This study is to investigate whether astaxanthin could alleviate the oxidative stress damages of follicles induced by BPA and improve the development of the cultured follicles and oocytes. Compared with BPA group, the survival rate, antrum formation rate, oocyte maturation rate and adherence area of the D8 and D10 follicles of the BPA+Asta group were significantly higher. The estrogen and progesterone in the culture medium of BPA+Asta group were significantly higher. PCNA in D8 and D10 granulosa cells and ERα in D10 granulosa cells of follicles in BPA+Asta group were significantly higher. The levels of malondialdehyde in the follicle culture medium, levels of ROS in the oocytes, the expression levels of caspase 3 and cathepsin B in the oocytes of the BPA+Asta group were significantly lower. However, the mitochondrial membrane potential, and the expression levels of antioxidant genes (CAT, SOD1 and SOD2) and anti-apoptotic gene Bcl-2 in the oocytes in the BPA+Asta group were significantly higher. Astaxanthin improves the development of follicles and oocytes through increasing the antioxidant capacity of follicles and oocytes, and relieving the BPA-induced oxidative stress during follicular development and oocyte maturation.
Bisphenol A (BPA) is an established environmental endocrine disruptor and can interfere with the development of female germ cells. However, the underlying mechanisms are still unclear. We investigated the effects of BPA on granulosa cell development and meiosis of oocytes using in vitro culture system of mouse preantral follicles. Preantral follicles from D14 mouse ovary were treated with 10 μg/mL BPA in vitro for 11 days. The adherent area of follicles was measured. On D11, cumulus cell expansion was observed. The meiosis recovery rate was calculated. Western blot detected P53, proliferating cell nuclear antigen (PCNA), estrogen receptor α (ERα), and cyclin B1. ELISA measured estrogen and progesterone levels. Immunofluorescence detected Cx37 on oocyte membrane. Gap junction communication was assessed. We found that BPA significantly promoted the expressions of PCNA and ERα in granulosa cells and the secretion of estrogen and progesterone by granulosa cells on D10 and significantly increased the attachment area of the follicles on D8 and D10. However, it reduced the expansion of cumulus cells, Cx37 expression, and the gap junction communication between cumulus cells and oocytes on D11. BPA promoted the recovery of oocytes from meiosis, interrupted the expression of cyclin B1 protein in arrested germinal vesicle breakdown (GVBD) oocytes, and reduced the in vitro maturation rate of oocytes. These GVBD oocytes were live without apoptosis or death. Conclusively, BPA disturbs the development of granulosa cells and the meiosis progression of oocytes by decreasing gap junction communication between oocytes and the granulosa cells as well as regulating cyclin B1 expression in GVBD oocytes.
Many ingredients in Wenshen Shengjing Decoction (WSSJD) can cause epigenetic changes in the development of different types of cells. It is not yet known whether they can cause epigenetic changes in sperms or early embryos. Here, we investigated the role of WSSJD in epigenetic modifications of sperms or early embryos and early embryo development. A mouse model with spermatogenesis disorders was established with cyclophosphamide (CPA). WSSJD was administrated for 30 days. The male model mice after the treatment were mated with the female mice treated with superovulation. The embryo development rate of each stage was calculated. Immunofluorescence staining was used to detect the expression of H3K27me3 in sperm, pronuclear embryos, and 2-cell embryos. Western blotting was used to detect the expression of histone demethylase KDM6A and methyltransferase EZH2 in 2-cell embryos with developmental arrest. The expressions of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) in 2-cell embryos with developmental arrest were analyzed with qRT-PCR. Comparing with the control group, CPA destroyed the development of seminiferous epithelium, significantly increased the expression level of H3K27me3 in sperm, reduced the expression ratio of H3K27me3 in female and male pronuclei, delayed the development of 2-cell embryos, and increased the developmental arrest rate and degeneration rate of 2-cell embryos. Moreover, the expressions of EZH2 and H3K27me3 were significantly increased in the 2-cell embryos with developmental arrest, and the expression of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) was significantly decreased. Compared with the CPA group, WSSJD promoted the development of seminiferous epithelium, maintained a low level of H3K27me3 modification in sperm and male pronucleus, significantly increased the development rate of 2-cell embryos and 3-4 cell embryos, and reduced the developmental arrest rate and degeneration rate of 2-cell embryos. WSSJD may promote early embryonic development by maintaining a low level of H3K27me3 modification in sperm and male pronucleus and regulating the zygotic genome activation in mice with spermatogenesis disorders induced by CPA.
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