study contribute to understand the molecular mechanism of HgCl2-induced reproductive impairment of T. fasciata.

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.

Effects of phenol on ovarian P450arom gene expression and aromatase activity in vivo and antioxidant metabolism in common carp Cyprinus carpio.

Ovarian cyp19a mRNA expression and P450 aromatase activity were measured in vivo in common carp Cyprinus carpio exposed to phenol for 96 h. Production of reactive oxygen species (ROS) and parameters of antioxidant defense system in serum ovary and liver of this fish after long-term phenol exposure were also studied. In vivo exposure of fish to sublethal dose of phenol for 96 h caused marked attenuation of ovarian cyp19a1a gene expression and P450 aromatase activity. Production of ROS like hydrogen peroxide and hydroxyl radicals in serum, liver and ovary in fish exposed to phenol for 15 days elevated significantly from day 1 to day 7 with no further significant increase thereafter compared to their respective control values. Total superoxide dismutase (SOD) and catalase activities in serum and ovary decreased gradually and significantly from day 1 to day 4, which then increased significantly for the rest of the exposure days. Liver SOD activity seemed to be distinctly responsive to phenol. SOD activity in liver of phenol-exposed fish started to increase gradually from day 1 to 4 with no further increase thereafter. Catalase activities in all the tissues showed significant inhibition up to day 4 which then increased gradually and significantly up to day 15 of phenol exposure compared to their respective control values. From our results, it appears that sublethal dose of phenol has the endocrine disruptive potential and effect is mediated via inhibition of ovarian P450arom gene expression and aromatase activity in vivo. Sublethal dose of phenol also caused oxidative stress, and antioxidant systems are very much effective to prevent the damages caused by the generation of ROS.

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.

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,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that 17,20ß-P-induced OM was significantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20 ß-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 17,20ß-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 17,20ß-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20ß-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species.

In vitro induction of oocyte maturation and steroidogenesis by gonadotropins, insulin, calcitonin and growth factor in an estuarine flat head grey mullet, Mugil cephalus L.

In this article, an in vitro investigation was carried out to ascertain the roles of hormones and growth factor in the inductions of oocyte maturation and steroidogenesis of the postvitellogenic follicles in an Indian estuarine grey mullet, Mugil cephalus L. Oocyte maturation was evaluated by scoring the germinal vesicle breakdown (GVBD) percent of the postvitellogenic follicles. All the sex [17α,20ß-dihydroxy-4-pregnane-3-one (DHP), estradiol 17ß (E2), progesterone (P), 17α-OH progesterone (17-OH-P) and testosterone] and other [bovine-insulin and salmon-calcitonin, human chorionic gonadotropin (hCG), luteinizing hormone (LH) or hCG+DHP] hormones and insulin-like growth factor-I (IGF-I) significantly increased GVBD% in 9 h culture. DHP had a maximum effect (75 %) compared to other effectors. Some effectors (hCG: 82.14 %, LH: 78.94 %, hCG plus DHP: 81.81 %, E2: 80 % and IGF-I: 74.19 %) including DHP (79 %) further increased GVBD% in 15-h culture. All the hormones (except DHP) and IGF-I increased DHP, E2 and testosterone productions by the postvitellogenic ovarian follicles in vitro. DHP and testosterone productions were increased with the increase of incubation time from 9 h through 15 h. E2 production was not further increased beyond 12 h. DHP production was highest by hCG compared to other effectors. The hCG of all the test compounds was most effective in both the induction of GVBD% and steroid production. DHP is the most potent inducer of oocyte maturation in Indian estuarine flat head grey mullet. Involvement of estrogen in mullet oocyte maturation is indicated. hCG, like DHP, is equally potent and induces oocyte maturation via DHP production in vitro. hCG with DHP has synergistic action on oocyte maturation in mullet ovary. Interplay of several hormones (hCG, LH, and probably E2 and testosterone) and IGF-I on oocyte maturation is suggested in the mullet.

Signaling pathways in insulin- and IGF-I mediated oocyte maturation in lower vertebrates.

