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目的:探讨骨形态发生蛋白4(BMP4)/Smad信号通路在小鼠原始卵泡发育中的作用及作用机制。方法:取3日龄昆明雌性小鼠卵巢体外培养,添加重组BMP4,并以不添加BMP4为对照,采用HE染色和TUNEL法,检测BMP4在原始卵泡存活和发育中的作用;采用免疫组织化学、Westernblotting与RT-PCR检测BMP4对原始卵泡中p-Smad1/5/8、sohlh2及c-kit表达的影响。结果:①BMP4组卵巢直径明显变大(1.17±0.24 mm vs 0.65±0.13 mm,P<0.05),原始卵泡数(45.3±4.2枚)明显低于对照组(55.0±5.0枚)(P<0.05),而初级卵泡的数(25.3±2.5枚)明显多于对照组(19.0±1.0枚)(P<0.05)。②TUNEL结果显示BMP4组凋亡卵母细胞比例明显低于对照组(P<0.05)。③免疫组织化学、Western blotting与RT-PCR结果显示,与对照组相比,BMP4组原始卵泡卵母细胞中p-Smad 1/5/8、Sohlh2及C-kit的表达均显著升高(P<0.01)。结论:BMP4/Smad信号通路可促进小鼠原始卵泡向初级卵泡转变,并抑制卵母细胞的凋亡;其可能通过上调Sohlh2及C-kit基因的表达而发挥作用。
Objective: To investigate the role of BMP4 / Smad signaling pathway in the development of mouse primordial follicles and its mechanism. METHODS: Ovary of 3-day-old Kunming female mice were cultured in vitro and recombinant BMP4 was added. BMP4 was added as a control. HE staining and TUNEL assay were used to detect the effect of BMP4 on the survival and development of primordial follicles. Immunohistochemistry, The effects of BMP4 on the expression of p-Smad1 / 5/8, sohlh2 and c-kit in primary follicles were detected by Western blotting and RT-PCR. RESULTS: The ovarian diameter of BMP4 group was significantly higher than that of control group (1.17 ± 0.24 mm vs 0.65 ± 0.13 mm, P <0.05), and the number of primordial follicles (45.3 ± 4.2) was significantly lower than that of control group (55.0 ± 5.0) , While the number of primary follicles (25.3 ± 2.5) was significantly higher than that of the control group (19.0 ± 1.0) (P <0.05). ②TUNEL results showed that the percentage of apoptotic oocytes in BMP4 group was significantly lower than that in control group (P <0.05). ③ Immunohistochemistry, Western blotting and RT-PCR results showed that compared with the control group, the expression of p-Smad 1/5/8, Sohlh2 and C-kit in the primordial follicular oocytes of BMP4 group were significantly increased (P <0.01). CONCLUSION: The BMP4 / Smad signaling pathway can promote the primary follicles to transform into primary follicles and inhibit the apoptosis of oocytes. It may play a role by up-regulating the expression of Sohlh2 and C-kit genes.