The Relationship Between SPEF2 Gene Expression And Semen Quality In Chinese Holstein Bulls

The widespread use of dairy bull semen requires high sperm quality, bull contribution more than 70% to the progress of cow population genetic advance, which is economically important in the artificial insemination industry. Normal sperm flagellum is essential for sperm motility and fertilization of the egg. This process involves the coordinated expression of many genes with unique cellular and temporal specificities. Identifying the genes specifically expressed in newborn and adult testes and sperm is crucial to understand testicular development and function, as well as spermatogenesis. It was reported that the sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we analysed the expression pattern of SPEF2 gene in testis, epididymis and sperm of Holstein bulls, identified function SNPs and analysed the DNA methylation pattern of the SPEF2 promoter. In order to understand the regulation mechanisms of SPEF2, and analysed the relationship between semen traits and SPEF2 gene, enriched the molecular mechanism of the high awareness of reproductive traits in bulls.1. Identification of SPEF2 gene splices variantFour novel splice variants were identified in the adult testes through RT-PCR and clone sequencing. We named SPEF2-SV1, SPEF2-SV2, SPEF2-SV3 and SPEF2-SV4, respectively. Western blot analysis showed that the SPEF2 proteion expressed in heart, liver, spleen, hung, kidney and testis of newborn bulls and adult bulls. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes. SPEF2-complete were differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV1 was expressed in all tissues of newborn bulls and adult bulls, except in lung of newborn bulls. SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis; SPEF2-SV4 expressed too low to detected in tissues. Our data suggest that alternative splicing is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis.2. Identification of functional SNP associated with alternative splicing isoformsUsing DNA secquencing methods, we screened two SNPs. An SNP (c.2851G>T) in exon 20 of SPEF2, we predicted the change in ESE using the c.2851G>T mutation for it near the splicing region of SPEF2-SV3 and found that it increased one binding site for the splicing factor SRp40, and it was involved in semen deformity rate and post-thaw cryopreserved sperm motility (P<0.05). Another SNP (g.11043C>T) near the splice sites around 140 bp was found in intron 1, using ESEfinder 3.0 software, we predicted that the SNP changed the SC35 combination with the target sequence and it might be the reason why the generation of the aberrant SPEF2-SV4 transcript. Association between the genotypes of the SNP (g.11043C<T) and sperm quality traits showed that the SNP had a significant effect on the semen deformity rate (P<0.05). Two SNPs can be used as potential novel molecular markers for the selection of fineness semen quality traits in Chinese Holstein bulls.3. Analysis of promoter activity and methylation level in promoter region of SPEF2 geneWe found a promoter and a CpG island in the 5’-flanking region of bovine SPEF2 gene using bioinformatics software. We amplified seven fragments through progressive deletion of nucleotides from the 5’-end, and each fragment was cloned into the pGL3-basic luciferase reporter vector and then transiently transfected into MLTC-1 cells respectively. This result indicates that the region from g.-586 to g.-157 is the core promoter responsible for most of the promoter activity in the bovine SPEF2 gene, and the CpG Island(g.-430-g.-161) was overlapping with core promoter. Using the nest BSP method, we amplified the 271 bp fragment with nine CG sites. The result showed that no differences in the sperm DNA methylation of the SFEF2 promoter were found between the high-performance (5.40%, n=30 clones) and low-performance (5.56%, n=30 clones) bulls, and the promoter methylation level of SPEF2 showed a hypomethylation level (mean value:10.26%, n=50 clones) in the testis of four adult bulls.In this study, the DNA methylation profile of the SPEF2 promoter was unrelated to bull sperm quality.