前培养对猪卵母细胞生发泡染色质构型和体外成熟的影响

Effect of pre-incubation of porcine COCSs in medium without gonadotropins on the chromatin configuration and quality of oocytes matured in vitro

在体外成熟培养时使卵母细胞过早接触高浓度激素可能影响体外成熟卵母细胞的质量。本实验研究了推迟卵母细胞与激素接触时间对卵母细胞GV染色质构型、卵母细胞核成熟质量及极体形态的影响。结果表明 :猪体外成熟卵母细胞的极体形态不同 ,分为完整、退化、碎裂和无极体四种。在不含激素培养液中前培养 12h ,A类卵母细胞的GV 1比例 (48 2 % )明显高于在含激素培养液中前培养 12h卵母细胞 (2 7 1% ) ;而前者的GV 3比例 (19 6 % )却明显低于后者 (5 0 8% ) ;前者成熟卵母细胞的极体完整率 (5 9 6 % )也明显高于后者(2 7 5 % )。这说明推迟卵母细胞与激素接触时间可能提高体外成熟卵母细胞的质量和发育同步水平 [动物学报49(1) :86～ 90 ,2 0 0 3]。

Although porcine oocytes matured and fertilized in vitro can develop to blastocyst stage, the rate of development is still low.Culture conditions and the heterogeneity of oocytes before culture may affect the quality of oocytes matured in vitro.The effect of pre-incubation of cumulus-oocyte complexes(COCs)in medium without gonadotropins on chromatin configuration and quality of porcine oocytes matured in vitro was, therefore, investigated in this experiment.Ovaries were collected from a local slaughterhouse, and the COCs were aspirated from 2～6 mm follicles on the ovarian surface and classified into A, B and C categories in accordance with cumulus morphology.Oocytes were cultured in TCM-199 supplemented with 10% FCS and 10% porcine follicular fluid, PMSG and hCG were added depending on experimental designs.Nuclear maturation and GV chromatin configuration of oocytes was evaluated by orecin and Hochest 33342 staining, respectively.The major findings are as follows: 1.the morphology of polar bodies in in vitro matured(IVM)oocytes varied and four types of oocytes could be distinguished, i.e.those with intact, degenerated, fragmented and no polar bodies.2.When type A oocytes were pre-cultured in the absence of gonadotropins for 12 hours, the proportion of germinal vesicle stage(GV-1) oocytes decreased significantly compared to those cultured in the presence of these hormones(48.2% vs.27.1%, P<0.05), but the proportion of GV-3 oocytes increased markedly(19.6% vs.50.8%).3.The proportion of oocytes with intact polar bodies after maturation was significantly higher when they were pre-cultured without hormones than when pre-cultured with hormones(59.6% vs.27.5%).Together, these results indicate that a delayed exposure of porcine COCs to gonadotropins led to an improved quality and a more synchronized development of oocytes matured in vitro [Acta Zoologica Sinica 49(1):86-90, 2003].