Unraveling Hypothalamus-Pituitary dysregulation: Hypergonadotropism in F1 progeny due to prenatal exposure to hexavalent chromium.

The endocrine disruptor hexavalent chromium [Cr(VI)] is a proven reproductive toxicant. We recently demonstrated that prenatal Cr(VI) exposure causes testicular resistance to gonadotropins, resulting in hypergonadotropic hypoandrogenism in F1 rats. However, the mechanism driving hypergonadotropism in F1 rats exposed to Cr(VI) prenatally remains an enigma. Therefore, we hypothesized that 'Prenatal Cr(VI) exposure may disrupt steroid hormones-mediated negative feedback regulation of the hypothalamic GnRH, and its receptor in the pituitary of F1 rats, leading to hypergonadotropism.' We administered potassium dichromate (50, 100, or 200 mg/L) to pregnant rats through drinking water between days 9 and 14, and their male F1 offspring were euthanized at 60 days of age. Prenatal Cr(VI) exposure in F1 rats resulted in the accumulation of Cr in the hypothalamus and pituitary. Western blot detected decreased hypothalamic GnRH, Kisspeptin1, and its receptor GPR54, along with diminished ERα, AR, aromatase, and 5α reductase, and GnRH regulatory transcription factors Pit-1 and GATA-4 proteins. Immunohistochemical studies revealed increased immunopositivity of GnRH receptor, AR, 5α reductase, ERα, ERß, and aromatase proteins in the pituitary, whereas decreased Kisspeptin1, GPR54, and inhibin ß. Our findings imply that Cr(VI) exposure during the prenatal period disrupts the hypothalamic Kisspeptin-GPR54-Pit-1/GATA4-GnRH network, boosting the pituitary GnRH receptor. We conclude that prenatal exposure to Cr(VI) alters GnRH expression in the hypothalamus and its receptor in the pituitary of F1 progeny through interfering with the negative feedback effect of androgens and estrogens.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Endocrine disruptors and endometriosis.

Endometriosis is a hormone-dependent inflammatory gynecological disease of reproductive-age women. It is clinically and pathologically characterized by the presence of functional endometrium as heterogeneous lesions outside the uterine cavity. The two major symptoms are chronic pelvic pain and infertility, which profoundly affect women's reproductive health and quality of life. This significant individual and public health concerns underscore the importance of understanding the pathogenesis of endometriosis. The environmental endocrine-disrupting chemicals (EDCs) are exogenous agents that interfere with the synthesis, secretion, transport, signaling, or metabolism of hormones responsible for homeostasis, reproduction, and developmental processes. Endometriosis has been potentially linked to exposure to EDCs. In this review, based on the robust literature search, we have selected four endocrine disruptors (i) polychlorinated biphenyls (PCB)s (ii) dioxins (TCDD) (iii) bisphenol A (BPA) and its analogs and (iv) phthalates to elucidate their critical role in the etiopathogenesis of endometriosis. The epidemiological and experimental data discussed in this review indicate that these four EDCs activate multiple intracellular signaling pathways associated with proinflammation, estrogen, progesterone, prostaglandins, cell survival, apoptosis, migration, invasion, and growth of endometriosis. The available information strongly indicates that environmental exposure to EDCs such as PCBs, dioxins, BPA, and phthalates individually or collectively contribute to the pathophysiology of endometriosis. Further understanding of the molecular mechanisms of how these EDCs establish endometriosis and therapeutic strategies to mitigate the effects of these EDCs in the pathogenesis of endometriosis are timely needed. Moreover, understanding the interactive roles of these EDCs in the pathogenesis of endometriosis will help regulate the exposure to these EDCs in reproductive age women.

Exposure to hexavalent chromium causes infertility by disrupting cytoskeletal machinery and mitochondrial function of the metaphase II oocytes in superovulated rats.