The endocrine control of oocyte maturation in fish and amphibians has proved to be a valuable model for investigating the rapid and non-genomic steroid actions at the cell surface. Considerable progress has made over the last decade in elucidating signaling pathways in steroid-induced oocyte maturation. In addition to steroids, various growth factors have also been reported to be involved in this process and progress being made to elucidate their mechanism of actions. Exposure of fully-grown oocytes to steroids or growth factors (insulin/IGFs) initiates various signaling cascade, leading to formation and activation of maturation-promoting factor (MPF), a key enzyme that catalyzes entry into M-phase of meiosis I and II. Whereas the function of MPF in promoting oocyte maturation is ubiquitous, there are differences in signaling pathways between steroids- and growth factors-induced oocyte maturation in amphibian and fish. Here, we have reviewed the recent advances on the signaling pathways in insulin- and IGF-I-induced oocyte maturation in these two groups of non-mammalian vertebrates. New findings demonstrating the involvement of PI3 kinase and MAP kinase in induction of oocyte maturation by insulin and IGF-I are presented.

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 17,20ß-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 17,20ß-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 3ß-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.

Effect of phenol on ovarian secretion of 17ß-estradiol in common carp Cyprinus carpio.

Phenol is a common substance present in many industrial wastewaters and in nonspecific pesticides. Due to its solubility and volatility phenol is often found in marine and freshwater environment. It is lipophilic compound and has a high potential for accumulating along the trophic chain. Phenol thus is not only a threat to natural environment but also to human health. The effects of phenol on the secretion of 17ß-estradiol were examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of phenol for 0, 24, 48 and 96 h. In the in vitro experiments, vitellogenic follicles were incubated with phenol and dose- and time-course effects on leuteinising hormone (LH) induced steroid production were examined. Exposure of fish with phenol gradually attenuated serum and ovarian 17ß-estradiol levels with increasing time and maximum inhibition was noticed after 96 h. Administration of phenol significantly inhibited LH-induced secretion of 17ß-estradiol by the ovarian follicles in vitro. To clarify the mechanism of attenuated production of 17ß-estradiol in phenol-treated follicles, stimulated by LH, in vitro effect phenol and LH on aromatase activity (conversion of testosterone to 17ß-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were investigated. Physiological safe dose of phenol significantly inhibited LH-stimulated aromatase activity and P450arom gene expression in ovarian follicles. The present study further demonstrated that LH-induced activation of ovarian steroidogenic factor-1 (SF-1) is strongly inhibited by phenol treatment. These results suggest that physiological safe dose of phenol as endocrine disruption (ED) potential and the effect can be mediated via several cellular pathways including the inhibition of SF-1 activity, aromatase activity and P450arom gene expression.

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 17ß-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.

Effect of cadmium chloride on secretion of 17ß-estradiol by the ovarian follicles of common carp, Cyprinus carpio.

Cadmium (Cd(2+)) is a common environmental pollutant present in wastes associated with mining, smelting and electroplating. It is a major constituent of the tobacco smoke. Exposure of this heavy metal has been linked to wide range of detrimental effects on mammalian reproduction particularly on ovarian steroidogenesis. Low doses of Cd(2+) are reported to stimulate ovarian luteal progesterone synthesis whereas high doses inhibited it. Cd(2+) exposure is also reported to inhibit gonadal function in fish. In the present study the effects of cadmium chloride (CdCl(2)) on the secretion of gonadotropin-induced 17ß-estradiol was examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of CdCl(2) for 0, 24, 48 and 96 h and serum and ovarian 17ß-estradiol levels were estimated. In the in vitro experiments, vitellogenic follicles were incubated with CdCl(2) and a dose- and time-dependent effects on steroid production were estimated induced by LH. Exposure of fish with CdCl(2) gradually attenuated serum and ovarian 17ß-estradiol levels with increasing time and maximum inhibition was noticed after 96 h. Administration of CdCl(2) to the incubations significantly inhibited LH-induced release of 17ß-estradiol in vitro. To clarify the mechanism of attenuated production of 17ß-estradiol, in vitro effects of CdCl(2) on LH induced P450 aromatase activity (conversion of testosterone to 17ß-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were evaluated. Results show that LH-stimulated P450 aromatase activity and P450arom gene expression in ovarian follicles were significantly inhibited by CdCl(2). The present study further demonstrated that LH-induced stimulation of ovarian steroidogenic factor-1 (SF-1) which activates aromatase enzyme, is strongly inhibited by cadmium chloride treatment.

Expression of LH receptor in nonpregnant mouse endometrium: LH induction of 3ß-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 3ß-hydroxy steroid dehydrogenase (3ß-hsd) mRNA and 3ß-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.