Multiple intersecting pathways are involved in CPEB1 phosphorylation and regulation of translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

Cellular senescence of granulosa cells in the pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, but its pathology has not been fully characterized and the optimal treatment strategy remains unclear. Cellular senescence is a permanent state of cell-cycle arrest that can be induced by multiple stresses. Senescent cells contribute to the pathogenesis of various diseases, owing to an alteration in secretory profile, termed 'senescence-associated secretory phenotype' (SASP), including with respect to pro-inflammatory cytokines. Senolytics, a class of drugs that selectively eliminate senescent cells, are now being used clinically, and a combination of dasatinib and quercetin (DQ) has been extensively used as a senolytic. We aimed to investigate whether cellular senescence is involved in the pathology of PCOS and whether DQ treatment has beneficial effects in patients with PCOS. We obtained ovaries from patients with or without PCOS, and established a mouse model of PCOS by injecting dehydroepiandrosterone. The expression of the senescence markers p16INK4a, p21, p53, γH2AX, and senescence-associated ß-galactosidase and the SASP-related factor interleukin-6 was significantly higher in the ovaries of patients with PCOS and PCOS mice than in controls. To evaluate the effects of hyperandrogenism and DQ on cellular senescence in vitro, we stimulated cultured human granulosa cells (GCs) with testosterone and treated them with DQ. The expression of markers of senescence and a SASP-related factor was increased by testosterone, and DQ reduced this increase. DQ reduced the expression of markers of senescence and a SASP-related factor in the ovaries of PCOS mice and improved their morphology. These results indicate that cellular senescence occurs in PCOS. Hyperandrogenism causes cellular senescence in GCs in PCOS, and senolytic treatment reduces the accumulation of senescent GCs and improves ovarian morphology under hyperandrogenism. Thus, DQ might represent a novel therapy for PCOS.

The Role of Cellular Senescence in Cyclophosphamide-Induced Primary Ovarian Insufficiency.

Young female cancer patients can develop chemotherapy-induced primary ovarian insufficiency (POI). Cyclophosphamide (Cy) is one of the most widely used chemotherapies and has the highest risk of damaging the ovaries. Recent studies elucidated the pivotal roles of cellular senescence, which is characterized by permanent cell growth arrest, in the pathologies of various diseases. Moreover, several promising senolytics, including dasatinib and quercetin (DQ), which remove senescent cells, are being developed. In the present study, we investigated whether cellular senescence is involved in Cy-induced POI and whether DQ treatment rescues Cy-induced ovarian damage. Expression of the cellular senescence markers p16, p21, p53, and γH2AX was upregulated in granulosa cells of POI mice and in human granulosa cells treated with Cy, which was abrogated by DQ treatment. The administration of Cy decreased the numbers of primordial and primary follicles, with a concomitant increase in the ratio of growing to dormant follicles, which was partially rescued by DQ. Moreover, DQ treatment significantly improved the response to ovulation induction and fertility in POI mice by extending reproductive life. Thus, cellular senescence plays critical roles in Cy-induced POI, and targeting senescent cells with senolytics, such as DQ, might be a promising strategy to protect against Cy-induced ovarian damage.

Temporal relationship between alterations in the gut microbiome and the development of polycystic ovary syndrome-like phenotypes in prenatally androgenized female mice.

It has been recently recognized that prenatal androgen exposure is involved in the development of polycystic ovary syndrome (PCOS) in adulthood. In addition, the gut microbiome in adult patients and rodents with PCOS differs from that of healthy individuals. Moreover, recent studies have suggested that the gut microbiome may play a causative role in the pathogenesis of PCOS. We wondered whether prenatal androgen exposure induces gut microbial dysbiosis early in life and is associated with the development of PCOS in later life. To test this hypothesis, we studied the development of PCOS-like phenotypes in prenatally androgenized (PNA) female mice and compared the gut microbiome of PNA and control offspring from 4 to 16 weeks of age. PNA offspring showed a reproductive phenotype from 6 weeks and a metabolic phenotype from 12 weeks of age. The α-diversity of the gut microbiome of the PNA group was higher at 8 weeks and lower at 12 and 16 weeks of age, and the ß-diversity differed from control at 8 weeks. However, a significant difference in the composition of gut microbiome between the PNA and control groups was already apparent at 4 weeks. Allobaculum and Roseburia were less abundant in PNA offspring, and may therefore be targets for future interventional studies. In conclusion, abnormalities in the gut microbiome appear as early as or even before PCOS-like phenotypes develop in PNA mice. Thus, the gut microbiome in early life is a potential target for the prevention of PCOS in later life.

