Reproductive Gene Expression in Male Sus scrofa: An examination of the differential gene expression of Divergent Testosterone selection and development of a Ribonucleic Acid extraction protocol from whole Porcine Spermatozoa

Abstract

The ability to characterize and enhance market traits in livestock has facilitated a greater interest in determining the genetic tool kit available for manipulation. In swine, using new approaches in genomics, such as microarray analysis and biological pathway analysis, we can show genes up and down regulated in a variety of processes and conditions. To this end, we identify the genes, pathways, and disease biomarkers affected by divergent selection of testosterone in boars. Testicular samples were taken from boars at 0, 30, 120, 150, and 180 days of age for lines of high (HT) and low testosterone (LT). Evidence that many of the differences in gene expression were at the pubertal period of 150 days led to a subsequent microarray study of the 150 day HT and LT animals. Microarray studies were followed by validation with real-time RT-PCR of 11 genes and extensive GeneGo pathway analysis (Metacore) of differentially expressed genes. While increased testosterone has long been associated with increased growth rates, we now have supporting genomic evidence of the genes and pathways up-regulated and down-regulated in these lines. To this end, this study has identified several disease biomarkers that may require further investigation and biological pathways associated with growth and metabolism that allow the recommendation of selective breeding for high testosterone to increase lean growth traits. The genetic blueprint contained in the spermatozoan transcriptome can also illuminate key issues in swine reproduction. By developing a procedure for effective RNA extraction of boar spermatozoa we are one step closer to elucidating the porcine sperm transcriptome and the genes implicated in growth and fertility. A viable protocol was developed to handle the complexities of large scale extraction of RNA from porcine semen utilizing an RNA carrier and Dnase treatment. This protocol was validated with PCR amplification of Sus scrofa prm1 in order to provide evidence of a successful RNA extraction from sperm.