Xiong Xian-rong, Wang Li-jun, Wang Yong-sheng, Hua Song, Zi Xiang-dong, Zhang Yong
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.
Zygote. 2014 Feb;22(1):1-9. doi: 10.1017/S0967199412000184. Epub 2012 Jul 11.
The preference of fertilized (IVF) and somatic cell nuclear transfer (SCNT) presumptive zygotes for different media when cultured in vitro to the blastocyst stage was evaluated in this study. The experiment comprised two zygote production methods (IVF and SCNT) × two culture media (mSOF and G1.5/G2.5) factorial design in which culture droplets that contained approximate 30 presumptive zygotes formed the experimental plots for the assessment of cleavage and blastocyst development. There were 15 to 20 replicates (culture droplets) per treatment combination. Sub-samples 30 to 41 of the blastocysts produced were assessed for cell number and cell apoptosis. A further 10 blastocysts per treatment combination were used for quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the relative abundance of Hsp70 and Bax mRNA. Presumptive zygotes produced by IVF were developmentally more competent than SCNT zygotes in terms of cleavage rate (66.9 vs. 57.0%; P < 0.05) and blastocyst development rates (blastocysts of presumptive zygotes 29.7 vs. 24.8%; blastocysts of cleaved zygotes 44.4 vs. 36.6%; P < 0.05). Over both zygote production systems, however, the results were similar whether culture was in mSOF or in G1.5/G2.5 media for cleavage rate (63.2 vs. 62.4%; P > 0.05) and blastocyst development rate (blastocysts of presumptive zygotes 26.4 vs. 25.7%; P > 0.05; blastocysts of cleaved zygotes 41.8 vs. 41.2%; P > 0.05). There was, however, a significant interaction between the method of zygote production and culture medium for the apoptotic index of blastocysts. The interaction was such that IVF-produced zygotes cultured in mSOF had a lower apoptotic index compared with those cultured in G1.5/G2.5 (4.7 ± 1.2% vs. 9.8 ± 0.9%; P < 0.05) whereas SCNT zygotes had a higher apoptotic index when cultured in mSOF compared with those cultured in G1.5/G2.5 (11.9 ± 1.5% vs. 4.5 ± 1.2%; P < 0.05). Moreover, RT-PCR analysis showed that embryos from IVF-produced zygotes cultured in mSOF had a lower expression level of stress-related and apoptosis genes (Hsp70 and Bax) than those cells cultured in G1.5/G2.5 medium, while SCNT-derived embryos cultured in mSOF had a higher expression level of these genes than those embryos cultured in G1.5/G2.5 medium. The results of this study show that bovine IVF- and SCNT-produced presumptive zygotes have different nutrient requirements for in vitro culture to the blastocyst stage of development. IVF-derived zygotes have a preference for mSOF as the culture medium whereas the G1.5/G2.5 medium is more suitable for the culture of bovine SCNT-derived zygotes.
本研究评估了体外培养至囊胚阶段时，受精（IVF）和体细胞核移植（SCNT）的假定受精卵对不同培养基的偏好。实验采用两种受精卵生产方法（IVF和SCNT）×两种培养基（mSOF和G1.5/G2.5）的析因设计，其中含有约30个假定受精卵的培养滴构成用于评估卵裂和囊胚发育的实验区组。每个处理组合有15至20个重复（培养滴）。对所产生囊胚的30至41个亚样本进行细胞数量和细胞凋亡评估。每个处理组合再取10个囊胚用于定量实时聚合酶链反应（RT-PCR），以评估Hsp70和Bax mRNA的相对丰度。就卵裂率（66.9%对57.0%；P<0.05）和囊胚发育率而言（假定受精卵的囊胚29.7%对24.8%；卵裂受精卵的囊胚44.4%对36.6%；P<0.05），IVF产生的假定受精卵在发育能力上比SCNT受精卵更强。然而，在两种受精卵生产系统中，无论在mSOF还是G1.5/G2.5培养基中培养，卵裂率（63.2%对62.4%；P>0.05）和囊胚发育率（假定受精卵的囊胚26.4%对25.7%；P>0.05；卵裂受精卵的囊胚41.8%对41.2%；P>0.05）的结果相似。然而，受精卵生产方法和培养基之间对于囊胚的凋亡指数存在显著交互作用。这种交互作用表现为，在mSOF中培养的IVF产生的受精卵与在G1.5/G2.5中培养的相比，凋亡指数更低（4.7±1.2%对9.8±0.9%；P<0.05），而在mSOF中培养的SCNT受精卵与在G1.5/G2.5中培养的相比，凋亡指数更高（11.9±1.5%对4.5±1.2%；P<0.05）。此外，RT-PCR分析表明，在mSOF中培养的IVF产生的受精卵来源的胚胎，其应激相关和凋亡基因（Hsp70和Bax）的表达水平低于在G1.5/G2.5培养基中培养的细胞，而在mSOF中培养的SCNT来源的胚胎，这些基因的表达水平高于在G1.5/G2.5培养基中培养的胚胎。本研究结果表明，牛IVF和SCNT产生的假定受精卵在体外培养至囊胚发育阶段时有不同的营养需求。IVF来源的受精卵偏好mSOF作为培养基，而G1.5/G2.5培养基更适合牛SCNT来源的受精卵的培养。