The possible effects of the Japan Society of Clinical Oncology Clinical Practice Guidelines 2017 on the practice of fertility preservation in female cancer patients in Japan.

Purpose: In 2017, the first guidelines for fertility preservation in cancer patients were published in Japan. However, the impact of the guidelines remains unknown. Therefore, the authors conducted a nationwide survey on cryopreservation procedures in the period from shortly before to after publication of the guidelines (2016-2019) and compared the results with our previous survey (2011-2015). The authors also surveyed reproductive specialists' awareness of the guidelines and implementation problems. Methods: The authors sent a questionnaire to 618 assisted reproductive technology facilities certified by the Japanese Society of Obstetrics and Gynecology. Results: The authors received responses from 395 institutions (63.8%). Among them, 144 institutions conducted cryopreservation for cancer patients (vs. 126 in 2011-2015) and performed 2537 embryo or oocyte and 178 ovarian tissue cryopreservation procedures (vs. 1085 and 122, respectively). Compared with the previous period, indications were more varied and protocols for controlled ovarian stimulation were more standardized. Reproductive specialists' interest in oncofertility was high, but many reported three main difficulties: selecting a treatment method, storing samples in the long term, and securing the necessary human resources. Conclusions: The practice of fertility preservation in cancer patients in Japan has been considerably affected by the first Japanese guidelines.

Endoplasmic reticulum stress: a key regulator of the follicular microenvironment in the ovary.

Intra-ovarian local factors regulate the follicular microenvironment in coordination with gonadotrophins, thus playing a crucial role in ovarian physiology as well as pathological states such as polycystic ovary syndrome (PCOS). One recently recognized local factor is endoplasmic reticulum (ER) stress, which involves the accumulation of unfolded or misfolded proteins in the ER related to various physiological and pathological conditions that increase the demand for protein folding or attenuate the protein-folding capacity of the organelle. ER stress results in activation of several signal transduction cascades, collectively termed the unfolded protein response (UPR), which affect a wide variety of cellular functions. Recent studies have revealed diverse roles of ER stress in physiological and pathological conditions in the ovary. In this review, we summarize the most current knowledge of the regulatory roles of ER stress in the ovary, in the context of reproduction. The physiological roles of ER stress and the UPR in the ovary remain largely undetermined. On the contrary, activation of ER stress is known to impair follicular and oocyte health in various pathological conditions; moreover, ER stress also contributes to the pathogenesis of several ovarian diseases, including PCOS. Finally, we discuss the potential of ER stress as a novel therapeutic target. Inhibition of ER stress or UPR activation, by treatment with existing chemical chaperones, lifestyle intervention, or the development of small molecules that target the UPR, represents a promising therapeutic strategy.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Activation of endoplasmic reticulum stress mediates oxidative stress-induced apoptosis of granulosa cells in ovaries affected by endometrioma.

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress-induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

Accumulation of advanced glycation end products in follicles is associated with poor oocyte developmental competence.

Advanced glycation end products (AGEs) affect the follicular microenvironment. The close relationship between AGEs, proinflammatory cytokine production and activation of the unfolded protein response (UPR), which involves activating transcription factor 4 (ATF4), is crucial for regulation of various cellular functions. We examined whether accumulation of AGEs in follicles was associated with proinflammatory cytokine production and activation of the UPR in granulosa cells and decreased oocyte developmental competence. Concentrations of AGEs, soluble receptor for AGE (sRAGE), interleukin (IL)-6 and IL-8 in follicular fluid (FF) were examined by ELISAs in 50 follicles. mRNA expression of ATF4, IL-6 and IL-8 in cumulus cells (CCs) were examined by quantitative RT-PCR in 77 samples. Cultured human granulosa-lutein cells (GLCs) were treated with AGE-bovine serum albumin (BSA) alone or following transfection of ATF4-targeting small interfering RNA. The AGE concentration and the AGE/sRAGE ratio in FF were significantly higher in follicles containing oocytes that developed into poor-morphology embryos (group I) than those with good-morphology embryos (group II). When compared with sibling follicles from the same patients, the AGE/sRAGE and concentrations of IL-6 and IL-8 in FF, as well as ATF4, IL-6 and IL-8 mRNA expression in CCs, were significantly higher in group I follicles than group II. AGE treatment increased mRNA expression of ATF4, IL-6 and IL-8 in cultured GLCs. Knockdown of ATF4 abrogated the stimulatory effects of AGE on mRNA expression and protein secretion of IL-6 and IL-8. Our findings support the idea that accumulation of AGEs in follicles reduces oocyte competence by triggering inflammation via activation of ATF4 in the follicular microenvironment.

