Effects Of Vitrification On Ca²⁺ And Its Regulatory Genes Of Bovine Oocytes

Mammalian oocyte vitrification plays an important role in the study of embryo biotechnology and the germplasm resources preservation of breeding livestocks and endangered animals.However,its application is severely limited by the low fertilization rate and poor developmental competence of thawed oocytes compared with that of fresh ones because vitrification causes abnormal increase of intracellular Ca²⁺ concentration,but the mechanism is not clear.Thus,the present study was to investigate the source of Ca²⁺ in vitrified bovine oocytes from the perspective of intracellular calcium pool（endoplasmic reticulum,mitochondria）and molecular level and the effects of BAPTA-AM（red,BAPTA）and RR（Ruthenium red）on the fertilization and development of vitrified bovine oocytes.The results were as below: 1.Bovine MII oocytes from in vitro maturation（IVM）were divided into fresh group,ethylene glycol（EG）group,dimethyl sulfoxide group（DMSO）,vitrification solution treatment group and vitrification group according to the different treatment and the concentrations of the intracellular Ca²⁺([Ca²⁺]i),endoplasmic reticulum Ca²⁺(ER Ca²⁺),mitochondrial Ca²⁺(m Ca²⁺),and the morphological distributions of endoplasmic reticulum and 1,4,5-trisphosphate receptor 1（IP3R1）of bovine oocytes in fresh group,vitrification solution treatment group and vitrification group were analyzed respectively using fluorescence staining.The results showed that the levels of [Ca²⁺]i,and m Ca²⁺ of vitrified bovine oocytes were increased and that of ER Ca²⁺ was decreased（mainly caused by the DMSO）,and vitrification caused the abnormal distributions of endoplasmic reticulum and IP3R1 of bovine oocytes（P<0.05）.2.Bovine MII oocytes were treated for 0.5 h in IVM medium containing 0.5,1 and 2 μM RR respectively.The effects of RR on the concentration of Ca²⁺,the content of ATP,the level of mitochondrial membrane potential（ΔΨm）,apoptosis and the developmental potential of bovine oocytes were analyzed with the fresh and vitrification control.The results indicated that RR treatment could significantly reduce the concentration of m Ca²⁺ by leaving more Ca²⁺ in the cytoplasm（P<0.05）,but it had no significant effect on the ER Ca²⁺,of which the ATP content,the level of ΔΨm and the survival rates of oocytes and the subsequent cleavage and blastocyst rates of parthenogenetic embryos in 1 μM RR group were significantly higher than those of vitrification group（P<0.05）while it had no significant difference with the fresh group.3.The bovine oocytes matured for 20～22 h were treated in the IVM medium with 5,10 and 20 μM BAPTA for 2 h,and the concentration of Ca²⁺,the distribution of cortical particles（CGs）and the fertilization and developmental competence were analyzed compared with the fresh and vitrification groups.The results showed that BAPTA treatment significantly decreased the levels [Ca²⁺]i of vitrified bovine oocytes（P<0.05）,but it had no significant effect on the concentrations of ER Ca²⁺ and m Ca²⁺.10 μM BAPTA significantly increased the CGs peripheral distribution,the normal fertilization and cleavage rates（P<0.05）after in vitro fertilization（IVF）.The co-treatment of 10 μM BAPTA and 1 μM RR significantly increased the cleavage and blastocyst rates,and blastocyst quality（P<0.05）of vitrified bovine oocytes after IVF.4.The bovine MII oocytes of fresh and vitrification groups were collected to do RNA-seq.The results showed that 102 differentially expressed genes（12 up-regulation and 90 down-regulation）were detected,and the m RNA expression levels of two Ca²⁺ regulatory genes（CALM and SSRG）were down regulated（P<0.05）.Functional analysis of GO revealed that these differentially expressed genes were mainly enriched in the membrane-bound organelles in the cell fractions（P<0.05）.The results of q RT-PCR and Western blotting analysis showed that the expression patterns of m RNA of 10 genes and CDK2 and UCHL3 proteins were consistent with those observed in Smart-seq2 analysis.In conclusion,this study found that vitrification could reduce the expression of Ca²⁺ regulatory genes（CALM,SSRG）,cause ER Ca²⁺ release（mainly caused by the DMSO）,lead to the increase of [Ca²⁺]i and m Ca²⁺ levels,the premature release of CGs,the damage of mitochondrial function and the increase of apoptosis rate in bovine oocytes.The combination of 10 μM BAPTA and 1 μM RR helped to alleviate the above abnormality and improve the fertilization and developmental competence of vitrified bovine oocytes.These findings provided a technical reference for improving the efficiency of vitrified oocytes.