FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响

doi:10.11843/j.issn.0366-6964.2018.09.011

摘要/Abstract

摘要：

旨在研究促卵泡素（FSH）对牦牛卵母细胞体外成熟，以及对表皮生长因子（EGF）、表皮生长因子受体（EGFR）的表达影响和与细胞凋亡的关系。在牦牛卵母细胞体外成熟培养液中添加不同浓度FSH （终浓度分别为0、2、5、10 μg·mL^-1），体外成熟培养后，运用实时荧光定量PCR （qRT-RCR）和免疫荧光染色技术检测EGF、EGFR表达情况，采用qRT-RCR和蛋白免疫印迹法检测Bax、Bcl-2表达变化。结果表明：1）成熟培养液中加入FSH可提高COCs成熟率，当成熟液中FSH为5 μg·mL^-1时，成熟率最高，达到76.84%，显著高于其他组（P<0.05）；2）随着FSH浓度的增加，EGF和EGFR表达逐渐升高，其中在5 μg·mL^-1FSH组中，EGF和EGFR的表达最高，显著高于其他组（P<0.05），而在10 μg·mL^-1FSH组中，EGF和EGFR的表达水平降低；EGF主要位于卵丘细胞中，而EGFR在卵丘细胞和卵母细胞均有表达；3）随着FSH浓度的增加，Bax和Bcl-2的表达显示出明显的逆向模式，Bax的表达水平逐渐降低，Bcl-2的表达水平逐渐升高，在5 μg·mL^-1FSH组中Bax的表达量最低，Bcl-2的表达量最高，而在10 μg·mL^-1FSH组中，Bax的表达水平升高。综上表明，在牦牛卵母细胞体外成熟过程中，FSH提高了卵母细胞发育能力，诱导EGF和EGFR表达，而且可能通过调控Bax、Bcl-2等凋亡相关因子的表达，抑制卵母细胞凋亡。

Abstract:

The aim of this study was to investigate the specific contribution of follicle-stimulating hormone (FSH) on maturation of yak oocytes in vitro, the effect of FSH on the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in yak oocytes, and the relationship between FSH and apoptosis. In this experiment, different concentrations of FSH (final concentration:0,2,5,10 μg·mL^-1) were added into the medium of the yak oocytes in vitro. The mRNA and protein expression of EGF and EGFR were detected by real-time PCR (qRT-RCR) and immunofluorescence staining, respectively. Meanwhile, the expression of Bax and Bcl-2 were detected by qRT-RCR and Western blotting. The results showed that:1) It was noteworthy that FSH promoted the maturation of oocytes, and the optimal concentration of FSH was 5 μg·mL^-1, and the rate of maturation oocytes was 76.84%, which was significantly higher than other groups (P<0.05). 2) The expression levels of EGF and EGFR depended on the doses of FSH. The expressions of EGF and EGFR were upregulated during the doses of FSH raised from 0 to 5 μg·mL^-1, which was significantly higher in 5 μg·mL^-1 FSH group than other groups (P<0.05). When the concentration of FSH was 10 μg·mL^-1, the expression level of EGF and EGFR was reduced. Immunofluorescence assay showed that EGF was mainly located in cumulus cells, while EGFR was expressed in cumulus cells and oocytes. 3) With the increase of FSH concentration, the expression of Bax and Bcl-2 showed the obvious reverse pattern, the expression level of Bcl-2 increased gradually, while the expression level of Bax decreased gradually. The expression of Bax was the lowest, and the expression of Bcl-2 was the highest when the dose of FSH was 5 μg·mL^-1, but the expression level of Bax increased in 10 μg·mL^-1 FSH group. The results showed that FSH increased the ability of oocyte development and induced the expression of EGF and EGFR during the process of yak oocyte maturation in vitro, and inhibited the apoptosis of oocytes by regulating the expression of apoptosis related factors such as Bax and Bcl-2.

中图分类号:

何翃闳, 潘阳阳, 张慧珠, 李秦, 王萌, 崔燕, 樊江峰, 余四九. FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响[J]. 畜牧兽医学报, 2018, 49(9): 1899-1907.

HE Hong-hong, PAN Yang-yang, ZHANG Hui-zhu, LI Qin, WANG Meng, CUI Yan, FAN Jiang-feng, YU Si-jiu. The Effects of Follicle-stimulating Hormone (FSH) on the Expression of EGF and EGFR in Yak Oocytes and Apoptosis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1899-1907.

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