Role of oxidative stress in follicular fluid on embryos of patients undergoing assisted reproductive technology treatment.

AIM: Oxidative stress (OS) is defined as an imbalance between oxidants and antioxidants in favor of the oxidants, and the disruption of redox signaling leads to molecular damages. This study was conducted to clarify the effect of OS in individual follicular fluid (FF) on oocyte fertilization and embryonic division. METHODS: A total of 124 patients who underwent assisted reproductive technology treatment in our hospital underwent intracytoplasmic sperm injection and 211 FF were collected. The ova were fertilized, and embryos were individually cultured until cleavage stage or blastocyst stage and classified according to the Veeck classification system or Gardner classification system. RESULTS: The follicular fluid corresponding to the ovum was analyzed by measuring the OS marker, the reactive oxygen metabolites (d-ROM test), and the antioxidant marker (biological antioxidant potential marker [BAP] test), and the relation between these markers and clinical parameters was analyzed. The value of d-ROM was correlated with the proper fertilization status and formation of good quality cleavage embryo, whereas the elevated value of BAP was observed in better embryonic development group. Oxidative stress index, defined as d-ROM/BAP × 100 clearly indicated that lower oxidative stress index was associated with better fertilization status and embryo development. CONCLUSION: Our results clearly indicate that the balance between OS and antioxidant capacity in FF at the time of oocyte retrieval is possibly important in the processes of fertilization and embryo division; thus, we propose that oxidative status and balance in FF might be used as novel biomarkers in assisted reproductive technology.

A Japanese nationwide survey on the cryopreservation of embryos, oocytes and ovarian tissue for cancer patients.

AIM: The survival rates of cancer patients have greatly improved owing to the advances in oncology. The preservation of fertility in cancer patients is an important task. To determine the reality of cryopreservation of embryos, oocytes and ovarian tissue in cancer patients, large-scale survey analysis was performed in Japan. METHODS: We sent 613 Japan Society of Obstetrics and Gynecology-certified assisted reproductive technology institutions a questionnaire about their experience of performing cryopreservation for cancer patients between January 2011 and December 2015. Subsequently, the institutions that conducted cryopreservation for cancer patients were sent a second questionnaire. RESULTS: We received replies from 481 (78.5%) institutions. Among them, 126 (26.2%) conducted cryopreservation for cancer patients. These 126 institutions were sent a second questionnaire. Of these, 108 (85.7%) institutions responded. At the 108 institutions, 1085 embryo or oocyte cryopreservation procedures and 122 ovarian tissue cryopreservation procedures were conducted for cancer patients. Cryopreservation was mainly performed for breast cancer patients (~70%), followed by patients with hematological malignancy. A total of 361 and 19 embryo transfer cycles were performed for patients whose embryos and oocytes were cryopreserved, respectively, and 42 and seven institutions reported pregnancy outcomes after embryo transfer in patients that underwent embryo and oocyte cryopreservation, respectively. However, pregnancy was not observed in the seven cases that underwent ovarian tissue transfer. CONCLUSION: Indications, age limits and ovarian stimulation protocols for cryopreservation widely varied between the institutions. A national registration system for oncofertility must be established to evaluate the safety and efficacy of the current system.

Multiple intersecting pathways are involved in the phosphorylation of CPEB1 to activate translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in the regulation of mRNA translation in oocytes. However, the nature of protein kinase cascades modulating the activity of CPEB1 is still a matter of controversy. Using genetic and pharmacological tools and detailed time courses, here we have reevaluated the relationship between CPEB1 phosphorylation and the activation of translation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on the phosphorylation of CPEB1 during prometaphase. Only inactivation of the CDK1/MAPK pathway disrupts translation, while inactivation of either pathway leads to CPEB1 stabilization. However, stabilization of CPEB1 induced by inactivation of the AURKA/PLK1 does not affect translation, indicating that destabilization/degradation can be dissociated from translational activation. The accumulation of the endogenous CCNB1 protein closely recapitulates the translation data. These findings support the overarching hypothesis that the activation of translation in prometaphase in mouse oocytes relies on a CDK1-dependent CPEB1 phosphorylation, and this translational activation precedes CPEB1 destabilization.

