Crozet N, Dahirel M, Gall L
Institut National de la Recherche Agronomique, Unité de Physiologie animale, 78352 Jouy-en-Josas cedex, France.
J Reprod Fertil. 2000 Mar;118(2):367-73.
The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.
本研究的目的是体外培养来自早期有腔卵泡的减数分裂无能的山羊卵母细胞，并使其具备经历生发泡破裂的能力。使用机械和酶促方法从直径0.35 - 0.45毫米的卵泡中分离出带有部分壁颗粒细胞的卵丘-卵母细胞复合体。根据卵母细胞直径将卵丘-卵母细胞复合体分为两组（A组：< 95微米；B组：> 95微米），并在颗粒细胞单层上培养8或9天。培养8天内，A组卵母细胞的平均直径从86 ± 0.4微米增加到95 ± 0.7微米，B组从106 ± 0.2微米增加到109 ± 0.5微米。培养9天后，A组和B组卵母细胞的平均直径分别为99 ± 0.5微米和112 ± 0.4微米。通过体外成熟评估体外培养的卵母细胞的减数分裂能力。培养8天内，A组只有3%的卵母细胞和B组6%的卵母细胞获得了经历生发泡破裂的能力。培养9天后，A组7%的卵母细胞和B组42%的卵母细胞有能力恢复减数分裂。通过蛋白质印迹技术分析体外培养的卵母细胞中p34(cdc2)的表达。在培养9天期间，两组生长中的卵母细胞中p34(cdc2)都有积累，但其浓度低于用作对照的完全成熟卵母细胞。结果首次表明，来自早期有腔卵泡的山羊卵母细胞在颗粒细胞单层上培养9天时能够生长、积累p34(cdc2)并获得恢复减数分裂的能力。
