丁内酯-I对小鼠卵母细胞体外成熟、染色质构型和超微结构的影响

The effect of butyrolactone-I on in vitro maturation, chromatin configuration andultrastructure of (mouse oocytes)

目的观察100μmol/L丁内酯-I(BL-I)对小鼠卵母细胞的体外成熟、染色质构型及超微结构的影响。方法实验组小鼠未成熟卵用"两步法"培养,先用BL-I培养液培养6 h,然后再用成熟培养液培养;对照组直接用成熟培养液培养,观察其生发泡破裂(GVBD)和第一极体形成的过程。将BL-I培养液培养6 h前后的卵母细胞分别固定行Hochest33342染色及制作电子显微镜标本,比较染色质构型及超微结构的变化。结果BL-I培养液培养小鼠未成熟卵6 h,实验组卵母细胞GVBD的发生率(21.5%)明显低于对照组(82.3%)(P=0.001)。BL-I的抑制作用消除后,实验组卵母细胞GVBD发生率(96.9%)和成熟率(86.1%)与对照组(分别为97.3%和83.3%)无差异,但GVBD和成熟过程明显加速。BL-I抑制卵母细胞核成熟6 h,部分卵母细胞(32.8%)的染色质凝集,染色质围绕核仁型(SN)和非围绕核仁型(NSN)卵母细胞的比例(50.8%和16.4%)较培养前(69.2%和30.8%)明显下降;卵丘颗粒细胞的伸展未受影响,卵母细胞胞质的超微结构未出现明显异常,皮质颗粒和线粒体的形态及分布与对照组相似。结论BL-I能可逆性抑制体外培养的小鼠卵母细胞的核成熟,抑制作用消除后,卵母细胞成熟能力不受影响,但GVBD和第一极体的形成过程明显加速;在BL-I抑制卵母细胞核成熟的过程中,染色质构型发生变化,部分NSN型转化为发育能力更高的SN型,而胞质的超微结构未出现明显异常,有待进一步研究。

Objective： This study was to investigate the effect of 100 μmol/L butyrolactone-Ⅰ （BL-Ⅰ） on in vitro maturation, chromatin configuration and ultrastructure of mouse oocytes. Methods. Mouse immature oocytes were cultured with a two-step culture system. Firstly immature oocytes were cultured in maturation medium with BL-Ⅰfor 6 h and then in maturation medium without BL-Ⅰ. Germinal vesicle breakdown （GVBD） and the first polar body extrusion were observed. Mouse oocytes cultured with BL-Ⅰfor 6 h were fixed and stained with Hochest 33342 or processed for transmission electron microscopy. The oocytes in control group were cultured in standard maturation medium. Results： The incidence of GVBD in experimental group （21.5%） was significantly lower than that in control group （82.3%） when the oocytes were cultured with BL-Ⅰ for 6 h. After 14 hrs of culture, the percentage of GVBD and maturation was similar in the two groups （96.9% vs 97.3% and 86.1% vs 83. 3%, respectively）, but the process of GVBD and the first polar body extrusion were accelerated in the oocytes of experiment group following the withdrawal of BL-Ⅰ. After oocytes were treated with BL-Ⅰ for 6 h,Chromatin in 32.8% of ooeytes condensed. The percentage of the ooeytes with ehromatin surrounded nueleolus type （SN type） and non-surrounded nueleolus type （NSN type） configuration in experiment group decreased significantly when compared with that of the control group （50.8% vs 69.2% and 16.4% vs 30.8%, respectively）. The expanding of cumulus cell, the structure and distribution of mitoehondria and the cortical granules in the ooplasm were similar between the two groups. Conclusions： BL-Ⅰ can reversibly inhibit nuclear maturation of mouse ooeytes. In the BL-Ⅰ-treated ooeytes, the mature competence was not affected but GVBD and the first polar body extrusion were accelerated following the withdrawal of BL-Ⅰ. Chromatin configuration of BL-Ⅰ-treated oeeytes changed, and some of NSN type could transform to SN type whose developmental competence is higher, The ultrastrueture of ooplasm did not appear abnormal.