The Effect And Physiological Mechanism Of Heat Stress On Interactions Between Oocytes And Cumulus Cells

The oocytes produced by female animals play important roles in livestock reproductions.Its quality is directly related to the fertilization,embryo development,pregnancy establishment and maintenance,and even the growth and health of offspring animals.The oocyte quality depends mainly on the fully maturation of nucleus and cytoplasm,which the latter is based on the nutrient and signal interactions between oocytes and their companion cumulus cells(CCs)through transzonal projections(TZPs)and gap junctions(GJs)intercellular structures by forming the cumulus-oocyte complexes(COCs).Heat stress(HS)is one kind of unavoidable stressors during the animal breedings,which may cause serious declines in animal reproductive capacities including pigs,cows and mouse,by inducing oocyte dysplasia during the first meiotic metaphase(MI),telophase(TI)and the second meiotic metaphase(MⅡ)periods.However,so far,the molecular mechanical explorations about the HS-induced oocyte quality damages are mainly established on the unilateral analyses of oocyte or CCs functions,yet little attention was paid to the intercellular interactions between oocytes and CCs under HS conditions.Therefore,in this study,two experimental models including the porcine ovarian HS model and the COCs HS model were firstly used to verify the adverse effects of HS on oocyte qualities.Secondly,the specific HS responses of oocytes and CCs in complexes or in isolation,as well as their correlations with intercellular communications were investigated in both models.Thirdly,the influence of HS on TZPs communication structures between oocyte and CCs was detected from gene and protein expression levels,and appropriate physiological regulators were selected on this basis to alleviate the damages of oocyte qualities caused by HS.1 Effect of different HS models on the porcine oocyte qualityHealthy ovaries obtained from the cross-bred prepubertal gilts(Duroc × Landrace ×Yorkshire;135～170 days of age;70～120 kg of body weight)slaughtered in a local abattoir located in Qixia district,Nanjing were transported to the laboratory within 3 hours.In ovarian HS experiment:ovaries were randomly divided into control group and HS group and then transferred into 38.5℃ and 41.5℃ water bath respectively for the experimental treatments.Subsequently,follicular fluid was collected from the follicles 3～6 mm in diameter,and COCs were aspirated under the stereoscopic microscope for further in vitro maturation(IVM)under normal conditions.COCs were then digested with 1 mg/mL hyaluronidase(Hya)to remove the CCs,the remaining oocytes were used for further experimental analyses.Results showed that the oocyte survival rate and the first polar body extrusion rate were significantly decreased(P<0.05)after 1,2 and 4 hours of ovarian HS,when compared to the control group.After 1 h ovarian HS and 24 h additional IVM,both CCs expansion rate and the ratio of mitochondrial cluster distribution pattern in oocytes decreased significantly(P<0.05).Meanwhile,ovarian HS made more than 85%of in vitro matured oocytes arrested in GV to MI phases,during which period more than 85%of the control group oocytes have entered the TI to MⅡ stages.In COCs HS experiment:ovaries were directly transferred into 38.5℃ water bath located in the sterile room to collect the follicular fluid and aspirate COCs.COCs were cultured and heat-stressed during the first IVM 0～24 h in vitro,The treated COCs were partially digested with Hya and oocytes were used for fluorescence staining and microimaging,the other part of COCs were cultured for another 20 h under normal conditions for further development potential detections after the parthenogenetic activation.Results revealed that COCs HS for 24 h in vitro remarkably reduced the mass of oocytes,characterized by significant decreases in the CCs expansion rate,oocyte survival rate,first polar body extrusion rate and the proportion of mitochondrial peri-pronuclear distribution(P<0.05).At the same time,COCs thermal stress still markedly inhibited the early embryonic development potential of oocytes after parthenogenetic activation,including significant decreases in 2-cell and 4-cell cleavage rate and blastocyst rate(P<0.05).Taken together,the present study show that under both ovarian HS and COCs HS models,hyperthermal stimulation significantly blocked the oocyte developmental processes,which thus resulted in the degradations of oocyte qualities and their subsequent embryonic developmental capacities.2 Distinct HS responses of porcine oocytes and CCsCOCs from ovarian and COCs HS models were obtained after 24 h IVM and digested with Hya to isolate the oocytes and CCs,which were used for further experimental analyses.Ovarian HS experiment showed that,mitochondrial DNA(mtDNA)-encoded genes and heat shock protein(HSPs)related genes expression levels as well as ATP content in oocytes and CCs were significantly down-regulated,accompanied by notable increases in the mitochondrial reactive oxygen species(mROS)levels(P<0.05).While,results from COCs IIS model showed that oocytes and CCs isolated from COCs displayed distinct responses to HS.The messenger