Follicle-stimulating hormone (FSH)-induced growth of ovarian follicles is independent of follicular vascularization. Recent evidence has indicated that follicular vascularization is critical to ovarian follicle development and survival. FSH, a gonadotropin that induces follicular growth and development, also acts as the major survival factor for antral follicles. FSH has been reported to stimulate angiogenesis in the theca layers mediated in part by the vascular endothelial growth factor A (VEGFA) and the transcription factor hypoxia inducible factor 1α (HIF-1α). However, it remains largely undetermined whether FSH-dependent growth and survival of antral follicles relies on FSH-induced vascularization. Here, we first demonstrated that induction of angiogenesis through the FSH–HIF–1α-VEGFA axis is not required for FSH-stimulated follicular growth in mouse ovary. FSH increased the total number of blood vessels in mouse ovarian follicles, which was correlated with elevated expression of VEGFA and HIF-1α in granulosa cells. In contrast, blocking of follicular angiogenesis using inhibitors against the HIF-1α-VEGFA pathway repressed vasculature formation in follicles despite FSH administration. Interestingly, by measuring follicular size and ovarian weight, we found that the suppression of angiogenesis via HIF-1α–VEGFA pathway did not influence FSH-mediated follicular growth. However, inhibition of FSH-induced follicular vascularization by PX-478, a small-molecule inhibitor that suppresses HIF-1α activity, blocked ovulation and triggered atresia in large follicles. On the other hand, PX-478 injection reduced oocyte quality via impairing the meiotic apparatus, showing a prominently defective spindle assembly and actin dynamics. Collectively, our findings unveiled a vascularization-independent effect of FSH on follicular growth, whereas follicular survival, ovulation, and oocyte development relies on FSH-mediated angiogenesis in the follicles.

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During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

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As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

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Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium fungi commonly found in corn, wheat, and other cereals which can infect food and feed commodities, and ZEA mainly has reproductive toxicity which causes widely reproductive disorders in pigs and other animals. However, the toxicity and the functional ways of ZEA on early embryo development is still unclear. In present study we showed that exposure to ZEA (10 μM) significantly decreased the 2-cell and blastocyst developmental rate in porcine early embryos in vitro . ZEA treatment resulted in the occurrence of oxidative stress, showing with increased reactive oxygen species (ROS) level, following with aberrant mitochondrial distribution. Moreover, we found positive signals of γH2A.X in the ZEA-treated embryos, indicating that ZEA induced DNA damage, and the increased autophagy confirmed this. These results suggested that ZEA induced oxidative stress, which further caused mitochondria dysfunction and DNA damage on early embryonic development. We next investigated the effects of melatonin on the ZEA-treated embryo development, and we found that melatonin supplementation could significantly ameliorate ZEA-induced oxidative stress, aberrant mitochondria distribution and DNA damage. In all, our results showed that ZEA was toxic for porcine embryos cultured in vitro and melatonin supplementation could protect their development from the effects of ZEA.