Previous studies from our laboratory showed that prenatal exposure to hexavalent chromium, Cr(VI), caused premature ovarian failure and decreased pregnancy rates and litter size. Exposure to the endocrine disrupting chemicals (EDCs) can cause X-chromosome aneuploidy of the oocytes, increasing chromosome missegregation and risk of infertility, autoimmune diseases, cancers, and various genetic disorders. Cr(VI) is an EDC that is widely used in numerous industries. Environmental exposure to Cr(VI) caused detrimental reproductive effects in women and health effects in infants from California. Women with occupational Cr(VI) exposure experienced infertility, pregnancy loss, spontaneous abortion, and stillbirth. However, the adverse effects of Cr(VI) on oocyte development and quality have not been reported. Mitochondrial membrane potential and function are the critical determinants of oocyte quality in natural pregnancies and successful assisted reproductive techniques. The cytoskeletal machinery of the oocytes orchestrates the meiotic division of the oocytes, whereas cortical granules (CGs) prevent polyspermy. Therefore, the objective of the current study was to examine whether the mechanism by which Cr(VI) compromises oocyte quality and morphology is by altering cytoskeleton dynamics and mitochondrial function of the metaphase II (MII) oocytes. Rats were treated with environmentally relevant doses of Cr(VI) (1 and 5 ppm potassium dichromate) in drinking water from postnatal day (PND) 22-28, followed by superovulation and retrieval of MII oocytes. The data indicate that Cr(VI) exposure disrupted F-actin structure and distribution pattern, compromised mitochondrial function, altered CGs distribution, increased dysmorphic and degenerated oocytes, delayed first polar body extrusion, and caused infertility.

Effects of selective inhibition of prostaglandin E2 receptors EP2 and EP4 on the miRNA profile in endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant, chronic inflammatory gynecological disease of reproductive-age women. Two major clinical symptoms of endometriosis are chronic pelvic pain and infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent disease recurrence. Prostaglandin E2 (PGE2) plays an important role in the survival and growth of endometriotic lesions. MicroRNAs (miRNAs) are small, noncoding RNAs that control gene expressions through multiple mechanisms and have important roles in the pathogenesis of endometriosis. The objective of the present study is to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on miRNA profile in endometriosis. The novel results collectively indicate that inhibition of PGE2-EP2/EP4 signaling regulated several miRNA clusters associated with cell adhesion, migration, invasion, survival and growth in cell-specific and the chromosome-specific manner and reverses the epigenetic silencing of proapoptotic miRNAs 15a and 34c in the human endometriotic epithelial and stromal cells and experimental endometriotic lesions. Thus, selective inhibition of EP2/EP4 receptors could emerge as a potential nonsteroidal therapy for endometriosis.

Inhibition of Sirtuin-1 hyperacetylates p53 and abrogates Sirtuin-1-p53 interaction in Cr(VI)-induced apoptosis in the ovary.

Environmental contamination with hexavalent chromium, Cr(VI), has been increasing in the United States as well as in developing countries. Exposure to Cr(VI) predisposes the human population to various diseases, including cancer, infertility, and developmental problems in children. Previous findings from our laboratory reported that prenatal exposure to Cr(VI) caused premature ovarian failure through p53-mediated mechanisms. Sirtuin 1 (SIRT1) is an NAD+ -dependent histone deacetylase class III. SIRT1 deacetylates several histones and non-histone proteins such as p53 and NFkB. The current study determines a role for the SIRT1-p53 network in apoptosis induced by Cr(VI) in the ovary and establishes physical interaction between SIRT1 and p53. Adult pregnant dams were given regular drinking water or Cr(VI) (10 ppm potassium dichromate in drinking water, ad libitum), and treated with SIRT1 inhibitor, EX-527 (50 mg/kg body weight, i.p.,), during 9.5 - 14.5 days post-coitum. On postnatal day-1, ovaries from F1 offspring were collected for various analyses. Results indicated that Cr(VI) increased germ cell and somatic cell apoptosis, upregulated acetyl-p53, activated the apoptotic pathway, and inhibited cell survival pathways. Cr(VI) decreased acetyl-p53-SIRT1 co-localization in the ovary. In an immortalized rat granulosa cell line SIGC, Cr(VI) inhibited the physical interaction between SIRT1 and acetyl-p53 by altering the p53:SIRT1 ratio. EX-527 exacerbated Cr(VI)-induced mechanisms. The current study shows a novel mechanism for Cr(VI)-induced apoptosis in the ovary, mediated through the p53-SIRT1 network, suggesting that targeting the p53 pathway may be an ideal approach to rescue ovaries from Cr(VI)-induced apoptosis.

