| In mammalian ovary, only a small amount of follicles can develop to maturation and ovulation, during follicular growth and development,most of follicles suffer degradation or atresia. Several studies have proved that follicular atresia is primarily induced by apoptosis of granulosa cells. Till now, there are a lot of reports about apoptosis of granulosa cells and its regulatory mechanism in human, rat, cow, pig, sheep, goat etc, but it has never been reported about the expression pattern of apoptosis-related genes and miRNAs in granulosa cells during buffalo follicular atresia.To understand the relationship between apoptosis of granulosa cells and follicular atresia, and the regulatory mechanism of buffalo follicular atresia, in this study, we first explored the expression of apoptosis-related genes and miRNAs in granulosa cells from buffalo follicles with different atretic status, in order. Then, the effect of E2on apoptosis rate, cell cycle, expression of apoptosis-related genes and miRNAs in granulosa cells cultured in vitro was investigated, it would help us to understand the role of E2on apoptosis of granulosa cells, and the regulatory mechanism of buffalo follicular atresia.The expression of apoptosis-related genes and miRNAs in granulosa cells of buffalo follicles with different atretic status1. Apoptotic status detection of granulosa cells from buffalo HF, EF and PF by HE staining, DNA ladder analysis and Tunel methods.There was no shedding granulosa cells in HF cavity, the genomic DNA of these kinds of granulosa cells didn’t appear DNA ladder, and their apoptosis rate was9.32%. For EF follicles, a few of shedding granulosa cells existed in EF cavity, DNA ladder appeared in the genomic DNA of granulosa cells, and the apoptosis rate was17.24%. There had a lot of shedding granulosa cells in PF cavity, clear and serious DNA ladder appeared in the genomic DNA of granulosa cells, and the apoptosis rate reached to20.26%.2. The expression of apoptosis-related genes and miRNAs in granulosa cells from buffalo follicles with different atretic status detected by qRT-PCR. The results revealed that all of Fas and other related genes in death receptor signal pathway expressed in granulosa cells from buffalo HF, EF and PF, the expression levels of them increased with the progress of follicular atresia. TNFa gene expressed in granulosa cells from EF and PF, but did not express in granulosa cells from HF. TRAIL gene didn’t express in granulosa cells from three kinds of follicles. Bcl-2and other genes related to mitochondrial signal pathway expressed in granulosa cells from three kinds of follicles, the expression of some of them, including Bcl-2, Bcl-xl and MCL-1genes, decreased with the follicular atresia. Genes, such as Bax, Bid and Bad, increased their expression with the progress of follicular atresia. All of genes related to endoplasmic reticulum signal pathway expressed in granulosa cells from three kinds of follicles, and their expression levels increased with the progress of follicular atresia. Total16miRNAs, including miR21-5P etc, were detected in granulosa cells from buffalo HF, EF and PF, the expression trend for10of them, like miR21-5P, was increased at first, then decreased with the progress of follicular atresia. The expression of other6miRNAs, like miR99a-5P, was decreased during the follicular atresia.3. The expression of Fas and Caspase3protein in granulosa cells by immunohistochemical technology. The results showed that the Fas and Caspase3protein expressed in granulosa cells from buffalo HF, EF and PF, and their expression levels increased with the progress of follicular atresia. This result was consistent with their mRNA expression level.The effect of E2on apoptosis of granulosa cells cultured in vitro and its regulatory mechanism.1. The effect of E2on apoptosis rate of granulosa cells was detected by Tunel method. The results showed that the E2concentration of10’5～10’9mol/L could effectively inhibit the apoptosis of granulosa cells with a dose-dependent manner. While etrogen receptor inhibitor, ICI182780could block the E2effect on apoptosis.2. The effect of E2on cell cycle of granulosa cells cultured in vitro was detected by flow cytometry. We found that the ratio of cells in S phase was increased, and the ratio of cells in G0/G1phase was decreased when treated with E2.There was no significant changes on the ratio of cells treated with ICI182780or E2+ICI182780.3. The effect of E2on expression of apoptosis-related genes and miRNAs in granulosa cells was detected by qRT-PCR. The results showed that ERβ, Fas and other detected genes expressed in granulosa cells of all experimemtal groups, the expression of ERβ and Bcl-2genes were increased after treated with E2, other genes and miRNAs, such as Fas and miR21-5P, decreased their expression after treated with E2. There had no significant expression changes in cells treated with ICI182780or E2+ICI182780.4. The effect of E2on expression of Fas, Bax and caspase9protein in granulosa cells was detected by western-blot method. The results showed that the expression of Fas, Bax and caspase9protein in granulosa cells were decreased after treated with E2, this result was consistent with the mRNA expression level.The above results indicated that a lot of apoptosis occurred in granulosa cells during follicular atresia in buffalo ovary, and the apoptosis rate of granulosa cells increased with the progress of follicular atresia. Some apoptosis-related genes in death receptor, mitochondrial and endoplasmic reticulum signal pathway, and partial miRNAs involved in the process and regulated the apoptosis of buffalo granulosa cells. E2could effectively inhibit the apoptosis of granulosa cells by a dose-dependent manner, and promoted the cell proliferation through the cell cycle, the expression of partial apoptosis-related genes and miRNAs mediated by ERβ... |
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