RNA abundance of HSP-related genes and mtDNA-encoded genes,together with ATP content,were significantly increased in CCs,yet decreased in oocytes,despite activation of caspase 3 detected in both CCs and oocytes(P<0.05).Meanwhile,to verify the effect of HS on the interactions between oocytes and CCs,oocytes and CCs were isolated directly after the collection of COCs and then heat stressed during the IVM 0～24 h period,individually.Similar results including the HSPs-related genes expression,ATP content and the caspase 3 activities were observed in the denuded oocytes and stripped CCs subjected to HS separately.However,interestingly,mROS was significantly increased only in oocytes from heat-stressed COCs,but when stripped CCs and denuded oocytes were heat stressed individually,mROS was only increased in CCs(P<0.01).The results hint us the different responses of oocytes and CCs to HS may due to the interference of intercellular communications under the hyperthermal environment.3 Influence of HS on intercellular TZPs structures between oocyte and CCsSimilarly,oocytes and CCs were obtained from both ovarian and COCs HS models to analyze the monomeric actin gene and protein expressions as well as the TZPs communication structural variations.Results from ovarian HS model showed that TZPs number and the filamentous actin(F-actin)fluorescence intensities decreased gradually with the development of oocytes in both control and HS group,and HS significantly reduced the TZPs numbers and F-actin intensities at IVM 24 h(P<0.05).In terms of the monomeric actins,the mRNA expression levels of β-actin and y-actin in control group oocytes increased firstly and decreased afterwards,and the protein expression levels were gradually decreased in control group,HS significantly down-regulated both β-actin and y-actin protein levels at IVM 24 h and IVM 44 h when compared with the control group(P<0.05).On the contrary,both gene and protein expression levels of β-actin and y-actin in control group CCs increased gradually with the advance of IVM time,and HS significantly down-regulated the gene expressions of both actins at IVM 24 h and IVM 44 h as well as their protein expressions at IVM 24 h(P<0.05),compared to the control group.The subsequent fluorescence staining experiments showed that the co-localization rates ofβ-actin and y-actin with F-actin were about 25%and 75%,respectively,indicating that F-actin is mainly composed by y-actin proteins.The co-localization results of TZPs and GJs structures showed that though fluorescence intensity of Connexin 45,which is a GJs marker protein,was markedly up-regulated after HS,the fluorescence intensity of F-actin as well as the costaining areas of TZPs and GJs on oolemma were significantly down-regulated(P<0.05).At the same time,COCs HS experiment demonstrated that HS also significantly reduced(P<0.05)the TZPs numbers and the F-actin intensities at IVM 24 h.Oocyte and CCs displayed opposite changing patterns in monomeric actins expressions during the IVM period,among which the mRNA and protein expression levels of β-actin and y-actin decreased gradually in control group oocytes,but increased gradually in control group CCs.HS significantly down-regulated the β-actin and y-actin protein levels at IVM 24 h in both oocytes and CCs,when compared with the control group(P<0.05).The results above indicated that the structure of TZPs was mainly composed by y-actin,and HS could affect the formation and stability of F-actin and the TZPs structures by reducing the y-actin protein expression in CCs.4 Alleviating measures of HS-induced oocyte quality reductionIn consideration of the changes of ATP,mROS and monomeric actin expressions as described above,physiological regulators including N-acetyl-L-cysteine(NAC),ATP and N,N,N-trimethylglycine(betaine)were added into the culture medium during the first 24 h HS treatment,effective physiological regulators were screened by the detection of oocyte survival rate and the first polar body extrusion rate,and the possible pathway involved here was also explored.Results revealed that HS significantly reduced(P<0.05)the oocyte survival rate and the first polar body extrusion rate at IVM 44 h,but no alleviating effect was observed after the NAC and betaine addition.Interestingly,when supplied with 200 nM ATP,the decreases of oocyte survival rate as well as the TZPs impairment caused by HS were obviously improved(P<0.05),and a mitigative trend(P=0.075)in the first polar body extrusion rate was also observed.Meanwhile,HS significantly up-regulated the HSP27 mRNA and protein expression levels,but markedly down-regulated the β-actin andγ-actin protein expression lever in CCs.Again,ATP addition observably alleviated(P<0.05)the high expression level of HSP27 protein in CCs caused by HS.In summary,the results above showed for the first time that HS disrupted the formation and homeostasis of F-actin and TZPs structures mainly through down-regulating the y-actin and up-regulating the HSP27 protein expressions in CCs.Therefore,by inhibiting the excessive expression of HSP27 protein in CCs,ATP addition can effectively alleviate the damages of F-actin and TZPs structures,and thus reverse the oocyte quality degradations induced by HS.