“两步法”体外培养山羊卵母细胞的超微结构观察

Ultrastructure of Goat Oocytes Cultured with "Two-step" Protocol

有假说认为,卵母细胞在体外培养过程中,如果延长GV期,可促进卵母细胞进一步成熟,因而提高发育潜能。采用山羊半卵泡和卵母细胞共培养,抑制卵母细胞GVBD发生,从而延长GV期。比较了共培养前后和恢复成熟培养后卵母细胞的超微结构变化,其目的从亚细胞水平寻找卵母细胞进一步成熟的证据。研究发现,常规成熟培养:有卵周隙存在,但不贯通,局部区域卵膜与透明带结合紧密;部分皮质区尚有细胞器存在;微绒毛大部分从透明带中撤出,倒伏于质膜表面,数量较多,形态较为粗大;皮层颗粒质膜下部分单层分布,部分散布于皮质区;线粒体均匀散布于卵质中央区。共培养前:卵母细胞的卵周隙尚未形成,微绒毛没有从透明带中撤出;线粒体等细胞器分布于皮质区,皮层颗粒成簇状分布,皮质区富含细胞器。共培养后:局部形成卵周隙,微绒毛已自透明带中撤出,数量较多,垂直或倒伏于卵膜表面;线粒体以簇状分批开始内移,皮层颗粒已部分单层分布于质膜下,部分皮质区缺乏细胞器。恢复成熟培养后:卵周隙进一步扩大并且贯通,微绒毛数量减少并且绝大多数垂直于卵膜;线粒体在卵质中央区均匀分布,皮层颗粒卵膜下单层分布,大部分皮质区无细胞器存在。利用“两步法”培养得到的卵母细胞与体外常规成熟培养的卵母细胞相比,更有利于皮层颗粒的质膜下单层分布,卵母细胞卵周隙的形成与贯通,微绒毛数量减少和垂直于卵膜表面,无细胞器皮层区的进一步形成。因此,更有利于卵母细胞胞质的进一步成熟。

It is hypothesized that if oocyte can be cultured in vitro under condition that maintains meiotic arrest at the germinal vesicle （GV） stage, then they may have the opportunity to acquire greater developmental competence. In this study, goat oocytes were cultured within follicle hemi-sections, which can inhibit GVBD and prolong the duration of GV stage. The changes of uhrastructure of goat oocyte before and ＇after co-culture with follicle hemi-section and ＂after resumption culture were observed. The purpose is to find out the proofs of further capacitation of ooeytes, and to observe the feasibility of two-step protocol for oocytes maturation. Before co-culture with follicle hemi-section： perivitelline space have not been formed in the oocyte, neither have the microvilli been released from the zona pellucida, the cortical region have a lot of oraganelles, clusters of cortical granules exit in the cortical region, mitochondrias amass in the cortical region, a number of vacuoles are presented. After co-culture with follicle hemi-section, the results are as follows ： perivitelline space is obvious, the microvilli have been released from the zona pellucida and pile up in stacks in the perivitelline space, mitochondria have begun to migrate toward the center of oocyte inclusters, the bulk of the cortical granules are distributed at solitary positions along the oolemma in the part, the number of vacuoles increase obviously, part of cortical region lack organelles. After resumption culture： perivitelline space develops further, the number of microvilli stacks have decreased and become more erect, mitochondria have distributed evenly in the central oolemma, cortical granules are distributed at solitary positions along the oolemma, the number of vacuoles decrease obviously, more parts of cortical region are free of organelles. In contrast to maturation in vitro, inhibition culture by follicle hemi-section contributes more to the formation of perivitelline space, decrease of number and erection of microvilli and the further formation of organelle-free cortex.