Effects of dual inhibition of AKT and ERK1/2 pathways on endometrial pro-inflammatory, hormonal, and epigenetic microenvironment in endometriosis.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. The prevalence of endometriosis is ~5-10% in reproductive-age women, increasing to 20-30% in women with subfertility. The current anti-estrogen therapies can be prescribed only for a short time because of the undesirable side effects on menstruation, pregnancy, bone health, and failure to prevent a recurrence. The causes of endometriosis-associated infertility are multifactorial and poorly understood. The objective of the present study was to determine the inhibitory effects of AKT and/or ERK1/2 pathways on the microenvironment of the endometrium in a xenograft mouse model of endometriosis of human origin. Results indicate that dual inhibition of AKT and ERK1/2 pathways, but not inhibition of either AKT or ERK1/2 pathway, suppresses the growth of the endometriotic lesions in vivo. Dual inhibition of AKT and ERK1/2 pathways suppresses the production of proinflammatory cytokines, decreases E2 biosynthesis and signaling, and restores progesterone receptor-B signaling components in the epithelial and stromal cells of the endometrium in a cell-specific manner. These results together suggest that dual inhibition of AKT and ERK1/2 pathways suppresses the estrogen-dominant state and concomitantly increases the progesterone-responsive state of the endometrium. Therefore, dual inhibition of AKT and ERK1/2 pathways could emerge as long-term nonsteroidal therapy for endometriosis.

Prenatal exposure to hexavalent chromium disrupts testicular steroidogenic pathway in peripubertal F1 rats.

We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis "transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs)." Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9-14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17ß estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3ß- and 17ß-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3ß and 17ß hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.

Dual inhibition of ERK1/2 and AKT pathways is required to suppress the growth and survival of endometriotic cells and lesions.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Current hormonal therapies targeting estrogen can be prescribed only for a short time. It indicates a need for non-hormonal therapy. ERK1/2 and AKT pathways control several intracellular signaling molecules that control growth and survival of cells. Objectives of the present study are to determine the dual inhibitory effects of ERK1/2 and AKT pathways: (i) on proliferation, survival, and apoptosis of human endometrioitc epithelial cells and stromal cells in vitro; (ii) on growth and survival of endometrioitc lesions in vivo in xenograft mouse model of endometriosis of human origin; and (iii) establish the associated ERK1/2 and AKT downstream intracellular signaling modules in the pathogenesis of endometriosis. Our results indicated that combined inhibition of ERK1/2 and AKT pathways highly decreased the growth and survival of human endometriotic epithelial cells and stromal cells in vitro and suppressed the growth of endometriotic lesions in vivo compared to inhibition of either ERK1/2 or AKT pathway individually. This cause-effect is associated with dysregulated intracellular signaling modules associated with cell cycle, cell survival, and cell apoptosis pathways. Collectively, our results indicate that dual inhibition of ERK1/2 and AKT pathways could emerge as potential non-hormonal therapy for the treatment of endometriosis.

Octyl gallate and gallic acid isolated from Terminalia bellarica regulates normal cell cycle in human breast cancer cell lines.

Herbal medicines stand unique and effective in treating human diseases. Terminalia bellarica (T. bellarica) is a potent medicinal herb, with a wide range of pharmacological activities. The present study was aimed to evaluate the effect of octyl gallate (OG) and gallic acid (GA) isolated from methanolic fruit extract of T. bellirica to inhibit the survival of breast cancer cells (MCF-7 & MDA-MB-231). Both OG & GA exhibited decreased MCF-7 & MDA-MB-231 survival and induced apoptosis, with IC50 value of OG and GA as 40 µM and 80 µM respectively. No toxic effect was observed on normal breast cells (MCF-10A). The compounds inhibited cell cycle progression by altering the expression of the cell cycle regulators (Cyclin D1, D3, CDK-4, CDK-6, p18 INK4, p21Waf-1 and p27 KIP). Octyl gallate was more effective at low concentrations than GA. In-silico results provided stable interactions between the compounds and target proteins. The present investigation proved the downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators inducing apoptosis in compound-treated breast cancer cells. Hence, both the compounds may serve as potential anticancer agents and could be developed as breast cancer drugs, with further explorations.

Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants.

In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants.

Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol.

Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in >50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50ppm potassium dichromate) from postpartum days 1-21 through drinking water with or without RVT (10mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E2) biosynthesis and enhancing metabolic clearance of E2, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E2. This is the first study to report the protective effects of RVT against any toxicant in the ovary.

A fetal whole ovarian culture model for the evaluation of CrVI-induced developmental toxicity during germ cell nest breakdown.

