Dual inhibition of AKT and ERK1/2 pathways restores the expression of progesterone Receptor-B in endometriotic lesions through epigenetic mechanisms.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Progesterone resistance, loss of progesterone receptor -B (PR-B) in the stromal cells of the endometrium, is one of the hallmarks of endometriosis and a major contributing factor for infertility in endometriosis patients. Loss of PR-B in the stromal cells of the endometriotic lesions poses resistance to the success of progesterone-based therapy. The working hypothesis is that PR-B is hypermethylated and epigenetically silenced, and inhibition of AKT and ERK1/2 pathways will decrease the hypermethylation, reverse the epigenetic silencing, and restore the expression of PR-B via DNA methylation and histone modification mechanisms in the endometriotic lesions. The objectives are to (i) determine the effects of dual inhibition of AKT and ERK1/2 pathways on the expression of PR-B and DNA methylation and histone modification protein machinery in the endometriotic lesions and (ii) identify the underlying epigenetic mechanisms of PR-B restoration in the endometriotic lesions. The results indicate that dual inhibition of AKT and ERK1/2 pathways decreases the hypermethylation, reverses the epigenetic silencing, and restores the expression of PR-B via DNA methylation and H3K9 and H3K27 methylation mechanisms in the endometriotic lesions or endometriotic stromal cells of human origin. These results support the novel concept that restored expression of PR-B in the endometriotic lesions and endometrium may improve the clinical outcome of progesterone therapy in endometriosis patients.

Alteration of epigenetic methyl and acetyl marks by postnatal chromium(VI) exposure causes apoptotic changes in the ovary of the F1 offspring.

Hexavalent chromium, Cr(VI), is a heavy metal endocrine disruptor used widely in various industries worldwide and is considered a reproductive toxicant. Our previous studies demonstrated that lactational exposure to Cr(VI) caused follicular atresia, disrupted steroid hormone biosynthesis and signaling, and delayed puberty. However, the underlying mechanism was unknown. The current study investigated the effects of Cr(VI) exposure (25 ppm) during postnatal days 1-21 via dam's milk on epigenetic alterations in the ovary of F1 offspring. Data indicated that Cr(VI) disrupted follicle development and caused apoptosis by increasing DNMT3a /3b and histone methyl marks (H3K27me3 and H3K9me3) along with decreasing histone acetylation marks (H3K9ac and H3K27ac). Our study demonstrates that exposure to Cr(VI) causes changes in the epigenetic marks, partially contributing to the transcriptional repression of genes regulating ovarian development, cell proliferation (PCNA), cell survival (BCL-XL and BCL-2), and activation of genes regulating apoptosis (AIF and cleaved caspase-3), resulting in follicular atresia. The current study suggests a role for epigenetics in Cr(VI)-induced ovotoxicity and infertility.

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.

Ex Vivo Fetal Whole Ovarian Culture Model: An Essential Tool for Studies in Reproductive Toxicology and Pharmacology.

Major limitations in understanding the direct effects of endocrine-disrupting chemicals (EDCs) and cell signaling events in ovarian cellular dynamics in mammals include a lack of proper and simple tools/techniques as well as gaps in knowledge regarding the critical window(s) of vulnerability. Identifying and validating such tools and evaluating the effects of EDCs on molecular dynamics and cellular events during the critical windows of ovarian development are very important to improve the fertility in women and preserve the future health of the developing fetuses. Therefore, we developed a fetal whole ovarian ex vivo culture model. Ex vivo ovary culture models allow varying culture parameters in a highly controlled manner and thus have the potential to allow a more thorough evaluation for reproductive toxicity studies and drug response. This chapter describes clear and thorough details for setting up and maintaining an ex vivo culture system from the rat ovaries and further analyses of mRNA and protein expressions and estimating follicle numbers.

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.

Sexually Dimorphic Impact of Chromium Accumulation on Human Placental Oxidative Stress and Apoptosis.

Environmental contamination with hexavalent chromium (CrVI) is a growing problem both in the United States and developing countries. Hexavalent chromium is widely used in numerous industries. Environmental exposure to CrVI adversely affects pregnancy outcomes and subsequent health of 2 generations, resulting in higher pregnancy loss, spontaneous abortion and low birth rate. Pregnant women exposed to CrVI through occupational settings experience increased risk of spontaneous abortion, stillbirth, preterm birth, and neonatal death. Children of the CrVI exposed women experience respiratory problems, perinatal jaundice, and increased birth defects. Because placental dysfunction may have a role in such adverse pregnancy outcome, we tested the hypothesis that environmental Cr exposure in pregnant women results in Cr accumulation in the human placenta, which could increase placental oxidative stress by disrupting antioxidant machinery and inducing apoptosis. Studies using frozen, deidentified human term placenta samples indicated that: (1) Cr accumulates in human term placenta tissues and (2) increase in Cr accumulation is positively correlated with oxidative stress and apoptotic markers, and altered antioxidants levels. Interestingly, there was a sexual dimorphism in the correlation between Cr accumulation and oxidative stress, and expression of apoptotic and antioxidant markers. Mechanistic in vitro studies using human trophoblast cells BeWo confirmed the detrimental effects of Cr in altering antioxidant genes. For the first time, this study provides evidence in support of a positive correlation between Cr accumulation in the human placenta and accelerated oxidative stress, with a gender bias toward the male sex.

