The genetic component of mammalian male fertility is complex and involves thousands of genes. The majority of these genes are distributed on autosomes and the X chromosome, while a small number are located on the Y chromosome. Human and mouse studies demonstrate that the most critical Y-linked male fertility genes are present in multiple copies, show testis-specific expression and are different between species. In the equine industry, where stallions are selected according to pedigrees and athletic abilities but not for reproductive performance, reduced fertility of many breeding stallions is a recognized problem. Therefore, the aim of the present research was to acquire comprehensive information about the organization of the horse Y chromosome (ECAY), identify Y-linked genes and investigate potential candidate genes regulating stallion fertility. To achieve theses goals, a direct cDNA (complementary DNA) selection procedure was used to isolate Y-linked genes from horse testes and 29 Y-specific genes were identified. All 29 genes were mapped to ECAY and their sequences were used to further expand the existing map. Copy number analysis identified 15 multicopy genes of which 9 were novel transcripts. Gene expression analysis on a panel of selected body tissues showed that some ECAY genes are expressed exclusively in testes while others show ubiquitous or intermediate expression. Quantitative Real-Time PCR using primers for 9 testis-specific multicopy genes revealed 5 genes with statistically significant differential expression in testis of normal fertile stallions and stallions with impaired fertility. Gene copy number analysis showed that the average copy number of 4 such genes was decreased in subfertile/infertile stallions compared to normal animals. Taken together, this research generated the first comprehensive physical gene map for the horse Y chromosome and identified a number of candidate genes for stallion fertility. The findings essentially expand our knowledge about Y chromosome genes in horses, open a new avenue for investigating the potential role of ECAY genes in stallion fertility which contribute to the development of molecular tools for the assessment of fertility in stallions.

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.