Effects And Molecular Mechanisms Of Postovulatory Aging Mouse Oocytes On Offspring Anxiety And Cognitive Behaviors

After ovulation,MII stage oocytes of mammals that remains in the oviduct（in vivo）or in culture medium（in vitro）,will undergo a time-dependent process of quality deterioration if they do not fertilized in time,this is referred to as postovulatory oocyte aging（POA）.POA is a complex and irreversible process,accompanied by a series of changes at the morphological,cellular,and molecular biology.It is reported that the fertilization of aged oocytes can lead to abnormalities in the offspring.In reality,the fertilization of aged oocytes with fresh spermatozoa occurs in human and some primate reproductive cycles as well as in oocytes used in assisted reproductive technology（ART）.Therefore,it is of great significance to explore the effects of postovulatory oocyte aging on the offspring.However,most of the studies on POA reported thus far have focused on its effects on oocyte quality and early embryo development,information about its long-term effects on offspring are limited.Aims of this study were to examine mouse oocytes aged in vivo and in vitro to test the hypothesis of whether there are effects on anxiety and cognitive behavior in offspring.In this study,mouse oocytes aged in vivo and in vitro were subjected to in vitro fertilization and their blastocysts were transferred to pseudopregnant recipients,and anxiety-like behavior,cognitive behaviors,and the expression of related genes in the F1 generation and F2generation were observed with the aim of obtaining information about the effects of POA on the behaviors of offspring and their molecular mechanisms.This experiment was conducted by in vivo and in vitro test models of oocyte aging.In the model in vivo,Freshly ovulated oocytes and in vivo aging oocytes were fertilized in vitro to observe the pre-implantation embryo development potential,respectively,the obtained blastocysts were transferred to pseudopregnant recipients,and the obtained offspring were used to test and analyze for anxiety-like behaviors and cognitive behaviors.In the model in vitro,in vitro aging oocytes that were obtained by culturing the freshly ovulated oocytes for 9h in media with different antioxidant levels.In vitro aging oocytes were fertilized in vitro to observe the embryo development,and the blastocysts obtained from the in vitro culture were also transferred to pseudopregnant recipients to obtain the F1 generation,and the F2generation（sexually mature female and male mice of the F1 generation mated with naturally born male and female mice in a combined cage,respectively）,and the anxiety-like behaviors and cognitive behaviors of the F1 and F2 generations were examined.The results of this study show that:1.POA affected blastocyst development and increased oxidative stress and mitochondrial damage.In vivo aging oocytes could be successfully fertilized in vitro with no significant difference in fertilization rate and 4-cell rates compared to that of freshly ovulated oocytes（p>0.05）;however,blastocyst rates were significantly lower（p<0.05）.Oxidative stress occurred with increased ROS levels in both in vivo and vitro aging oocytes and their resultant blastocysts and a significant decrease in GSH/GSSG;meanwhile,the mitochondrial membrane potential was significantly decreased（p<0.05）.The expression levels of mitochondrial function-related genes Pgc1-α,Sirt1,Cox1 and Cox2 were significantly down-regulated（p<0.05）,and the expression levels of mitophagy genes Pink1 and Parkin were elevated,suggesting the occurrence of mitochondrial function impairment.The results of POA in vitro in culture medium with different antioxidant potential showed that freshly ovulated oocytes cultured for 9 h in different antioxidant potential medium levels could be successfully fertilized in vitro,and there was no significant difference in fertilization rate and 4-cell rate,but the blastocyst rate tended to decrease with the weakening of antioxidant potential of the culture medium,and the blastocyst rate was significantly lower than that in the high antioxidant potential culture medium（p<0.05）.The ROS level of oocytes aged in vitro showed a tendency to increase with the decrease of antioxidant potential,while the mitochondrial membrane potential level and the expression of mitochondrial function-related genes were significantly decreased,and the expression of mitophagy-related genes was significantly up-regulated,which suggests that the mitochondrial function is impaired.Blastocysts derived from oocytes aged in vitro in low antioxidant potential culture medium showed similar results to oocytes in vivo aged,also exhibited increased H₂O₂ levels,suggesting elevated ROS levels.2.POA increases anxiety-like behavior of offspringIn order to investigate whether POA affects the anxiety-like behavior of offspring,the anxiety-like behavior of the offspring was tested using elevated