Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls

Abstract

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851GOT) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, 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. The exonic SNP (c.2851GOT) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

Detection of one large insertion/deletion (indel) and two novel SNPs within the <i>SPEF2</i> gene and their associations with male piglet reproduction traits

“…The SPEF2 gene participated in spermatogenesis, sperm tail formation and ciliary function (Sironen et al, 2002(Sironen et al, , 2011Guo et al, 2013). A reproductive problem within the Finnish Yorkshire boars was first detected at the end of 1990s, referred to as a "short-tail" defect, which is caused by a large insertion within the SPEF2 gene in chromosome 16; this conclusion has been further proved by several experiments (Andersson et al, 2000;Sironen et al, 2002Sironen et al, , 2006Sironen et al, , 2007.…”

“…The sperm flagella 2 (SPEF2, also known as KPL2) gene is essential for normal sperm tail development and male fertility (Sironen et al, 2002;Guo et al, 2013). In rats, the SPEF2 gene was found to be stage-specific and intensive in spermatocytes and round spermatids in the seminiferous tubules (Ostrowski et al, 1999).…”

“…The loss of SPEF2 function in mice resulted in spermatogenesis defects and primary ciliary dyskinesia, which caused a decline in elongating spermatids and faults in the formation of sperm tail (Sironen et al, 2011). In cattle, alternative splicing (AS) has been suggested to be involved in the regulation of SPEF2 expression in the testes and sperm, and it was one of the determinants of sperm motility during bull spermatogenesis (Guo et al, 2013). In diploid and triploid cyprinid fish, RNA-seq was used to examine fertility-related molecular mechanisms and found that SPEF2 was expressed at higher levels in the testis of diploid fish than in those of triploids, which indicated that there is a positive correlation between the level of SPEF2 expression and male fertility (Xu et al, 2015).…”

A g.-1256 A>C in the promoter region of CAPN1 is associated with semen quality traits in Chinese Holstein bulls

“…Western blot analysis was performed according to the method of our previous report (Guo et al 2014). The tissue samples from the adult bulls were homogenized using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China).…”

“…In the past, many similar studies were conducted on goats and boars (Huang et al 2002, Lin et al 2005, Wimmers et al 2005. Recently, several studies focused on candidate marker genes in bulls (Pan et al 2013, Gao et al 2014, Guo et al 2014, 2015.…”