Development to the blastocyst stage of parthenogenetically activated in vitro matured porcine oocytes after solid surface vitrification (SSV)
Development to the blastocyst stage of parthenogenetically activated in vitro matured porcine oocytes after solid surface vitrification (SSV)
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DOI：
10.1016/j.theriogenology.2005.11.023
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发表时间：
2006-07-15
期刊：
影响因子：
2.8
通讯作者：
Niemann, Heiner
中科院分区：
文献类型：
--
作者：
Somfai, Tamas;Dinnyes, Andras;Niemann, Heiner
研究了固体表面玻璃化冷冻（SSV）对猪体外成熟卵母细胞活力和孤雌发育的影响。对无卵丘IVM猪卵母细胞进行SSV或SSV与细胞松弛素B（CB）预处理（SSV + CB）或SSV的所有步骤但不冷却（毒性对照= TC; CB预处理的毒性对照= TC + CB）。卵母细胞的活力进行了评价质膜的完整性和酯酶活性测定的荧光素二乙酸酯，碘化丙啶和Hoechst 33342的组合染色。将存活的卵母细胞孤雌激活，然后在体外（IVC）培养6天。无论CB预处理与否，玻璃化冷冻后的活卵比例均显著低于TC、TC + CB和对照组。用冷冻保护剂处理卵母细胞不会降低卵母细胞的存活率。卵母细胞激活后，SSV + CB组的卵裂率显著高于SSV组（P < 0.05）。而TC组、TC + CB组和对照组的卵裂率显著高于对照组（P < 0.05）。第6天，SSV和SSV + CB组的囊胚率无显著差异。TC组、TC + CB组和对照组的卵母细胞发育至囊胚的数量和每个胚胎的平均卵裂球数均显著高于SSV组和SSV + CB组（P < 0.05）。据我们所知，这是第一个报告孤雌发育的猪卵母细胞玻璃化冷冻中期II阶段的囊胚。结果表明，高浓度的冷冻保护剂对猪卵母细胞的体外发育无不良影响，CB预处理可提高猪SSV玻璃化冷冻卵母细胞的存活率和发育率。(c)2006年爱思唯尔公司All rights reserved.
The effects of solid surface vitrification (SSV) on viability and parthenogenetic development of in vitro matured (IVM) porcine oocytes was investigated in the present study. Cumulus-free IVM porcine oocytes were subjected either to SSV or SSV combined with a cytochalasin B (CB) pre-treatment (SSV + CB) or all steps of SSV but without cooling (toxicity control = TC; toxicity control with CB pre-treatment = TC + CB). Oocyte viability was evaluated by plasma membrane integrity and esterase activity measured by a combined staining with fluorescein diacetate, propidium iodide and Hoechst 33342. Surviving oocytes were parthenogenetically activated then cultured in vitro (IVC) for 6 days. The proportion of live oocytes after vitrification was significantly lower than that of the TC, TC + CB and the control groups, regardless of the CB pre-treatment. Treatment of oocytes with cryoprotectants did not decrease the rates of surviving oocytes. After activation of oocytes, the proportion of cleaved embryos was significantly higher in the SSV + CB (P < 0.05) than that of the SSV group. Nevertheless, significantly more oocytes cleaved (P < 0.05) in the TC, TC + CB and the control groups. On Day 6, the rate of blastocysts in the SSV and SSV + CB groups did not differ significantly. The number of oocytes developing to blastocyst and the mean number of blastomeres per embryo were significantly higher (P < 0.05) in the TC, TC + CB and the control compared with that of the SSV and SSV + CB groups. To our knowledge, this is the first report on parthenogenetic development to blastocysts of porcine oocytes vitrified at the metaphase-II stage. Results indicate that the high concentrations of cryoprotectants were not harmful for in vitro development, and that CB pretreatment may increase survival and development of SSV vitrified porcine oocytes. (c) 2006 Elsevier Inc. All rights reserved.