Wu Caihong, Rui Rong, Dai Jianjun, Zhang Chunyan, Ju Shiqiang, Xie Bing, Lu Xiao, Zheng Xiaofeng
College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu, China.
Mol Reprod Dev. 2006 Nov;73(11):1454-62. doi: 10.1002/mrd.20579.
The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.
本研究的目的是确定猪生发泡（GV）期和减数分裂II（MII）期卵母细胞玻璃化冷冻后超微结构和细胞骨架的变化。为了研究玻璃化冷冻对发育能力的影响，将卵母细胞分为三组：新鲜GV期卵母细胞（对照组）、玻璃化冷冻的GV期卵母细胞和玻璃化冷冻的MII期卵母细胞。在GV期和MII期卵母细胞中，玻璃化冷冻后形态正常的比例都很高（分别为92.4%和94.2%，P>0.05），而玻璃化冷冻的GV期卵母细胞的存活率高于玻璃化冷冻的MII期卵母细胞（分别为56.8%和41.9%，P<0.05）。在玻璃化冷冻的GV期卵母细胞中，154个卵母细胞中有12个在体外受精后发生了卵裂，其中6个发育到8细胞期，3个发育到16细胞期，3个发育成桑葚胚。玻璃化冷冻的MII期卵母细胞未发生卵裂。为了对卵母细胞进行超微结构分析，随机选择新鲜的和玻璃化冷冻复温后的GV期和MII期卵母细胞进行透射电子显微镜（TEM）观察。结果显示，玻璃化冷冻对GV期卵母细胞造成了不同程度的冷冻损伤。一些卵母细胞的卵丘细胞与卵丘-卵母细胞复合体（COC）分离，与卵丘细胞相邻的透明带破裂。卵丘细胞之间的缝隙连接破裂，许多微绒毛被破坏或消失。只观察到均匀的脂滴。玻璃化冷冻后，MII期卵母细胞的皮质颗粒仍排列在卵母细胞膜上。只发现围绕大液泡的形态不规则、不均匀的脂滴。为了检测细胞骨架结构，通过激光扫描共聚焦显微镜（LSCM）对新鲜的和玻璃化冷冻复温后的MII期卵母细胞进行分析；玻璃化冷冻复温后的GV期卵母细胞在进行LSCM分析前培养42-44小时。在58个对照卵母细胞中，79.5%显示纺锤体正常，染色体沿赤道板排列。在玻璃化冷冻的卵母细胞中，玻璃化冷冻的GV期和MII期卵母细胞中纺锤体组织正常的比例均显著降低（分别为10.1%和12.9%，P<0.05）。玻璃化冷冻的GV期和MII期卵母细胞中F-肌动蛋白分布正常的卵母细胞比例低于对照组（分别为16.9%和37.2%，对照组为72.3%）。本实验结果表明，猪GV期和MII期卵母细胞玻璃化冷冻后细胞骨架的不可逆损伤可能是影响发育能力的一个重要因素。
