猪卵母细胞玻璃化冷冻后细胞骨架的变化
Cytoskeletal Changes of Vitrified Porcine Oocytes
-
摘要: 为研究玻璃化冷冻后猪卵母细胞纺锤体、染色体和微丝的变化，从屠宰猪卵巢表面直径2—5 mm卵泡中采集未成熟(GV)期卵母细胞，由GV期卵母细胞经成熟培养获得体外成熟(MⅡ)期卵母细胞。GV期和MⅡ期卵母细胞各分为3组：对照组、冷冻保护剂处理组和玻璃化冷冻组。MⅡ期卵母细胞经分组处理后直接用于激光扫描共聚焦显微镜(LSCM)观察样本；而GV期卵母细胞处理后先经44 h成熟培养，再用作LSCM观察样本。供试卵母细胞经固定、免疫荧光染色后，于LSCM下观察。结果表明，冷冻保护剂处理组GV期卵母细胞经成熟培养后，其纺锤体结构、染色体排列与微丝分布正常率分别为42.9%、89.6%和28.6%；玻璃化冷冻组此3项指标的正常率分别为10.1%、36.4%和16.9%，两组间差异显著(P<0.05)；除冷冻保护剂处理组染色体正常率与对照组无较大差异外，两试验组的其他指标均明显低于对照组(分别为79.5%、93.1%和72.3%，P<0.05)。MⅡ期卵母细胞冷冻保护剂处理组的纺锤体结构、染色体排列与微丝分布正常率分别为34.4%、61.3%和47.9%，而冷冻组分别为12.9%、56.7%和37.2%，两组均显著低于对照组(分别为78.3%、90.1%和72.8%，P<0.05)。结果表明，猪GV期和MⅡ期卵母细胞经冷冻保护剂处理或玻璃化冷冻保存后，均造成了纺锤体、染色体和微丝不可逆的损伤，这可能是影响卵母细胞成熟、受精与发育的重要原因。Abstract: This experiment was designed to examine the spindle organization, and chromosomal and microfilament distribution of vitrified porcine oocytes. The germinal vesicle -stage (GV stage) oocytes were aspirated from antral follicles (2-5 mm in diameter). All Metaphase Ⅱ-stage (MⅡ stage) oocytes used in the experiment were derived from GV oocytes matured in vitro. Either GV or MⅡ stage oocytes were divided into three groups：the control group, the group treated with vitrification solution, and the vitrified group. Vitrified-warmed MⅡ oocytes derived from maturation in vitro were directly used for Laser-Scanning Confocal Microscopy (LSCM); vitrified-warmed GV oocytes were firstly cultured for 44 h and then used for LSCM. Oocytes used for the experiment were fixed and stained by immunofluorescence and were then observed by LSCM. The percentage of GV stage oocytes treated with vitrification solution with normal spindle organization, chromosome alignment and actin filaments (F-actin) distribution was 42.9%, 89.6% and 28.6%, respectively. These were significantly higher than those from the vitrified group of GV stage oocytes (10.1%, 36.4% and 16.9%, respectively; P<0.05). The values for GV oocytes treated with vitrification solution were all significantly lower than those from the control, except for the percentage with normal chromosome alignment (79.5%, 93.1% and 72.3%, respectively, P<0.05). The percentage of MⅡ stage oocytes from the group treated with vitrification solution and from the vitrified group with normal spindle organization, chromosome alignment and actin filaments (F-actin) distribution were 34.4% versus 12.9%, 61.3% versus 56.7%, and 47.9% versus 37.2%, respectively. These were significantly lower than those from the control (78.3%, 90.1% and 72.8%, respectively; P<0.05). Results from this experiment suggest that irreversible damage to the cytoskeleton of porcine GV and MⅡ oocytes after vitrification could be an important factor affecting the maturation, fertility and subsequent development of the oocytes.
下载: