Elamaran G, Singh K P, Singh M K, Singla S K, Chauhan M S, Manik R S, Palta P
Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
Reprod Domest Anim. 2012 Dec;47(6):1027-36. doi: 10.1111/j.1439-0531.2012.02009.x. Epub 2012 Mar 27.
This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.
本研究检测了体外成熟（IVM）、受精（IVF）和培养（IVC）过程中氧气浓度（5% 与 20%），或在 IVM 和 IVC 培养基中添加半胱胺（分别为 50 和 100 μM；IVM、IVF 和 IVC 在 20% 氧气浓度下进行），对水牛未成熟及体外成熟卵母细胞以及 2 - 细胞、4 - 细胞、8 - 16 细胞、桑葚胚和囊胚阶段胚胎的囊胚率以及通过实时定量 PCR 检测的一些凋亡相关基因的相对 mRNA 丰度的影响。在 5% 氧气浓度下，囊胚率显著更高（p < 0.05），而 TUNEL 阳性细胞百分比显著更低（p < 0.05）。在许多胚胎阶段，抗凋亡基因 BCL - 2 和 MCL - 1 的 mRNA 表达在 5% 氧气浓度下显著更高（p < 0.05），而促凋亡基因 BAX 和 BID 的表达更低（p < 0.05）。添加半胱胺后，在胚胎发育的许多阶段，囊胚率以及 BCL - XL 和 MCL - 1 的相对 mRNA 丰度显著更高（p < 0.05），BAX 的表达更低（p < 0.05），但 BID 的表达未受影响，尽管它对囊胚中 TUNEL 阳性细胞百分比无显著影响。这些基因在胚胎发育过程中的 mRNA 表达模式在 5% 与 20% 氧气浓度组以及添加半胱胺组与对照组之间存在差异。在水牛胚胎发育阻滞发生的 8 - 16 细胞阶段，BCL - 2 和 MCL - 1 的相对 mRNA 丰度在 5% 氧气浓度下最高，而 BAX 和 BID 的相对 mRNA 丰度在 20% 氧气浓度下最高（p < 0.05）。这些结果表明，在体外胚胎生产过程中，低氧气浓度和添加半胱胺产生有益作用的机制之一是通过增加抗凋亡基因的表达和降低促凋亡基因的表达来实现的。
