The Effects Of Boar Spermatozoa Epigenetic Related Genes Expression Induced By Cryopreservation

During sperm cryopreservation, the process of freezing leads to the structural and functional damages of boar sperm and reduces sperm motility and fertilizing ability, farrowing rate and litter size. Compared with fresh sperm, only 1% artificial insemination was conducted with frozen-thawed boar sperm. Although numerous studies have focused on the constituent of freezing extenders and freezing procedures, the underlying mechanism remains unknown. Studies have demonstrated that freezing can cause sperm DNA methylation, histone acetylation and other epigenetic changes, but there are some controversial opinions. The epigenetic modifications play an important roles in maintaining the function of sperm during spermatogenesis, embryogenesis. Abnormal epigenetic modifications are thought to be closely associated with male infertility. Furthermore, there were no any reports whether the process of frozen can cause the changes of epigenetics in boar sperm. Therefore, in this study, we investigate the change of epigenetic-related genes during sperm cryopreservation. The results will benefit to explain the cryoinjury mechanism and provide the scientific basis for further optimizing boar freezing extenders.In this study, the ejaculates of five landrace boars with normal fertility were used. The total RNA in fresh, no LEY and program-controlled freezing (LEY,3% glycerol,250mM trehalose, 1mM glutathione) of boar sperm were extracted to evaluate the mRNA and protein expression levels of epigenetic-related genes (Dnmt3a, Dnmt3b, Jhdm2a, Kat8, Prml, Prm2 and IGF2) by qRT-PCR and ELISA. Results were as follows:1. The sperm viability and motility in program freezing treatments was significant lower than program-controlled freezing treatments. Meanwhile,3% glycerol had the best post-thawed sperm viability and motility, and significant higher than other treatments in the program-controlled freezing treatments.2. The results of qRT-PCR showed that there were significant differences of epigenetic-related genes between fresh and freezing treatments. Furthermore, the expression of epigenetic-related genes in program freezing treatments was the lowest. 3. ELISA analysis indicated there were significant difference of the expression levels of all proteins between fresh and LEY treatment. Furthermore, significant difference of the expression level of proteins Dnmt3a, Dnmt3b, Jhdm2a and Prm were found among fresh, no LEY and 1mM glutathione treatments. There were significant difference of the expression levels of proteins Kat8, Prm, IGF2, Jhdm2a between fresh and 250mM trehalose treatment. For Jhdm2a, there were significant differences between fresh and other treatments.In conclusions, our study confirmed that freezing can cause the changes of mRNA and protein expression levels of epigenetic-related DNA methyltransferases, histone demethylase, histone acetylation and protamine gene, imprinted gene during boar sperm cryopreservation.