解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (3): 375-382.doi: 10.3969/j.issn.0529-1356.2014.03.015

• 组织学胚胎学发育生物学 • 上一篇    下一篇

骨形态发生蛋白4/Smad信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用

张海玉 张晓丽 纪淑芳 邴鲁军 郝晶*   

  1. 山东大学医学院组织学与胚胎学教研室，教育部实验畸形学重点实验室，济南 250012
  • 收稿日期:2013-10-30 修回日期:2013-12-12 出版日期:2014-06-06 发布日期:2014-06-06
  • 通讯作者: 郝晶 E-mail:haojing@sdu.edu.cn
  • 基金资助:

    国家自然科学基金资助项目

Regulation of bone morphologenetic protein 4/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes

ZHANG Hai-yu  ZHANG Xiao-li  JI Shu-fang  BING Lu-jun  HAO Jing*   

  1. Key Laboratory of the Ministry of Education for Experimental Teratology, Department of Histology and Embryology, School of Medicine, Shandong University, Ji’nan 250012, China
  • Received:2013-10-30 Revised:2013-12-12 Online:2014-06-06 Published:2014-06-06
  • Contact: HAO Jing E-mail:haojing@sdu.edu.cn

摘要：

目的 探讨骨形态发生蛋白（BMP）4/Smad 信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用。方法 取出生3d的雌性昆明小鼠卵巢，采用两步酶消化法，分离纯化卵母细胞，进行原代培养。将卵母细胞分为3组:正常对照组(Con组)、添加重组人BMP4组(BMP4组)、添加BMP4和BMP4抑制剂6-{4-［2-（1-呱啶基）乙氧基］苯基}-3-（4-吡啶基）吡唑并（1，5-A）嘧啶（dorsomorphin）组（BMP4+抑制剂组)。采用TUNEL法, 检测BMP4对卵母细胞凋亡的影响；采用免疫组织化学染色与实时定量PCR，检测BMP4对卵母细胞中p-Smad1/5/8、sohlh2、c-kit及foxo3a表达的影响。采用RNA干扰技术、转染sohlh2质粒和LY294002处理，探讨BMP4/Smad 信号通路在小鼠原始卵泡卵母细胞凋亡中的调控机制。结果 TUNEL 结果显示，BMP4组凋亡卵母细胞比例明显低于Con组(P<0.05)和BMP4+抑制剂组(P<0.05)；加入BMP4可明显促进细胞Smad入核，上调 sohlh2和c-kit表达，抑制foxo3a入核; 转染sohlh2 siRNA 后，可明显阻断BMP4抑制卵母细胞凋亡的作用；转染sohlh2质粒后，p-foxo3a表达量显著增加，LY294002可明显抑制sohlh2促进foxo3a磷酸化的作用。 结论 激活BMP4/Smad信号通路可抑制小鼠原始卵泡卵母细胞的凋亡，其作用机制可能是通过上调sohlh2和c-kit基因表达，进而调控foxo3a入核而实现的。

关键词: 骨形态发生蛋白4/Smad信号通路, 卵母细胞, Sohlh2, 原位缺口末端标记, 免疫组织化学, 小鼠

Abstract:

Objective To explore the effect and mechanism of the bone morphologenetic protein 4 (BMP4)/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes. Methods Three-day-old Kunming mouse ovarine tissues were digested by the two-step enzymatic method to extract and purify oocytes. The cultured oocytes were divided into three groups: the normal culture medium (Con group), the medium with BMP4 (BMP4 group), and the medium with BMP4 and BMP4 inhibitor (BMP4+inhibitor group). TUNEL was used to examine the effects of BMP4 on the survival of the primordial follicle oocyte; Immunohistochemical staining and Real-time quantitative PCR were performed to investigate the expressions of p-Smad1/5/8, sohlh2, c-kit and foxo3a; siRNA interference, sohlh2 plasmid transfection and LY294002 treatments were performed to explore the mechanism of the BMP4/Smad signaling pathway on the apoptosis of oocytes. Results TUNEL results demonstrated that the ratio of apoptotic oocytes in BMP4 group was significantly lower than that in the Con group (P<0.05) and the BMP4+inhibitor group(P<0.05); BMP4 significantly promoted the nuclear translocation of Smad and inhibited the nuclear translocation of foxo3a, the mRNA and protein levels of sohlh2 and c-kit remarkably increased in BMP4 group. The effect of BMP4 on the oocyte survival was significantly repressed after sohlh2 siRNA transfection. Sohlh2 overexpression up-regulated the expression of p-foxo3a, and this activity was abolished by LY 294002. Conclusion BMP4/Smad signaling pathway may inhibit primordial follicle oocyte apoptosis, via up-regulation of the expression of sohlh2 and c-kit, and then down-regulation of the nuclear translocation of foxo3a.

Key words: Bone morphologenetic protein 4/Smad signaling pathway, Oocyte, Sohlh2, TUNEL, Immunohistochemistry, Mouse