plus maze and open field tests,and their serum cortisol levels were also detected.The results showed that the ratio of the time spent in the open arm of the elevated plus maze to the sum of the time spent in the open and closed arms（OT/（OT+CT））was significantly decrease in female and male offspring derived from in vivo aging oocytes,and the time spent in the central region of the open field was significantly decrease.After restraint stress for 5 min,cortisol levels were significantly higher in F1 generation derived from aged oocyte than that of in F1 generation derived from freshly ovulated oocyte.These changes are suggestive of a significant increase in the level of anxiety-like behavior in both female and male offspring（F1 generation）derived from fertilization of oocytes aged in vivo.There was no statistically significant difference in the results of the anxiety behavior test in the F2 generation.The results of anxiety behavior tests on oocytes aged in vitro showed that the OT/（OT+CT）values of female and male offspring derived from oocytes aged in low antioxidant-potential medium were significantly lower;and the time spent in the central zone of the open field was significantly reduced.Given 5 min of restraint stress,cortisol levels were significantly higher in F1 littermates than that of in F1 littermates from oocytes aged in high antioxidant potential medium.This suggests that both female and male offspring（F1generation）produced by fertilization of oocytes aged in vitro have the same anxiety behavior.3.POA impairs cognitive behavior of the offspringIn order to investigate the effects of POA on the cognitive behavior of their offspring,passive avoidance and Morris water maze experiments were used to test their fear memory,spatial learning and memory abilities.The results showed that the cognitive behavior（fear memory,spatial learning and memory abilities）of both female and male F1 generations produced by oocytes aged in vivo were significantly decreased.And it did not affect the cognitive behavior of their F2 generation mice.The results from oocytes aged in vitro showed that the cognitive behavior of the F1 generation derived from in the culture medium with low antioxidant potential decreased significantly than that in F1 generation derived from in the culture medium with high antioxidant potential,and there was no significant difference in the cognitive behavior of their F2 generation mice,which suggests that POA in vitro does not have a significant effect on the cognitive behavior of the F2 generation.4.Preliminary investigation of the mechanism underlying POA on anxiety-like and cognitive behavior of offspringIn order to explore the mechanisms by which POA leads to behavioral abnormalities in their offspring,we examined the levels of oxidative stress,mitochondrial function,and the expression of anxiolytic and cognitive behavior-related genes in the hippocampus of the offspring.The results showed that the hippocampus of adult F1 generation derived from in vivo aging oocytes had significantly increased H₂O₂ levels and significantly decreased ATP and COX levels,which indicated enhanced oxidative stress and impaired mitochondrial function in the hippocampus of adult F1 generation derived from in vivo aging oocytes.Related gene expression tests showed that the m RNA expression levels of anxiolytic genes and cognitive behavior-related genes,such as Bdnf,Gr and Nr2a,as well as mitochondrial function-related genes,Pgc1-α,Sirt1,Cox1,and Cox2 were significantly down-regulated.These results suggest that the behavioral abnormalities of the offspring littermates may be caused by the changes in these indicators.The results of the POA in vitro equivalent assay showed that the changes in H₂O₂,ATP and COX contents in the hippocampus of adult F1 generation derived from oocytes in vitro aging in low antioxidant potential medium were consistent with the results of in vivo aging.The m RNA expression levels of anxiolytic and cognitive behavior-related genes such as Bdnf,Gr and Nr2a,as well as mitochondrial function-related genes Pgc1-α,Sirt1,Cox1,and Cox2were also significantly down-regulated,which was consistent with the results from POA in vivo.In summary,under the conditions of this study,the rate of blastocysts derived from after fertilization of vivo and vitro aging oocytes was reduced,and the blastocysts showed increased oxidative stress and damage mitochondrial function;the F1 generation produced by aged oocytes all showed significant increases in anxiety behaviors,markedly damage cognitive behaviors,and an increase in cellular mitochondrial functional damage and oxidative stress in the hippocampus,accompanied by a significant decrease in m RNA expression levels of anxiolytic and cognitive behavior-related genes,as well as mitochondrial function-related genes in the hippocampus.The results of this study provide a theoretical basis for improving the adverse effects of POA on offspring.