Mahesh Yelisetti Uma, Gibence Henderson Rose Winnie, Shivaji Sisinthy, Rao Brahmasani Sambasiva
CSIR- Centre for Cellular and Molecular Biology (CCMB), Hyderabad 500007, India.
CSIR- Centre for Cellular and Molecular Biology (CCMB), Hyderabad 500007, India.
Cryobiology. 2017 Apr;75:106-116. doi: 10.1016/j.cryobiol.2017.01.004. Epub 2017 Jan 16.
The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.
本研究的目的是确定一种最适合水牛未成熟卵丘-卵母细胞复合体（COCs）玻璃化冷冻的冷冻装置，这是根据玻璃化-解冻后卵母细胞的活力、减数分裂能力及其体外受精（IVF）后的发育能力来判断的。在体外成熟后，比较了玻璃化-解冻卵母细胞中卵母细胞分泌因子及其受体（GDF9、BMP15、BMPR2、TGFBR1）和凋亡相关基因（BCL2、BAX、P53、C-MYC）的表达。使用常规细管（CS）、开放式拉制细管（OPS）、冷冻环（CL）、半细管（HS）或冷冻载玻片（CT），将屠宰后水牛卵巢中的COCs在二甲基亚砜、乙二醇和蔗糖的组合中进行玻璃化冷冻。新鲜的COCs像在其他玻璃化方法中一样暴露于玻璃化和解冻溶液中，但不投入液氮（EC）。HS和CT组在解冻后2小时玻璃化-解冻COCs的活力与新鲜组和EC组相似，但显著高于CS和OPS方法。HS和CT组在体外成熟24小时后具有第一极体的卵母细胞比例显著高于CS、OPS和CL方法。所有玻璃化组中这些玻璃化-解冻卵母细胞在IVF后发育到囊胚阶段的能力显著低于对照组和EC组。在玻璃化组中，HS、CT和CL组的囊胚率显著高于OPS和CS组。还观察到CT、HS和CL组中玻璃化-解冻COCs中GDF9、BMP15、BMPR2、TGFBR1、BCL2、BAX、P53和C-MYC基因的表达水平与对照组相似。结果表明，HS、CT和CL是更适合水牛未成熟卵母细胞玻璃化冷冻的冷冻装置。
