Ferré Pilar, Bui Tra Mi Thi, Wakai Takuya, Funahashi Hiroaki
Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan.
Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan.
Theriogenology. 2016 Oct 15;86(7):1705-10. doi: 10.1016/j.theriogenology.2016.05.024. Epub 2016 May 30.
The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes.
本研究旨在检测小卵泡（SF；直径0.5 - 2毫米）和中卵泡（MF；直径3 - 6毫米）来源的卵母细胞在体外成熟（IVM）前、IVM期间及IVM后去除卵丘细胞时的凋亡状态和减数分裂进程。从青春期前母猪卵巢的SF或MF中收集卵丘 - 卵母细胞复合体（COC）。在IVM培养开始前或开始20小时后，将部分卵母细胞去除卵丘细胞并进行IVM培养。在IVM结束时，通过膜联蛋白V/碘化丙啶（Annexin-V/PI）检测和4',6-二脒基-2-苯基吲哚（DAPI）染色，将卵母细胞的凋亡状态和减数分裂进程与在卵丘细胞（CCs）存在下成熟的卵母细胞进行比较。卵母细胞的凋亡状态仅受去除卵丘细胞时间的影响。在SF和MF来源的卵母细胞中，尽管在IVM前去除CCs时早期和晚期凋亡卵母细胞的发生率显著更高，但在IVM开始20小时和44小时后去除CCs时，该比率显著更低。成熟卵母细胞的发生率受COC来源和从COC中去除卵丘细胞的时间两者的显著影响。尽管MF中成熟卵母细胞的百分比高于SF，但与IVM开始后0小时和44小时相比，在IVM开始20小时去除卵丘细胞时成熟率显著更高。然而，在IVM 44小时而非22小时去除卵丘细胞时，具有形态正常纺锤体的成熟卵母细胞百分比显著更高。总之，即使COC来自SF，在IVM开始20小时后去除CCs似乎也能促进卵母细胞减数分裂进程至中期II阶段，尽管在IVM期间来自CCs的因子或与CCs的通讯可能需要在成熟卵母细胞中获得形态正常的纺锤体。