Prenatal exposure to endocrine disrupting chemicals (EDCs), including bisphenol A, dioxin, pesticides, and cigarette smoke, has been linked to several ovarian diseases such as premature ovarian failure (POF) and early menopause in women. Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries. As one of the world's leading producers of Cr compounds, the U.S. is facing growing challenges in protecting human health against adverse effects of CrVI. Our recent findings demonstrated that in vivo CrVI exposure during gestational period caused POF in F1 offspring. Our current research focus is three-fold: (i) to identify the effect of CrVI on critical windows of great vulnerability of fetal ovarian development; (ii) to understand the molecular mechanism of CrVI-induced POF; (iii) to identify potential intervention strategies to mitigate or inhibit CrVI effects. In order to accomplish these goals we used a fetal whole ovarian culture system. Fetuses were removed from the normal pregnant rats on gestational day 13.5. Fetal ovaries were cultured in vitro for 12 days, and treated with or without 0.1 ppm potassium dichromate (CrVI) from culture day 2-8, which recapitulated embryonic day 14.5-20.5, in vivo. Results showed that CrVI increased germ cell/oocyte apoptosis by increasing caspase 3, BAX, p53 and PUMA; decreasing BCL2, BMP15, GDF9 and cKIT; and altering cell cycle regulatory genes and proteins. This model system may serve as a potential tool for high throughput testing of various drugs and/or EDCs in particular to assess developmental toxicity of the ovary.

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis.

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 modulates DNA methylation and histone modification machinery proteins in human endometriotic cells.

Endometriosis is an inflammatory gynecological disease of reproductive-age women. The prevalence of endometriosis is 5-10% in reproductive-age women. Modern medical treatments are directed to inhibit the action of estrogen in endometriotic cells. However, hormonal therapies targeting estrogen can be prescribed only for a short time because of their undesirable side effects. Recent studies from our laboratory, using human endometriotic epithelial cell line 12Z and stromal cell line 22B derived from red lesion, discovered that selective inhibition of prostaglandin E2 (PGE2) receptors EP2 and EP4 inhibits adhesion, invasion, growth, and survival of 12Z and 22B cells by modulating integrins, MMPs and TIMPs, cell cycle, survival, and intrinsic apoptotic pathways, suggesting multiple epigenetic mechanisms. The novel findings of the present study indicate that selective pharmacological inhibition of EP2 and EP4: (i) decreases expression of DNMT3a, DNMT3b, H3K9me3, H3K27me3, SUV39H1, HP1a, H3K27, EZH2, JMJD2a, HDAC1, HDAC3, MeCP2, CoREST and Sin3A; (ii) increases expression of H3K4me3, H3H9ac, H3K27ac; and (iii) does not modulate the expression of DNMT1, hSET1, LSD1, MBD1, p300, HDAC2, and JMJD3 epigenetic machinery proteins in an epithelial and stromal cell specific manner. In this study, we report for the first time that inhibition of PGE2-EP2/EP4 signaling modulates DNA methylation, H3 histone methylation and acetylation, and epigenetic memory machinery proteins in human endometriotic epithelial cells and stromal cells. Thus, targeting EP2 and EP4 receptors may emerge as long-term nonsteroidal therapy for treatment of active endometriotic lesions in women.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Edaravone mitigates hexavalent chromium-induced oxidative stress and depletion of antioxidant enzymes while estrogen restores antioxidant enzymes in the rat ovary in F1 offspring.

Environmental contamination of drinking water with chromium (Cr) has been increasing in more than 30 cities in the United States. Previous studies from our group have shown that Cr affects reproductive functions in female Sprague Dawley rats. Although it is impossible to completely remove Cr from the drinking water, it is imperative to develop effective intervention strategies to inhibit Cr-induced deleterious health effects. Edaravone (EDA), a potential inhibitor of free radicals, has been clinically used to treat cancer and cardiac ischemia. This study evaluated the efficacy of EDA against Cr-induced ovarian toxicity. Results showed that maternal exposure to CrVI in rats increased follicular atresia, decreased steroidogenesis, and delayed puberty in F1 offspring. CrVI increased oxidative stress and decreased antioxidant (AOX) enzyme levels in the ovary. CrVI increased follicle atresia by increased expression of cleaved caspase 3, and decreased expression of Bcl2 and Bcl2l1 in the ovary. EDA mitigated or inhibited the effects of CrVI on follicle atresia, pubertal onset, steroid hormone levels, and AOX enzyme activity, as well as the expression of Bcl2 and Bcl2l1 in the ovary. In a second study, CrVI treatment was withdrawn, and F1 rats were injected with estradiol (E2) (10 µg in PBS/ethanol per 100 g body weight) for a period of 2 wk to evaluate whether E2 treatment will restore Cr-induced depletion of AOX enzymes. E2 restored CrVI-induced depletion of glutathione peroxidase 1, catalase, thioredoxin 2, and peroxiredoxin 3 in the ovary. This is the first study to demonstrate the protective effects of EDA against any toxicant in the ovary.

