Promoter DNA methylation and expression analysis of PIWIL1 gene in purebred and crossbred cattle bulls

Abstract

Major credit for India being the largest producer of milk in the world, goes to crossbred cows produced by inseminating low-producing indigenous cattle with semen from high producing exotic bulls. However, over the years, the policy of crossbreeding has been confronted with a major problem of subfertility in crossbred male progenies, culminating into disposal of a major fraction of mature bulls. Many studies have demonstrated relationship between epigenetic alterations and male fertility across different species. PIWIL1 is an important candidate gene for spermatogenesis and germ line development. Negative correlation between DNA methylation and expression of this gene has been highlighted in inter species hybrids of cattle and yaks. The present study envisaged elucidating promoter methylation status and expression profile of PIWIL1 gene in exotic Holstein Friesian cattle, indigenous Sahiwal cattle and their crossbreds with varying semen motility parameters. Semen samples were collected from bulls for isolation of DNA and RNA from spermatozoa. Bisulfite converted DNA was used to amplify promoter of PIWIL1 gene using methylation specific primers. The amplified products were sequenced after cloning in pTZ57R/ T vector. The degree of methylation of the PIWIL1 promoter region was significantly higher in poor motility crossbred bulls (7.17%) as compared to good motility crossbreds (1.02%), Sahiwal (1.02%) and Holstein Friesian bulls (0.77%). PIWIL1 expression was 1.75, 1.71 and 1.59 folds higher in HF, Sahiwal and good motility crossbreds, respectively as compared to poor motility crossbreds.