Van Blerkom J, Davis P W
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347, USA.
Hum Reprod. 1998 May;13(5):1317-24. doi: 10.1093/humrep/13.5.1317.
This study determined the occurrence of two molecular markers of apoptosis, chromosomal DNA strand breaks and oolemma phosphatidylserine redistribution, in >200 uninseminated and unfertilized human oocytes, and >800 newly ovulated and cultured mouse oocytes. DNA breaks were analysed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and phosphatidylserine by annexin V staining, with imaging by conventional epifluorescence and scanning laser confocal fluorescence microscopy. More than 300 intact and 500 fragmented mouse oocytes were examined at 24 h intervals during 6 days of culture in three different types of medium. For the human, 205 oocytes were examined at retrieval or at 24 h intervals during 7.5 days of culture in two types of medium. The perifollicular vascularity and the dissolved oxygen content of follicular fluid were determined for most of the follicles from which human oocytes were derived. The results demonstrate that TUNEL fluorescence of metaphase II (MII) chromosomes and annexin V staining of the oolemma in newly ovulated and cultured mouse and human oocytes are rare, and, when detected, are not spatially or temporally related. This finding also applied to mouse oocytes that fragmented during culture and exhibited morphological features that grossly resembled apoptotic body formation. In contrast, TUNEL but not annexin V staining occurred in the first polar body of a relatively high proportion of newly ovulated mouse oocytes, but was rarely detected in newly aspirated human oocytes. For the human, the occurrence of MII chromosomal TUNEL fluorescence was patient-specific and unrelated to perifollicular vascularity or dissolved oxygen content of the corresponding follicular fluid. The pattern of chromosomal TUNEL fluorescence observed in the first polar body and in the MII chromosomes of a very small number of mouse and human oocytes, especially after many days of culture, suggests that DNA strand breaks may not arise by apoptosis-associated endonuclease digestion. The results with these two markers suggest that it is premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects.
本研究测定了凋亡的两种分子标志物——染色体DNA链断裂和卵膜磷脂酰丝氨酸重分布——在200多个未受精和未受精卵母细胞以及800多个新排卵并培养的小鼠卵母细胞中的发生情况。通过末端脱氧核苷酸转移酶介导的dUDP缺口末端标记法（TUNEL）分析DNA断裂情况，通过膜联蛋白V染色分析磷脂酰丝氨酸情况，采用传统落射荧光显微镜和扫描激光共聚焦荧光显微镜成像。在三种不同类型的培养基中培养6天期间，每隔24小时检查300多个完整的和500个破碎的小鼠卵母细胞。对于人类，在两种类型的培养基中，在取卵时或培养7.5天期间每隔24小时检查205个卵母细胞。测定了大多数来源人类卵母细胞的卵泡周围血管情况和卵泡液中的溶解氧含量。结果表明，新排卵并培养的小鼠和人类卵母细胞中期II（MII）染色体的TUNEL荧光和卵膜的膜联蛋白V染色很少见，而且，当检测到时，在空间或时间上并无关联。这一发现也适用于培养过程中破碎并呈现出与凋亡小体形成极为相似的形态特征的小鼠卵母细胞。相比之下，相当比例的新排卵小鼠卵母细胞的第一极体中出现了TUNEL染色，但在新抽吸的人类卵母细胞中很少检测到。对于人类，MII染色体TUNEL荧光的发生具有患者特异性，与相应卵泡液的卵泡周围血管情况或溶解氧含量无关。在极少数小鼠和人类卵母细胞的第一极体和MII染色体中观察到的染色体TUNEL荧光模式，尤其是在培养许多天后，表明DNA链断裂可能并非由凋亡相关的核酸内切酶消化引起。这两种标志物的结果表明，断定排卵的卵母细胞中发生凋亡或这种机制参与消除或防止有细胞质或染色体缺陷的卵母细胞受精还为时过早。