Effects of the prenatal and postnatal nurturing environment on the phenotype and gut microbiota of mice with polycystic ovary syndrome induced by prenatal androgen exposure: a cross-fostering study.

The gut microbiome is implicated in the pathogenesis of polycystic ovary syndrome (PCOS), and prenatal androgen exposure is involved in the development of PCOS in later life. Our previous study of a mouse model of PCOS induced by prenatal dihydrotestosterone (DHT) exposure showed that the reproductive phenotype of PCOS appears from puberty, followed by the appearance of the metabolic phenotype after young adulthood, while changes in the gut microbiota was already apparent before puberty. To determine whether the prenatal or postnatal nurturing environment primarily contributes to these changes that characterize prenatally androgenized (PNA) offspring, we used a cross-fostering model to evaluate the effects of changes in the postnatal early-life environment of PNA offspring on the development of PCOS-like phenotypes and alterations in the gut microbiota in later life. Female PNA offspring fostered by normal dams (exposed to an abnormal prenatal environment only, fostered PNA) exhibited less marked PCOS-like phenotypes than PNA offspring, especially with respect to the metabolic phenotype. The gut microbiota of the fostered PNA offspring was similar to that of controls before adolescence, but differences between the fostered PNA and control groups became apparent after young adulthood. In conclusion, both prenatal androgen exposure and the postnatal early-life environment created by the DHT injection of mothers contribute to the development of PCOS-like phenotypes and the alterations in the gut microbiota that characterize PNA offspring. Thus, both the pre- and postnatal environments represent targets for the prevention of PCOS and the associated alteration in the gut microbiota in later life.

Roles of endoplasmic reticulum stress in the pathophysiology of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women, affecting up to 15% of women in this group, and the most common cause of anovulatory infertility. Although its etiology remains unclear, recent research has revealed the critical role of endoplasmic reticulum (ER) stress in the pathophysiology of PCOS. ER stress is defined as a condition in which unfolded or misfolded proteins accumulate in the ER because of an imbalance in the demand for protein folding and the protein-folding capacity of the ER. ER stress results in the activation of several signal transduction cascades, collectively termed the unfolded protein response (UPR), which regulates various cellular activities. In principle, the UPR restores homeostasis and keeps the cell alive. However, if the ER stress cannot be resolved, it induces programmed cell death. ER stress has recently been recognized to play diverse roles in both physiological and pathological conditions of the ovary. In this review, we summarize current knowledge of the roles of ER stress in the pathogenesis of PCOS. ER stress pathways are activated in the ovaries of both a mouse model of PCOS and in humans, and local hyperandrogenism in the follicular microenvironment associated with PCOS is responsible for activating these. The activation of ER stress contributes to the pathophysiology of PCOS through multiple effects in granulosa cells. Finally, we discuss the potential for ER stress to serve as a novel therapeutic target for PCOS.

Notch Signaling Induced by Endoplasmic Reticulum Stress Regulates Cumulus-Oocyte Complex Expansion in Polycystic Ovary Syndrome.

Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.

Inhibition of autophagy in theca cells induces CYP17A1 and PAI-1 expression via ROS/p38 and JNK signalling during the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a clinical syndrome characterized by hyperandrogenism, oligo/anovulation, and polycystic ovary. Autophagy is an intracellular system that degrades cytosolic proteins and organelles. The relationship between autophagy and PCOS has not been clarified. We found that p62 and ubiquitin were significantly increased in theca cells of women with PCOS using immunohistochemistry. Autophagy inhibition by palmitic acid and chloroquine in bovine theca cells increased p62 and ubiquitin and induced the expression of cytochrome P450 17A1 (CYP17A1) and plasminogen activator inhibitor-1 (PAI-1) mRNA. Furthermore, palmitic acid and chloroquine exposure significantly increased reactive oxygen species (ROS) and activated p38 and c-Jun N-terminal kinase (JNK). Inhibition of p38 and JNK significantly reduced CYP17A1 and PAI-1 mRNA expression. We showed that inhibition of autophagy in theca cells may have contributed to the pathogenesis of PCOS, based on CYP17A1 and PAI-1 mRNA expression via the ROS/p38 and JNK signalling pathways.

