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Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients
journal contribution
posted on 2015-09-04, 00:00 authored by Diba Ahmadi Rastegar, Mehdi Sharifi Tabar, Mehdi Alikhani, Pouria Parsamatin, Fazel Sahraneshin
Samani, Marjan Sabbaghian, Mohammad
Ali Sadighi Gilani, Ali Mohammad Ahadi, Anahita Mohseni
Meybodi, Abbas Piryaei, Naser Ansari-Pour, Hamid Gourabi, Hossein Baharvand, Ghasem Hosseini SalekdehThe
human Y chromosome has an inevitable role in male fertility
because it contains many genes critical for spermatogenesis and the
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 nonobstructive azoospermia (NOA) (40 Sertoli-cell-only
syndrome (SCOS) and 28 premiotic 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 those of men with
complete spermatogenesis. In contrast, the expression of five transcripts
increased significantly in NOA patients. Furthermore, to confirm the
qPCR results at the protein level, we performed immunoblotting and
IHC experiments (based on 24 commercial and homemade antibodies) that
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 those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed 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 the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.
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WB5D5C41 alternative transcriptsPRYCDYUSP 9Y DDX 3Y XKRYHSFYSCOSIHCPCRHuman Y Chromosome Azoospermia Factor GenesNOAsperm retrieval28 premiotic maturation arrestexpressionX Chromosome ParalogsAZFc region genes1ARBMXKDMX chromosome homologue transcriptsCYORF12 MA patientsY chromosome genesTESEEIFBPYisoform level signaturetesticular sperm extractionRPS15B4Yspermatogenesiprotein levelX chromosome counterpartsDAZL 1 transcripts1AYtestis cell typesRBMYazoospermic men15A
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