下调 Hsp27 的基因表达和活性均可改善小鼠卵母细胞的体外成熟。
Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.
机构信息
Department of life science and technology, China Pharmaceutical University, Nanjing 210038, China.
出版信息
Reprod Biol Endocrinol. 2010 May 14;8:47. doi: 10.1186/1477-7827-8-47.
BACKGROUND
Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified.
METHODS
The expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot.
RESULTS
Hsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9).
CONCLUSIONS
Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.
背景
热休克蛋白 27（Hsp27）是小分子热休克蛋白家族的一员，是一种凋亡调节因子。我们之前的蛋白质组学研究表明，Hsp27 主要在人卵母细胞中表达，并且在多囊卵巢综合征（PCOS）患者来源的卵巢中表达下调，PCOS 是一种众所周知的内分泌紊乱疾病，其凋亡活性和卵泡发生异常。然而，Hsp27 下调对卵母细胞发育的确切影响尚未阐明。
方法
使用 siRNA 腺病毒感染体外培养的小鼠卵母细胞，下调 Hsp27 基因的表达，同时通过向生发泡期（GV）卵母细胞的细胞质中注射多克隆 Hsp27 抗体来降低 Hsp27 的活性。通过形态学观察评估卵母细胞成熟率。使用 Annexin-V 染色分析确定早期凋亡，并用实时 RT-PCR 监测关键凋亡因子和细胞因子的 mRNA 水平，并用免疫荧光和 Western blot 监测其蛋白表达水平。
结果
Hsp27 在成熟卵母细胞中表达水平较高。用 AdshHsp27 感染和向 GV 卵母细胞中注射 Hsp27 抗体，可提高卵母细胞的发育和成熟。与对照组（AdGFP 组为 76.2%，IgG 注射组为 64.4%）相比，两个 AdshHsp27 处理组（88.7%，86.0%）和 Hsp27 抗体注射组（77.0%）的卵母细胞的生发泡破裂（GVBD）率显著升高。此外，两个 AdshHsp27 处理组（73.8%，76.4%）和 Hsp27 抗体注射组（67.3%）的中期 II（MII）发育率均高于对照组（AdGFP 组为 59.6%，IgG 注射组为 55.1%）。我们还发现，在 IVM 8 小时后，Hsp27 下调组（46.5%和 45.6%）的早期凋亡率高于对照组（34.1%）。同样，下调 Hsp27 后，通过激活外源性 caspase 8 介导的途径，导致凋亡因子（caspase 8、caspase 3）和细胞因子（bmp15 和 gdf9）的表达显著增强。
结论
下调 Hsp27 可改善小鼠卵母细胞的成熟，同时通过诱导外源性 caspase 8 介导的途径激活，增加卵母细胞的早期凋亡。
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