Editor's Highlight: Exposure to CrVI during Early Pregnancy Increases Oxidative Stress and Disrupts the Expression of Antioxidant Proteins in Placental Compartments.

Epidemiologic studies document relationships between chromium VI (CrVI) exposure and increased risk of spontaneous abortion, stillbirth, preterm birth, and neonatal death in pregnant women. Environmental contamination with CrVI is a growing problem both in the United States and developing countries. CrVI is widely used in numerous industries. This study was designed to understand the mechanism of CrVI toxicity on placental oxidative stress and antioxidant (AOX) machinery. Pregnant mother rats were treated with or without CrVI (50 ppm K2Cr2O7) through drinking water from gestational day (GD) 9.5-14.5, and placentas were analyzed on GD 18.5. Results indicated that CrVI reduced the trophoblast cell population. CrVI increased reactive oxygen species (ROS) and decreased the expression of AOX proteins. CrVI disrupts the trophoblast proliferation of the placenta. This study provides insight into the critical role of AOXs in placental function.

Chromium VI - Induced developmental toxicity of placenta is mediated through spatiotemporal dysregulation of cell survival and apoptotic proteins.

Environmental contamination with hexavalent chromium (CrVI) is a growing problem both in the U.S and developing countries. CrVI is a heavy-metal endocrine disruptor; women working in Cr industries exhibit an increased incidence of premature abortion and infertility. The current study was designed to understand the mechanism of CrVI toxicity on placental cell survival/death pathways. Pregnant mothers were treated with or without CrVI (50ppmK2Cr2O7) through drinking water from gestational day (GD) 9.5-14.5, and placentas were analyzed on GD 18.5. Results indicated that CrVI increased apoptosis of trophoblasts, vascular endothelium of the metrial glands and yolk sac epithelium through caspase-3 and p53-dependent pathways. CrVI increased apoptosis in labyrinth and basal zones in a caspase-3-independent manner via AIF, and through an ATM-p53-NOXA-PUMA-p27 network. CrVI downregulated cell survival proteins Bcl-2, Bcl-XL and XIAP in the placenta. CrVI disrupts placental histoarchitecture and increases cell death by spatiotemporal modulation of apoptotic signaling.

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.

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.

Early pregnancy modulates survival and apoptosis pathways in the corpus luteum in sheep.

The corpus luteum (CL) is a transient endocrine gland. Functional and structural demise of the CL allows a new estrous cycle. On the other hand, survival of CL and its secretion of progesterone are required for the establishment of pregnancy. Survival or apoptosis of the luteal cells is precisely controlled by interactions between survival and apoptosis pathways. Regulation of these cell signaling components during natural luteolysis and establishment of pregnancy is largely unknown in ruminants. The objective of the present study was to determine the regulation of survival and apoptosis signaling protein machinery in the CL on days 12, 14, and 16 of the estrous cycle and pregnancy in sheep. Results indicate that: i) expressions of p-ERK1/2, p-AKT, ß-catenin, NFκB -p65, -p50, -p52, p-Src, p-ß -arrestin, p-GSK3ß, X-linked inhibitor of apoptosis protein (XIAP), and p-CREB proteins are suppressed during natural luteolysis; in contrast, their expressions are sustained or increased during establishment of pregnancy; ii) expressions of cleaved caspase-3, apoptosis inducing factor (AIF), c-Fos, c-Jun, and EGR-1 proteins are increased during natural luteolysis; in contrast, their expressions are decreased during establishment of pregnancy; and iii) expressions of Bcl-2, Bcl-XL, Bad, and Bax proteins are not modulated during natural luteolysis while expressions of Bcl2 and Bcl-XL proteins are increased during establishment of pregnancy in sheep. These proteomic changes are evident in both large and small luteal cells. These results together indicate that regression of the CL during natural luteolysis or survival of the CL during establishment of pregnancy is precisely controlled by distinct programmed suppression or activation of intraluteal cell survival and apoptosis pathways in sheep/ruminants.

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.