Characterization of a Gene Abundantly Expressed in Stallion Testis

Abstract

NMES1 is a gene of unknown function first characterized in 2002. Reduction of the expression of this gene has been implicated in skin tumorigenesis in mice. Expression of NMES1 is observed in epithelial tissue but expression in the testis is significantly higher than in epidermis. Because stallion fertility is an economically important trait, we decided to characterize the NMES1 gene in stallions. We screened the CHORI241 library and obtained the full length equine NMES1 genomic sequence by direct sequencing off of clone CH241-11J8. In order to experimentally determine the 5’ and 3’ untranslated regions (UTRs) we conducted RLM-RACE experiments using stallion testis RNA. The equine NMES1 mRNA is 534 nt long and contains 5 exons. Fluorescence in situ hybridization mapped NMES1 to chromosome Eca1q23. In situ experiments to testis tissue sections were inconclusive and yielded no data confirming the physical expression pattern of NMES1 in stallion testis tissue. In order to determine the expression pattern of NMES1 mRNA we conducted qRT-PCR assays on a panel of stallion testis samples from horses with normal and abnormal fertility. We found that expression was variable among both groups, with significantly less expression in some individuals. We also conducted the qRT-PCR assay on a panel of five equine tissues and found that the expression of NMES1 was more than 100-fold greater in testis than in other tissues examined. miR-147b is a miRNA of unknown target found within the 3’ UTR of NMES1. We conducted a miRNA qRT-PCR assay to determine the expression levels in stallion testis samples from fertile and sub-fertile stallions. We observed similar expression among both groups and the ratio of mRNA to miRNA did not appear constant. We also investigated miR-147b expression in a panel of five equine tissues and found that equine spleen had more than 8-fold greater expression than testis.