| Heat stress caused by high temperature in summer leads to the decline of animal reproductive performance,which brings huge economic losses to animal husbandry.The reproductive performance of sows determines the efficiency of pig industry,and the quality of oocytes is the key to determine the reproductive performance of sows.In the long development process of oocytes,the maturation process before ovulation is the most critical,and it is also the most sensitive to heat stress.In this process,the cumulus oocyte complexes(COCs)which are composed of oocytes and cumulus cells,can resist all kinds of external stimuli to ensure the quality of oocyte maturation.However,it is not clear whether there are differences in the sensitivity of oocytes and cumulus cells to heat stress at different stages of maturation,and whether the two kinds of cells respond to heat stress are consistent.In addition,cumulus cells can protect oocytes from short-term heat stress,which fails when COCs are exposed to long-term heat stress.What is the difference between the response of cumulus cells to short-term and long-term heat stress?Can we explore the cellular and molecular mechanisms of heat stress affecting oocyte quality by analyzing differentially expressed genes?How are the key target genes regulated by heat stress?Is the expression of key target genes mediated by RNA modification(m6A)involved in stress regulation?Based on the above scientific problems,we have carried out the following four aspects of research.1 Transcriptome analysis of oocytes and cumulus cells at different stages during in vitro maturation and their response to short-term heat stressThe follicular fluid of young sows was extracted,and then COCs were collected for in vitro maturation culture.COCs were divided into two groups:control group(38.5℃)and short-term heat stress group(20-24 h,42℃).After cultured for 44 h in vitro,the quality of oocytes maturation and cell response to heat stress at different maturation stages(24 h and 44 h)were compared by transcriptome sequencing.The results showed that the short-term heat stress for 4 h had no significant effect on the survival rate of oocytes,the first polar body excretion rate and the proportion of oocytes developed to 2/4-cells and blastocysts.The nuclear and cytoplasmic maturation of oocytes did not change significantly,but the mitochondrial membrane potential of oocytes significantly decreased at 24 h which recovered at 44 h.These results suggest that short-term heat stress has no significant effect on oocyte quality,only slight disturbance occurs at 24 h.Transcriptome analysis of oocytes at different maturation stages showed that more gene expression was down regulated at 44 h than at 24 h,which was consistent with the suppression of genomic transcription during oocyte maturation.However,the cumulus cells did not show similar phenomenon.When the two kinds of cells were exposed to short-term heat stress,cumulus cells showed more significant response than oocytes and the transcriptome at 24 h was more significant than that at 44 h.The enriched DEGs were related to apoptosis,cytoskeleton and cell connectivity.The response of oocytes to heat stress was relatively slow,and the enrichment of DEGs was not significant.These results suggest that cumulus cells are more sensitive and responsive to heat stress,and the response of cumulus cells to heat stress is more obvious at 24 h than 44 h.Therefore,in order to further study the mechanism of heat stress affecting the maturation of COCs,we chose cumulus cells at 24 h during maturation.2 Effect of short and long-term heat stress on maturation and the transcriptome response of cumulus cells to heat stressThe results of the previous chapter show that cumulus cells can effectively protect oocytes from short-term thermal stimulation during in vitro maturation of pig COCs.However,when COCs were exposed to heat stress for a longer period of time,this protective effect failed.In this study,we compared transcriptome changes of cumulus cells in response to short-term and long-term heat stress to explore the cellular pathway,gene network and potential target genes which were involved in the protection process.The COCs were randomly divided into three groups:con group(CON),COC4 group(20-24 h,42℃)and COC24 group(0-24 h,42℃).The effects of different treatments on oocyte maturation quality were detected,and the transcriptome of cumulus cells after short and long-term heat stress treatment were analyzed.The results showed that the oocyte maturation quality was not affected in the short-term heat stress group but was significantly decreased in the long-term heat stress group.The oocyte survival rate and the first polar body excretion rate were significantly decreased(P<0.05 and P<0.05),and the structure of the cumulus extracellular matrix(ECM)was seriously damaged.The results showed that the accumulation of DEGs in cumulus cells after short-term heat stress treatment was not significant(P>0.05)However,in the long-term heat stress group,DEGs were significantly enriched in the pathways related to cell proliferation and ECM,and several genes related to the formation of cumulus ECM including PTX3 were only changed in COC24 group.These results indicate that the different effects of short and long-term heat stress on the maturation quality of oocytes may be caused by the differences of ECM production and structure.However,the specific mechanism of heat stress affecting ECM structure is not clear,and further