Hewitt D A, England G C
Department of Farm Animal and Equine Medicine and Surgery, Royal Veterinary College, University of London, Hatfield, UK.
Anim Reprod Sci. 1999 Feb 12;55(1):63-75. doi: 10.1016/s0378-4320(98)00162-6.
The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.
母犬体外成熟技术尚未完善，而这是濒危犬种配子挽救计划的一项重要前提条件。与大多数哺乳动物不同，母犬排出的是未成熟卵母细胞，该细胞在输卵管内进行减数分裂成熟。因此，输卵管环境模型可能有助于进行体外成熟培养。本研究旨在探究将输卵管成分引入培养环境，首先使用合成输卵管液（SOF），其次与分离出的犬输卵管上皮细胞共培养，对体外卵母细胞成熟率的影响。结果发现，培养48小时后，含0.3%牛血清白蛋白（BSA，45%）的SOF、含4% BSA（36%）的SOF与对照培养基199（27%）之间，发生生发泡破裂（GVBD）的卵母细胞比例并无差异。培养48小时后，含0.3% BSA（5%）的SOF、含4% BSA（7%）的SOF与对照培养基199（6%）之间，卵母细胞核成熟至中期I/后期I/中期II（MI/AI/MII）的情况也无差异。此外，培养96小时后，含0.3% BSA（0）的SOF、含4% BSA（7%）的SOF与对照培养基199（11%）之间，卵母细胞核成熟至MI/AI/MII的情况同样无差异。相比之下，培养96小时后，发生GVBD的卵母细胞比例受所用处理方式影响，含0.3% BSA的SOF中为27%，含4% BSA的SOF中为62%，培养基199中为63%。结果发现，共培养处理方式（199、199 +细胞、共培养基、共培养基 +细胞）之间，发生GVBD的卵母细胞比例并无差异；培养48小时后，对于卵母细胞核成熟至MI/AI/MII的情况，培养基199（2%）、199 +细胞（0）、共培养基（6%）和共培养基 +细胞（2%）之间也无差异。此外，培养96小时后，199（61%）、199 +细胞（59%）、共培养基（65%）和共培养基 +细胞（53%）之间，卵母细胞核成熟至GVBD的情况无差异。相比之下，培养96小时后，成熟至MI/AI/MII的卵母细胞比例受所用处理方式影响，培养基199（0）、199 +细胞（9%）、共培养基（0）和共培养基 +细胞（0）之间存在显著差异。因此，结果表明，在SOF中培养卵母细胞时，补充高浓度蛋白质可改善卵母细胞核成熟，而在输卵管上皮细胞存在的情况下培养可改善卵母细胞成熟，但这仅在延长培养时间后才会出现。
