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We have shown that STK35 and IFT27 genes are differentially expressed in spermatozoa from boars with good and poor semen freezability (GSF and PSF, respectively). STK35 is a stress-related gene that is implicated in spermatogenesis, whereas IFT27 is a motility-related gene that is mainly involved in intracellular protein transport. In this study we hypothesized that polymorphic variants in the 5'-flanking regulatory regions of STK35 and IFT27 genes could contribute to differences in semen freezability. We also predicted the interactions of the polymorphic variants with transcription factors on the gene promoter activity, using bioinformatics. The 5'-flanking region sequences of the STK35 and IFT27 were PCR amplified and analyzed by Sanger sequencing method. Protein expression in STK35 and IFT27 was determined in pre-freeze (PF) and frozen-thawed (FT) spermatozoa, using western blotting analysis. Sanger sequencing revealed a single nucleotide polymorphism (SNP) rs327863835 (C T) in STK35 promoter, while two SNPs (rs337563873, A T; rs331520020, T C) were detected in IFT27 promoter. STK35 and IFT27 promoter polymorphisms showed significant allele frequency differences between the GSF and PSF groups. Using bioinformatics approaches, we predicted that SNPs resulted in the generation of additional transcription factor binding sites for NFATC2, ELK1 and GR-β, which appeared to enhance or repress the promoter activity of STK35 or IFT27 in either freezability group. Wide variations in STK35 and IFT27 protein expression were observed among the boars, however, significantly higher protein expression was detected in IFT27 in FT spermatozoa of the GSF group. We suggest that the upstream variants, detected in STK35 and IFT27 promoters, might regulate the transcriptional activity of the genes by affecting their potential binding of transcription factors. The results indicate that the allelic variants in STK35 and IFT27 could be considered as potential genetic markers for predicting boar sperm freezability. |
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