Androgens Increase Accumulation of Advanced Glycation End Products in Granulosa Cells by Activating ER Stress in PCOS.

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, and we previously found that androgens activate endoplasmic reticulum (ER) stress in granulosa cells from patients with PCOS. In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to the pathology. Therefore, we hypothesized that androgens upregulate the receptor for AGEs (RAGE) expression in granulosa cells by activating ER stress, thereby increasing the accumulation of AGEs in these cells and contributing to the pathology. In the present study, we show that testosterone increases RAGE expression and AGE accumulation in cultured human granulosa-lutein cells (GLCs), and this is reduced by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response factor activated by ER stress, inhibits testosterone-induced RAGE expression and AGE accumulation. The expression of RAGE and the accumulation of AGEs are upregulated in granulosa cells from PCOS patients and dehydroepiandrosterone-induced PCOS mice. Administration of the RAGE inhibitor FPS-ZM1 or TUDCA to PCOS mice reduces RAGE expression and AGE accumulation in granulosa cells, improves their estrous cycle, and reduces the number of atretic antral follicles. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress, and that targeting the AGE-RAGE system, either by using a RAGE inhibitor or a clinically available ER stress inhibitor, may represent a novel approach to PCOS therapy.

Endoplasmic Reticulum Stress Activated by Androgen Enhances Apoptosis of Granulosa Cells via Induction of Death Receptor 5 in PCOS.

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and growth arrest of antral follicles. Previously, we found that endoplasmic reticulum (ER) stress is activated in granulosa cells of antral follicles in PCOS, evidenced by activation of unfolded protein response (UPR) genes. Based on this observation, we hypothesized that ER stress is activated by androgens in granulosa cells of antral follicles, and that activated ER stress promotes apoptosis via induction of the UPR transcription factor C/EBP homologous protein (CHOP) and subsequent activation of death receptor (DR) 5. In this study, we found that testosterone induced expression of various UPR genes, including CHOP, as well as DR5, in cultured human granulosa-lutein cells (GLCs). Pretreatment with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) inhibited testosterone-induced apoptosis and expression of DR5 and CHOP. Knockdown of CHOP inhibited testosterone-induced DR5 expression and apoptosis, and knockdown of DR5 inhibited testosterone-induced apoptosis. Pretreatment with flutamide, as well as knockdown of androgen receptor, decreased testosterone-induced DR5 and CHOP expression, as well as apoptosis. Expression of DR5 and CHOP was upregulated in GLCs obtained from patients with PCOS, as well as in granulosa cells of antral follicles in ovarian sections obtained from patients with PCOS and dehydroepiandrosterone-induced PCOS mice. Treatment of PCOS mice with TUDCA decreased apoptosis and DR5 expression in granulosa cells of antral follicles, with a concomitant reduction in CHOP expression. Taken together, our findings indicate that ER stress activated by hyperandrogenism in PCOS promotes apoptosis of granulosa cells of antral follicles via induction of DR5.

Activation of Endoplasmic Reticulum Stress in Granulosa Cells from Patients with Polycystic Ovary Syndrome Contributes to Ovarian Fibrosis.

Recent studies report the involvement of intra-ovarian factors, such as inflammation and oxidative stress, in the pathophysiology of polycystic ovary syndrome (PCOS), the most common endocrine disorder of reproductive age women. Endoplasmic reticulum (ER) stress is a local factor that affects various cellular events during a broad spectrum of physiological and pathological conditions. It may also be an important determinant of pro-fibrotic remodeling during tissue fibrosis. In the present study, we showed that ER stress was activated in granulosa cells of PCOS patients as well as in a well-established PCOS mouse model. Pharmacological inducers of ER stress, tunicamycin and thapsigargin, were found to increase the expression of pro-fibrotic growth factors, including transforming growth factor (TGF)-ß1, in human granulosa cells, and their expression also increased in granulosa cells of PCOS patients. By contrast, treatment of PCOS mice with an ER stress inhibitor, tauroursodeoxycholic acid or BGP-15, decreased interstitial fibrosis and collagen deposition in ovaries, accompanied by a reduction in TGF-ß1 expression in granulosa cells. These findings suggest that ER stress in granulosa cells of women with PCOS contributes to the induction of pro-fibrotic growth factors during ovarian fibrosis, and that ER stress may serve as a therapeutic target in PCOS.