Isoform Level Gene Expression Profile of Human Y Chromosome Azoospermia Factor Genes and their X Paralogues in the Testicular Tissue of Non-obstructive Azoospermia Patients

The human Y chromosome has an inevitable role in male fertility as it contains many genes critical for spermatogenesis and development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with non-obstructive azoospermia (NOA) (40 Sertoli cell only syndrome (SCOS) and 28 pre-miotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level, and western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1 and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm qPCR results at the protein level, immunoblotting and IHC experiments, based on 24 commercial and homemade antibodies, detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to 12 MApatients with failed sperm retrieval to predict success of sperm retrieval in azoospermic men. We show that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2 and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS and normal testicular tissues and suggests the possibility of diagnosing presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.

Genetic Molecular Markers, Alternative Transcript Variants, AZF, Human Y chromosome, Human Proteome Project (HPP), TESE, Sperm retrieval, Azoospermia