Somfai Tamás, Kikuchi Kazuhiro, Onishi Akira, Iwamoto Masaki, Fuchimoto Dai-ichiro, Papp Agnes Bali, Sato Eimei, Nagai Takashi
Developmental Biology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Zygote. 2003 Aug;11(3):199-206. doi: 10.1017/s0967199403002247.
We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.
我们研究了侵袭性腺苷酸环化酶（iAC）、3-异丁基-1-甲基黄嘌呤（IBMX）和二丁酰环磷腺苷（dbcAMP）对猪卵母细胞体外成熟（IVM）、体外受精（IVF）及后续胚胎发育的影响。在补充或不补充0.1微克/毫升iAC和0.5毫摩尔/升IBMX的Hepes缓冲NCSU-37中收集猪卵母细胞。在补充或不补充1毫摩尔/升dbcAMP的改良NCSU-37中进行IVM 22小时，然后在不添加dbcAMP的情况下再培养24小时。IVF后，将卵母细胞体外培养6天。IVM 12小时后，无论在收集和IVM过程中是否添加这些化学物质，核状态均未观察到差异。在22小时时，用dbcAMP培养的卵母细胞大多数（95%）仍处于生发泡（GV）期，而未用dbcAMP培养的卵母细胞中有44.3%发生了GV破裂。在36小时时，用dbcAMP培养的卵母细胞已进展到前中期I或中期I（MI）（分别为32.6%和49.3%），而未处理的卵母细胞已进一步进展到后期I、末期I或中期II（MII）（分别为13.6%、14.3%和38.0%）。在46小时时，用dbcAMP培养的卵母细胞中处于MII期的成熟卵母细胞比例（81%）高于未用dbcAMP培养的卵母细胞（57%），而用dbcAMP培养时停滞在MI期的卵母细胞比例（15%）低于未用dbcAMP培养时（31%）。用dbcAMP培养的卵母细胞单精子受精率（21%）高于未用dbcAMP培养的卵母细胞（9%），总穿透率无差异（分别为58%和52%）。用dbcAMP培养的卵母细胞囊胚率（32%）高于未用dbcAMP培养的卵母细胞（19%）。这些结果表明，卵母细胞收集过程中细胞内cAMP水平的变化不影响猪卵母细胞的成熟和发育能力，并且使用dbcAMP使减数分裂成熟同步化可通过促进MI向MII的转变来增强卵母细胞的减数分裂潜能，并通过单精子受精产生高发育能力。
