Smitz J, Cortvrindt R, Van Steirteghem A C
Centre for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University, Belgium.
Mol Reprod Dev. 1996 Dec;45(4):466-75. doi: 10.1002/(SICI)1098-2795(199612)45:4<466::AID-MRD9>3.0.CO;2-P.
This study compares the effects of reduced (5%) or normal (5% CO2 in air; 20% O2) oxygen tension on the in vitro maturation of early preantral ovarian follicles isolated from 14-day-old (C57BI/6J x CBAca) F1 mice. Intact follicles (100-130 microns) are singly cultured in 20 microliters droplets alpha-MEM enriched with FCS and rFSH under mineral oil at 37 degrees C and 100% humidity. In this culture system the follicles are allowed to attach to the bottom of the petri dishes. Follicle in vitro growth, hormone secretory capacity, and in vitro ovulation were studied under the two oxygen tensions. Spontaneous oocyte release from the follicle during a 16-day culture period was observed significantly more under 5% oxygen. Antral-like cavity formation was not observed under 5% O2. The follicles in the 5% O2 cultures reaching day 16 were stripped of their granulosa cell layers, and 83% of the retrieved oocytes had already undergone spontaneous germinal-vesicle breakdown (GVBD). Under 20% O2, the GV stage was maintained until day 16 in 77% of the oocytes. Under 5% O2, intact follicle survival up to day 12 was significantly reduced as compared to the 5% CO2 in air conditioning. The hCG stimulus on day 12 induced mucification in a significantly larger proportion of follicles cultured under 20% O2 (79% vs. 47%). Germinal-vesicle breakdown (20% O2:95, 5%, O2:42%) and first polar body extrusion (20% O2:40%, 5% O2:15%) were significantly more prevalent under normal oxygen tension. A reduced secretory capacity of E2 and inhibin was demonstrated for follicles cultured under 5% O2. The histological study of serially sectioned follicles showed increased areas of centrally located granulosa cell necrosis and pyknosis in the cumulus cells. Gassing follicle cultures using 5% CO2 in air provided appropriate conditions for normal growth, enhanced whole-follicle survival, differentiation, and hormone production, and improved the yield of meiotic competent oocytes.
本研究比较了低氧张力（5%）或正常氧张力（空气中5%二氧化碳；20%氧气）对从14日龄（C57BI/6J×CBAca）F1小鼠分离的早期腔前卵泡体外成熟的影响。完整卵泡（100 - 130微米）在富含胎牛血清（FCS）和重组促卵泡激素（rFSH）的α - MEM 20微升液滴中，于37℃、100%湿度条件下在矿物油中进行单卵泡培养。在该培养系统中，卵泡可附着于培养皿底部。在两种氧张力条件下研究卵泡的体外生长、激素分泌能力及体外排卵情况。在16天培养期内，5%氧条件下观察到卵泡自发释放卵母细胞的情况显著增多。5%氧气条件下未观察到类腔形成。培养至第16天的5%氧气培养的卵泡去除颗粒细胞层后，83%回收的卵母细胞已自发发生生发泡破裂（GVBD）。在20%氧气条件下，77%的卵母细胞在第16天仍维持在GV期。与空气中5%二氧化碳培养条件相比，5%氧气条件下完整卵泡存活至第12天的比例显著降低。第12天的人绒毛膜促性腺激素（hCG）刺激在20%氧气培养的卵泡中诱导黏液形成的比例显著更高（79%对47%）。正常氧张力下生发泡破裂（20%氧气：95%，5%氧气：42%）和第一极体排出（20%氧气：40%，5%氧气：15%）更为普遍。5%氧气培养的卵泡显示出雌二醇（E2）和抑制素分泌能力降低。对连续切片的卵泡进行组织学研究显示，中央部位颗粒细胞坏死区域增加，卵丘细胞出现核固缩。使用空气中5%二氧化碳对卵泡培养进行通气可为卵泡正常生长、提高整个卵泡存活率、分化及激素产生提供适宜条件，并提高减数分裂能力正常的卵母细胞产量。
