| Autophagy plays an important role in embryonic development.In the female mammalian reproductive tract,the physiological oxygen concentration is lower than that in vitro embryo culture.However,there are few or controversial studies on how autophagy specifically regulates oocyte maturation and early embryonic development,especially under hypoxic culture conditions.Yaks live in hypoxic and alpine areas,which can be used as a typical representative for studying hypoxia.Therefore,the purpose of this study was to explore the correlation between autophagy regulation and hypoxia adaptation during yak oocyte maturation and early embryonic development,so as to provide new ideas and theoretical basis for further optimizing oocytes in vitro maturation and early embryonic development in yaks.This study conducted the following experiments:1.Oocytes were cultured with different concentrations of rapamycin,oocyte maturation was evaluated by the expansion of COCs and the extrusion of the first polar body,counting the cleavage rate and blastocyst rate of embryos after parthenogenetic activation of yak oocytes.The ROS level and mitochondrial membrane potential of yak oocytes were also detected.The expression of autophagy and apoptosis-related genes and proteins in COCs was detected by q RT-PCR and Western Blotting,to clarify the effects of rapamycin treatment on in vitro oocyte maturation and early embryonic developments.The results showed that when the concentration of rapamycin was 10n M,the maturation rate of oocytes was significantly higher than other concentration treatment groups（P<0.05）,when the concentration of rapamycin was 10o n M,the maturation rate of oocytes was significantly lower than other concentration treatment groups（P<0.05）.The relative gene expression of LC3b and Beclin-1 in 1 n M,10 n M,and 100 n M rapamycin were significantly increased compared with that in the 0 n M rapamycin treatment（P<0.05）,and were increased in a concentration-dependent manner;correspondingly,the relative protein expression of LC3-Ⅱand Beclin-1 in 1n M,10 n M,and 100 n M rapamycin were significantly increased compared with that in the 0 n M rapamycin treatment（P<0.05）,however,there were no significant changes between 0 n M and 1 n M（P>0.05）.Therefore,10 n M rapamycin was selected for subsequent experiments.10 n M rapamycin treatment significantly reduced ROS level and increased mitochondrial membrane potential,compared with the 0 n M group.The m RNA and protein expressions of Bcl-2 were up-regulated and the expression of Bax was down-regulated in mature COCs（P<0.05）.When parthenogenetic embryos were treated with different concentrations of rapamycin,10 n M rapamycin treatment showed higher 8-cell and blastocyst rates.In addition,the m RNA levels of CDX2,POU5F1,SOX2 and Nanog in blastocysts were also up-regulated（P<0.05）.2.Oocyte maturation was evaluated by the expansion of COCs and the extrusion of the first polar body,counting the cleavage rate and blastocyst rate of embryos,the ROS level and mitochondrial membrane potential of yak oocytes were detected,to clarify the correlation between autophagy induction and hypoxia adaptation during in vitro oocyte maturation in yaks.The results showed that the maturation rate of oocytes in the rapamycin treatment group was significantly higher than that in the control group under 1%O2 conditions（P<0.05）.Under 1%O2 conditions,the cleavage rate and blastocyst rate of subsequent parthenogenetic embryos in the rapamycin treatment group were also significantly higher than those in the control group（P<0.05）.At the same time,rapamycin treatment increased mitochondrial membrane potential and decreased ROS levels in each group under 1%O2,5%O2 and 20%O2 conditions（P<0.05）.The results showed that rapamycin-induced autophagy was beneficial to the in vitro maturation of yak oocytes under extreme hypoxia conditions（1%O2）.At different O2 concentrations,rapamycin-induced autophagy is involved in regulating mitochondrial membrane potential,ROS,and apoptosis,thereby further regulating oocyte maturation and subsequent embryonic development.3.Oocyte maturation was evaluated by the expansion of COCs and the extrusion of the first polar body,counting the cleavage rate and blastocyst rate of embryos,the ROS level and mitochondrial membrane potential of yak oocytes were detected,the expression of related genes in COCs was detected by q RT-PCR,to investigate the correlation between autophagy inhibition and hypoxia adaptation during in vitro maturation of yak oocytes.The results showed that when 3-MA inhibited autophagy under 1%O2 and 5%O2 conditions,the maturation rate of oocytes was significantly lower than control group（P<0.05）.When autophagy was inhibited under 1%O2 and5%O2 conditions,the cleavage rate and blastocyst rate of subsequent parthenogenetic embryos were significantly reduced（P<0.05）.Under 1%O2,5%O2 and 20%O2conditions,3-MA treatment reduced the mitochondrial membrane potential and increased ROS levels in each group.The results showed that inhibition of autophagy under extreme hypoxia（1%O2）and physiological normoxia（5%O2）conditions significantly reduced the maturation rate of yak oocytes and the development ability of subsequent parthenogenetic embryos.4.Counting the cleavage rate and blastocyst rate of embryos.The apoptosis rate of blastocysts was detected by TUNEL kit,and the expression of related genes in blastocysts was detected by q RT-PCR,to investigate the correlation between autophagy induction and hypoxia adaptation during early embryonic development of yak.The results showed that the cleavage rate and blastocyst rate of parthenogenetic embryos were significantly higher than those of 1%O2control group（P<0.05）.Autophagy was induced under 1%O2 conditions,and the blastocyst apoptosis rate was significantly reduced（P<0.05）.The results of q RT-PCR showed that after rapamycin induced autophagy,the relative expression of pro-apoptotic gene Bax m RNA in blastocysts cultured under 1%O2 condition was lower than control group（P<0.05）,and the relative expression of anti-apoptotic gene Bcl-2 m RNA was higher than control group（P<0.05）.The relative expression of pro-apoptotic gene Bax m RNA in blastocysts cultured under 5%O2 condition had no significantly change（P>0.05）,and the relative expression of Bcl-2 m RNA was significantly higher than control group under 5%O2condition（P<0.05）.The results showed that rapamycin-induced autophagy under extreme hypoxia（1%O2）could improve the development ability of early parthenogenetic embryos of yak,indicating that autophagy was involved in the early embryonic development of yak under extreme hypoxia（1%O2）.The above results indicate that autophagy protects yak oocytes and early embryos from quality loss and developmental delay caused by extreame hypoxia（1%O2）,and participates in the regulation of mitochondrial membrane potential,ROS level and apoptosis,thereby further regulating oocyte maturation. |
