Appeltant R, Somfai T, Nakai M, Bodó S, Maes D, Kikuchi K, Van Soom A
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan.
Theriogenology. 2015 Mar 1;83(4):567-76. doi: 10.1016/j.theriogenology.2014.10.026. Epub 2014 Nov 4.
The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development.
本研究的目的是在使用能够对卵母细胞进行个体追踪的系统，在化学成分明确的培养基中对卵丘 - 卵母细胞复合体（COCs）进行体外成熟（IVM）期间，阐明卵母细胞与卵丘细胞（CCs）在卵丘扩展和卵母细胞成熟水平上的相互作用。特别是，通过添加裸卵（DOs）来研究卵母细胞分泌因子的影响，作为改善IVM系统的一种可能方法。基本成熟培养基是猪卵母细胞培养基，仅在IVM的前20小时添加促性腺激素。在IVM期间，通过粘性Cell - Tak涂层将COCs固定在培养皿底部，这使得在IVM期间能够对COCs进行个体追踪。通过数字图像分析测量IVM期间COCs的大小变化。完整COCs在猪卵母细胞培养基中的卵丘扩展在20小时前以典型方式增加，随后在IVM的48小时内大小减小（P < 0.05）。通过卵母细胞切除术从COCs中去除卵母细胞可使CCs在一定程度上扩展，尽管其扩展能力低于COCs（P < 0.05）。添加DOs（COCs与DOs的比例为9:16）并未提高完整COCs的卵丘扩展和卵母细胞成熟率（P > 0.05），但确实增强了卵母细胞切除复合体的卵丘扩展（P < 0.05）。此外，与COCs相比，IVM前去除CCs可提高卵母细胞成熟率（分别为52.3%和32.9%）（P < 0.05），并且当在IVM培养基中添加缝隙连接抑制剂羧苄青霉素时，在COCs中观察到类似的效果：羧苄青霉素在IVM 20小时时抑制了COCs的扩展。总之，猪卵母细胞通过缝隙连接通讯以及可能通过卵母细胞分泌因子的产生来增强卵丘扩展。然而，卵母细胞的存在并非此过程的先决条件。作为回报，CCs在初始培养期间通过缝隙连接维持卵丘包裹的卵母细胞的减数分裂停滞。基于这些发现，未来的研究可以调查IVM期间与DOs共培养是否有利于受精和胚胎发育。