research is needed.3 Effects of heat stress on cumulus ECM and mechanism involvedIn order to explore the possible mechanism of heat stress on porcine cumulus cell ECM,we divided the COCs into three groups:CON(38.5℃),COC4(20-24 h,42℃)and COC24(0-24 h,42℃)groups,The results showed that there was no significant difference between the short-term heat treatment group and the control group,while the long-term heat stress treatment led to a large amount of HA escaping from COCs cell mass into the medium,and the PTX3 transcript content decreased after long-heat stress(P<0.01),but the PTX3 protein content increased significantly(P<0.01),and the increased PTX3 did not stay in the COCs to play a stabilizing role,but entered into medium.In order to explore whether the regulation of PTX3 content occurs at the transcriptional level or at the post transcriptional level,we added transcription inhibitors actinomycin D(ActD)and translation inhibitor cyclooxygenamide(CHX)into the culture system respectively.It was found that after adding CHX,the level of PTX3 mRNA only in the long-term heat stress group was significantly increased(P<0.01),suggesting that the long-term heat stress treatment may increase the RNA stability of PTX3 or increase the efficiency of protein translation.Meanwhile,the total RNA of cells was detected by dot-blot,and the level of total RNA m6A modification in cumulus cells decreased significantly after long-term heat stress treatment(P<0.01).Therefore,we speculate that m6A may be involved in the regulation of PTX3 expression in porcine cumulus cells facing long-term heat stress,but the specific regulatory mechanism still needs to be further studied.4 Effects of heat stress on PTX3 and mechanism involved in KGN cellsDue to the small sample size of porcine COCs and the inconvenience of gene manipulation,we used human ovarian granulosa cell tumor(KGN)cell line as the model to simulate the changes of PTX3 expression under heat stress,and to explore the regulation mechanism of PTX3 expression.But surprisingly,the same heat stress treatment did not replicate the results of COCs in KGN cells.PTX3 in KGN cells did not show a decrease in mRNA but an increase in protein content after heat stress,but both mRNA and protein levels were significantly up regulated(P<0.01).Considering the interaction between two kinds of cells in COCs,and extracellular heat stress is oxidative stress in cells.We used H2O2 treatment(100 μM,1 h)to simulate the increase of ROS content in cumulus cells after heat stress and found that it can well simulate the changes of PTX3 after heat stress.After H2O2 treatment,ROS content increased significantly(P<0.01),PTX3 mRNA content decreased significantly(P<0.05),while protein content increased significantly(P<0.01).By adding transcription inhibitor ActD and translation inhibitor CHX,we found that CHX caused a significant increase in PTX3 mRNA content in H2O2 treated group,suggesting that there is post transcriptional or translational regulation.Further RNA stability experiments showed that H2O2 treatment significantly increased the stability of PTX3 mRNA.At the same time,H2O2 treatment significantly reduced the level of RNA m6A modification(P<0.01),and the expression of m6A demethylase FTO protein was significantly increased(P<0.01).Single base extension and ligation based PCR amplification method(SELECT)was used to detect the level of m6A at the specific site of PTX3 mRNA 3’UTR,which found that the abundance of m6A decreased significantly in H2O2 group(P<0.01).Over expression of mettl3 increased the level of m6A modification.The mRNA and protein contents of PTX3 in the control group were significantly decreased(P<0.05).However,in the H2O2 treatment group,over expression of METTL3 did not change the mRNA content of PTX3,but its protein content decreased significantly(P<0.05).The above results showed that H2O2 treatment increased ROS content and FTO protein level,decreased m6A abundance at PTX3 3’UTR,increased mRNA stability and increased PTX3 protein level.In conclusion,in vitro culture of porcine COCs,the maturation of oocytes was accompanied by oocyte genomic transcription inhibition,and the response to short-term heat stress was not obvious.While in cumulus cells,the transcriptome was highly active,especially at 24 h,the transcriptome showed significant response to short-term heat stress,and the differentially expressed genes were enriched in cell proliferation and ECM related pathways.The adaptive changes of cumulus cells to short-term heat stress protected the oocytes.By comparing the effects of long-term heat stress on the transcriptome of cumulus cells at different times,combined with the determination of oocyte quality,it was found that the damage of oocyte quality caused by long-term heat stress was related to the cumulus cell ECM,and PTX3 was the key target gene.By studying the mechanism of heat stress affecting the expression of PTX3,it was found that heat stress increased ROS content,up-regulated FTO protein,reduced the modification level of PTX3 mRNA 3’UTR specific m6A site,increased the stability of PTX3 mRNA and increased the translation of PTX3 protein.However,the up-regulated PTX3 protein was released into the medium instead of remaining in COCs in the form of polymerization to stabilize the ECM structure,which may involve PTX3’s post-translational modification and interaction with other ECM components,and it still needs further study. |
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