小鼠胚胎卵巢体外培养中卵细胞的生长及其数量的调节

Mouse Embryo Oocyte Growth Pattern and Number Regulation in Vitro

利用胚胎卵巢体外长期培养技术研究了小鼠卵巢中卵细胞的生长发育状况。材料取自B6D2雌小鼠F1代胚胎，服龄为11．5日和13．5日。用Orcein染色法鉴别雌雄。分离出的卵巢培养12～14d。5d后换一次培养液，以后每2d换1次。培养8d后，13．5日龄胚胎卵巢中卵细胞已形成与体内相似的有规则的分布。在卵巢皮质部有大量的卵母细胞和少量体细胞，而在髓质部只有少量的卵母细胞和大量的体细胞。大量的未生长的小卵母细胞（直径＜25μm）紧密地分布在皮质的外侧，而生长印母细胞则主要分布在皮质的内侧和髓质。11．5日龄胚胎卵巢经过10～12d培养，没有形成上述分布规律，所有观察到的卵细胞都是生长中的卵母细胞。卵细胞数量也显著低于13．5日龄胚胎的卵巢（34．4±6．53比566．7±49．5）。用IGF－I和FSH处理11．5日龄胚胎卵巢可显著增加其卵细胞的数量（分别为88对55，P＜0．01和140对20，P＜0．005）。较老龄的胚胎卵巢组织对于11．5日龄胚胎卵巢卵细胞数量均有促进作用，其中以16．5日龄胚胎卵巢的作用较强（165对65，P＜0.005）。实验结果表明小鼠胚胎发育到达11．5～13．5日龄时期对于卵巢来说是获得形成卵巢内卵细胞分布规律能力的重要时期。11．0日龄胚胎卵巢缺乏某些因子的作用，因而不能在体外正常发育。

The present works are conducted to observe oocyte growth/initiation in vitro, using a cul ture system of fetal mouse ovaries aged 11. 5 days (morphological undifferentiated), 13. 5 days (ovary recognizable). The ovaries from B6D2F1 embryo were cleaned from extraovarian mesonephric tissue and cultured for 12 to 14 days in minimum essential medium with 2% fetal calf serum. The medium was changed 5 days latter at first time,then renewed every second days. All ovaries flattened out during growth allowing inspection for small and growth oocyte under an inverted microscope during the culture period. When the 13. 5 day-old embryo ovary was cultured for 8 days, a cortex and a medulla developing with small nongrowing oocyte in the outer part of the cortex and growing oocyte at inner part was observed. During the culture, the growing oocyte at inner part of cortex,usually surrounded by several layers of somatic cells. It was revealed that,in culture of 11. 5 days p. c., the geographic growgh patten of oocyte was not appeared and the number of oocyte is significantly reduced compared with 13. 5 day-old embryo ovary (34. 4 ± 6. 53to 566. 7±49. 50). FSH and IGF-1 could stimulate the oocyte number increase by cultured 11. 5day-old embryo ovary when the medium was supplemented with FSH and IGF-1, respectively (140±17. 21; 90±14. 95 to 38. 6±9. 5 of control). The coculture with older embryo ovaries could also significantly increase oocyte number of 11. 5 day-old embryo ovaries. The older the enbryo was, the stronger the stimulatory action was observed. Our results indicate it seems that the period from 11. 5 to 13. 5 p. c. is a crucial time for ovary to gain the ability to form a cortex with a growth gradient and for how many oocytes can enter folliculogenesis.