The Mechanism And Regulation Of The Effect Of Vitrification On The Plasma Membrane Sperm Fusion Protein JUNO Of Bovine Oocytes

Cryopreservation of oocytes is the preservation of oocytes in a low-temperature environment.This technology maintains the integrity of the oocytes and stops their biological activities through low temperatures.Vitrification involves an ice-free approach to convert a liquid containing living cells or tissues into a glass-like amorphous solid either using high concentrations of cryoprotectants(CPAs)and rapidly cooling.Although vitrification is widely used in various fields,it greatly improves the application efficiency of oocytes.However,the significantly reduced fertilization ability of oocytes after vitrification has become a huge obstacle restricting the application of vitrified oocytes.In 2014,Bianchi et al.discovered that folate receptor 4 on the plasma membrane of oocytes is a key protein in the fertilization process and named it "Juno".But at present,there are few reports on the study of the effect of vitrification on the JUNO protein of bovine oocytes.In response to the above problems,in this study,from the perspectives of JUNO protein expression related events(gene methylation and m RNA and expression detection)and protein function(sperm fusion,embryonic development ability),the mechanism of the effect of vitrification on the JUNO protein of bovine oocytes was explored.At the same time,JUNO m RNA was microinjected in the GV phase to develop effective regulatory measures.The results of the study are as follows:1.Taking bovine oocytes as the research object,perform vitrification of mature bovine oocytes.The levels of JUNO gene methylation,JUNO m RNA levels and JUNO protein expression levels,sperm binding ability and embryo quality in vitrified bovine oocytes were studied by using sulfite sequencing,fluorescent quantitative PCR,and immunofluorescence staining methods.The results of the study showed that vitrification significantly increased the expression level DNMT1 gene m RNA(P < 0.05)and the methylation level of the JUNO gene promoter(73.58 ? 0.02% vs.43.57 ? 0.02%;P < 0.05),and significantly reduced the expression level of JUNO and TET1 gene m RNA(P < 0.05)in bovine oocytes,JUNO protein level(P < 0.05),sperm binding ability(9.17 ? 0.92 vs.20.62 ? 1.84;P < 0.05),normal fertilization rate(61.29 ? 4.82% vs.81.03 ? 7.25%;P < 0.05),the cleavage rates(56.50 ? 5.28% vs.84.97 ? 7.62%;P < 0.05),the blastocyst rates(14.55 ? 1.34% vs.44.62 ? 4.02%;P < 0.05),and blastocyst quality(74.69 ? 7.17 vs.105.83 ? 9.29;P < 0.05).2.In this experiment,JUNO m RNA was prepared by in vitro transcription,and JUNO m RNA was injected into GV-stage oocytes by microinjection.After IVM 24 h,MII-stage bovine oocytes were selected for vitrification.To study the effects of JUNO m RNA microinjection on the expression level of JUNO protein,sperm binding ability,normal fertilization rate and embryonic development ability.The results of the study showed that microinjection of 200 pg JUNO m RNA can significantly increase the expression level of JUNO protein(P < 0.05),sperm binding ability(18.78 ± 1.37 vs.9.53 ? 0.97;P < 0.05),normal fertilization rate(76.19 ± 6.75% vs.57.62 ± 4.87%;P < 0.05),the cleavage rate(70.34 ? 5.92% vs.54.54 ? 4.39%;P < 0.05)and the blastocyst rate(26.47 ? 2.45% vs.11.11 ? 1.02%;P < 0.05)in vitrified bovine oocytes.In summary,studies have shown that vitrification affects the ability of oocytes to fertilize and embryonic development by reducing the expression level of JUNO protein in bovine oocytes.In addition,microinjection of 200 pg of JUNO m RNA can up-regulate the expression level of JUNO protein in vitrified bovine oocytes,and improve the fertilization ability and embryonic development ability of bovine oocytes.These studies revealed the reasons for the decrease in fertilization rate of bovine oocytes after vitrification and provided a new method of protein regulation in oocytes.