Intrauterine coadministration of ERK1/2 inhibitor U0126 inhibits interferon TAU action in the endometrium and restores luteolytic PGF2alpha pulses in sheep.

In ruminants, prostaglandin F2 alpha (PGF2alpha) is synthesized and released in a pulsatile pattern from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau (IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts locally on endometrial LE cells to inhibit pulsatile releases of PGF2alpha and thus establish an endocrine environment for recognition of pregnancy. Cell signaling pathways through which IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely unknown. Results of the present investigation indicate that intrauterine administration of IFNT inhibits pulsatile release of PGF2alpha, while coadministration IFNT and ERK 1/2 inhibitor U0126 restores luteolytic PGF2alpha pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2 pathways inhibits IFNT action, decreases pERK1/2 and PGT protein interactions, and re-establishes the spatial expression of the oxytocin receptor protein completely and the estrogen receptor protein partially without modulating the expression of interferon regulatory factor-2 (IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling and the molecular endocrine control of PGF2alpha release at the time of establishment of pregnancy in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies to understand molecular and cellular mechanisms of IFNT action in endometrial LE in ruminants.

Prenatal exposure to chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing germ cell cyst breakdown in the F1 offspring.

Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries such as chrome plating, welding, wood processing and tanneries. As one of the world's leading producers of chromium compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. CrVI is rapidly converted to CrIII intracellularly, and can induce apoptosis through different mechanisms. Our previous studies demonstrated postnatal exposure to CrVI results in a delay or arrest in follicle development and puberty. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD 20, and total Cr was estimated in the placentae; ovaries were removed from the F1 offspring on postnatal day (PND)-1 and various analyses were performed. Our results show that gestational exposure to CrVI resulted in (i) increased Cr concentration in the placenta, (ii) increased germ cell apoptosis by up-regulating p53/p27-Bax-caspase-3 proteins and by increasing p53-SOD-2 co-localization; (iii) accelerated germ cell cyst (GCC) breakdown; (iv) advanced primordial follicle assembly and primary follicle transition and (v) down regulation of p-AKT, p-ERK and XIAP. As a result of the above events, CrVI induced early reproductive senescence and decrease in litter size in F1 female progeny.

PPARγ activation inhibits growth and survival of human endometriotic cells by suppressing estrogen biosynthesis and PGE2 signaling.

Endometriosis is a chronic inflammatory disease of reproductive age women leading to chronic pelvic pain and infertility. Current antiestrogen therapies are temporizing measures, and endometriosis often recurs. Potential nonestrogenic or nonsteroidal targets are needed for treating endometriosis. Peroxisome proliferator-activated receptor (PPAR)γ, a nuclear receptor, is activated by thiazolidinediones (TZDs). In experimental endometriosis, TZDs inhibit growth of endometriosis. Clinical data suggest potential use of TZDs for treating pain and fertility concurrently in endometriosis patients. Study objectives were to 1) determine the effects of PPARγ action on growth and survival of human endometriotic epithelial and stromal cells and 2) identify the underlying molecular links between PPARγ activation and cell cycle regulation, apoptosis, estrogen biosynthesis, and prostaglandin E2 biosynthesis and signaling in human endometriotic epithelial and stromal cells. Results indicate that activation of PPARγ by TZD ciglitazone 1) inhibits growth of endometriotic epithelial cells 12Z up to 35% and growth of endometriotic stromal cells 22B up to 70% through altered cell cycle regulation and intrinsic apoptosis, 2) decreases expression of PGE2 receptors (EP)2 and EP4 mRNAs in 12Z and 22B cells, and 3) inhibits expression and function of P450 aromatase mRNA and protein and estrone production in 12Z and 22B cells through EP2 and EP4 in a stromal-epithelial cell-specific manner. Collectively, these results indicate that PGE2 receptors EP2 and EP4 mediate actions of PPARγ by incorporating multiple cell signaling pathways. Activation of PPARγ combined with inhibition of EP2 and EP4 may emerge as novel nonsteroidal therapeutic targets for endometriosis-associated pain and infertility, if clinically proven safe and efficacious.