BackgroundThe ovulatory dysfunction mechanisms underlying polycystic ovary syndrome (PCOS) are not completely understood. And the roles of EPHA7 and EPHA7-regulated pathway factors in the pathogenesis of anovulation remain to be elucidated.MethodsWe used human granulosa cells (hGCs) of PCOS and non-PCOS patients to measure EPHA7 and other target gene expressions. We performed in vitro experiments in KGN cells to verify the molecular mechanisms. Additionally, we conducted in vivo loss- and gain-of-function studies using EPHA7 shRNA lentivirus and recombinant EPHA7-Fc protein injection to identify the ovulation effects of EPHA7.FindingsEPHA7 functions as a critically positive upstream factor for the expression of ERK1/2-mediated C/EBPβ. This protein, in turn, induced the expression of KLF4 and then ADAMTS1. Moreover, decreased abundance of EPHA7 was positively correlated with that of its downstream factors in hGCs of PCOS patients. Additionally, a 1-week functional EPHA7 shRNA lentivirus in rat ovaries contributed to decreased numbers of retrieved oocytes, and a 3-week functional lentivirus led to menstrual disorders and morphological polycystic changes in rat ovaries. More importantly, we found that EPHA7 triggered ovulation in rats, and it improved polycystic ovarian changes induced by DHEA in PCOS rats.InterpretationOur findings demonstrate a new role of EPHA7 in PCOS, suggesting that EPHA7 is an effective target for the development of innovative medicines to induce ovulation.FundNational Key Research and Development Program of China, National Natural Science Foundation, Shanghai Municipal Education Commission--Gaofeng Clinical Medicine, and Shanghai Commission of Science and Technology.

Ovulatory disorder is common in patients with hyperprolactinemia or polycystic ovary syndrome (PCOS). Previous studies have shown that ATF4 plays critical role in apoptosis and glucose homeostasis, but its role in regulating reproductive function was not explored. The present study investigated the role of ATF4 in ovarian ovulatory function. Human granulosa cells (hGCs) from 48 women newly diagnosed with PCOS and 37 controls were used to determine ATF4 expression. In vitro cultured hGCs were used to detect the upstream and downstream genes of ATF4. A shRNA- Atf4 lentiviral vector (shAtf4) was injected into rat ovaries to establish an in vivo gene knockdown model to further assess the in vivo relevance of the results from PCOS women. We found that ATF4 expression was lower in hGCs from PCOS patients than in hGCs from non-PCOS women. Many pivotal transcripts involved in cumulus-oocyte complex (COC) expansion, extracellular matrix (ECM) remodeling, and progesterone production were significantly down-regulated after ATF4 knockdown. ChIP-qPCR assays indicated that ATF4 could directly bind to the COX2 promoter and that ATF4 knockdown could attenuate human chorionic gonadotropin (hCG)-induced COX2 expression and PGE2 production. The in vivo study showed that shRNA-lentivirus mediated Atf4 knockdown in rat ovaries led to reduced number of retrieved oocytes. Collectively, these findings suggested previously unknown roles of ATF4 in ovulation. Furthermore, ATF4 malfunction in PCOS patients may impact the ovulation process, which could contribute, in part, to the pathogenesis of PCOS.

Background: The interface between environmental risk factors and genetic factors could contribute to the pathogenesis of hyperandrogenism and insulin resistance in polycystic ovary syndrome (PCOS); however, the underlying complex mechanism remains to be elucidated. Methods: We used dehydroepiandrosterone (DHEA)-induced PCOS-like rat model to measure circadian clock genes and insulin resistance-related genes. Additionally, we performed in vitro experiments in mature adipocytes to verify the molecular mechanisms. Results: DHEA-induced PCOS-like rats exhibited insulin resistance and arrhythmic expression of circadian clock genes in the liver and adipose tissues, particularly showing decreased brain and muscle ARNT-like protein 1 (BMAL1) expression. In addition, hyperandrogenism gave rise to negative regulation of BMAL1 expression to nicotinamide phosphoribosyltransferase and sirtuin 1, which further inhibited downstream glucose transporter type 4, leading to insulin resistance in mature adipocytes, which was consistent with our previous results in HepG2 cells. Conclusions: Decreased BMAL1 expression in the liver and adipose played a potentially novel role in the contribution of hyperandrogenism to insulin resistance, which might be a possible mechanism accounting for the pathogenesis of PCOS.