Study On The Occurrence Of Autophagy In Pig Parthenogenesis Activates Blastocysts Vitrification And The Effect Of Autophagy On Apoptosis After Freezing

Autophagy is an intracellular metabolic degradation process that can deliver endogenous or exogenous cytoplasmic material into lysosomes for degradation through autophagosomes.In general,autophagy promotes cell survival;However,autophagy activation may lead to cell death under stress conditions.Studies have found that cryogenic freezing promotes excessive activation of autophagy in germ cells,leading to cell death,and inhibition of autophagy can improve its survival rate.At present,the occurrence and effect of autophagy in early embryo freezing have not been discovered,and it is unclear whether freezing will lead to excessive activation of early autophagy,so this study carried out relevant studies on blastocysts activated by pig parthenogenesis and obtained the following results:1.In order to establish and optimize the vitrified freezing system of porcine parthenoblastocysts,the formula with the highest blastocyst cavity recovery rate and blastocyst cell number at 24 h after thawing was selected from three commonly used vitrification cryogenic solution formulations（p<0.05）.Then,after incubation with 7.5μg/ml of cytochalasins B（CB）for 45 minutes before freezing,it was found that CB could not significantly improve the recovery rate of blastocyst cavity and blastocyst cell number（p>0.05）24 h after thawing;finally,using the stepwise freezing method,the glass equilibration process was split into two to three steps,and a lower concentration of ES was used to pre-equilibrate first,thereby improving the cryopreservation effect by increasing the liquid replacement rate of the blastocyst cavity,and the results showed that 1/2 ES was equilibrated for 10 min,followed by a step-by-step freezing method of ES equilibration for 3-5 min,and the blastocyst cavity recovery rate was highest for 24 h（p<0.05）.In summary,pig parthenogenetic blastocysts were equilibrated with 1/2 concentration of vitrified equilibrium solution ES:HM+7.5%（DMSO+EG）for 10 min,then ES equilibrated for 3-5 min,and finally vitrified freezing method with vitrified cryogenic solution VS:HM+15%（DMSO+EG）+0.5M Su penetration for 40-60 s,and the cryopreservation effect was better.Pretreatment with CB before freezing does not improve cryopreservation results.2.In order to explore the changes of autophagy within 0-12 h of porcine parthenoblastocysts after thawing,the immunofluorescence staining of the blastocysts LC3B,P62 and ATG16L1 proteins phosphorylated at the serine 278 site was immunofluorescence-stained(p ATG16L1^s278),and it was found that the relative fluorescence intensity of LC3B and P62 changed in the blastocyst after thawing,showing a trend of increasing first and then decreasing The relative fluorescence intensity 0 h after thawing was the same as that of the unfrozen control group（p>0.05）,then gradually increased,reaching the highest point（p<0.05）at 6 h after thawing,and then gradually decreasing to the same level as the unfrozen control group（p>0.05）at about 12 h.The relative fluorescence intensity of p ATG16L1^s278 decreased first and then increased within 0-12 h after thawing,except for a significant decrease in fluorescence intensity at 6 h（p<0.05）,and there was no significant difference between the fluorescence intensity at other time points and the unfrozen control group（p>0.05）.In summary,vitrification of porcine parthenoblastocysts does not activate autophagy,but affects the degradation process of autophagy within 6 h after thawing,resulting in the accumulation of intracellular LC3B and P62,and negative feedback regulation reduces the occurrence level of autophagy.3.The results of the previous test showed that autophagy was not activated after freezing and thawing,but the autophagy process was blocked.In order to explore the effect of this change on apoptosis after early blastocyst thawing in pig parthenosperm,the autophagy inhibitor hydroxychloroquine（HCQ）was used for the study.LC3B immunofluorescence staining screening showed that 100μM HCQ significantly inhibited autophagy activity within0-6 h after thawing.Thaw blastocysts with this concentration of HCQ for 6 h after TUNEL staining and q RT-PCR.The results showed that the apoptosis level（p<0.05）of porcine parthenoblastocysts after 6 h treatment with HCQ of 100μM significantly reduced the apoptosis level（p<0.05）and the relative transcription levels（p<0.05）of autophagy-related genes LC3B,ATG5 and apoptosis-related genes were down-regulated.However,after continuing to culture for 6 h after HCQ treatment,the apoptosis level of blastocysts was significantly increased（p<0.05）,and the relative expression levels of LC3B,P62 and Bax were significantly increased（p<0.05）.In conclusion,the treatment of thawed porcine parthenoblastocysts with 100μM HCQ significantly inhibited their autophagy levels,and HCQ treatment for 6 h significantly reduced the apoptosis level of thawed blastocysts and improved the survival rate of blastocyst cells.In summary,pig parthenogenetic blastocysts were equilibrated by 1/2 concentration of vitrification equilibrium solution ES:HM+7.5%（DMSO+EG）for 10 min,then ES equilibrated for 3-5 min,and finally vitrified freezing solution VS:HM+15%（DMSO+EG）+0.5M Su penetrated for 40-60 s,and the cryopreservation effect was better.Vitrification of porcine parthenoblastocysts does not activate autophagy,but affects the degradation process of autophagy within 6 h after thawing,resulting in the accumulation of intracellular LC3B and P62,and negative feedback regulation reduces the occurrence level of autophagy.Treatment with 100μM HCQ of thawed porca can significantly inhibit the autophagy level,and treatment with HCQ for 6 h can significantly reduce the apoptosis level of thawed blastocysts and improve the survival rate of blastocyst cells.