title: 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.
abstract: The 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.
authors.affiliation: Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.
authors.forename: Diba
authors.initials: D
authors.lastname: Ahmadi Rastegar
authors.affiliation: Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Sharifi Tabar
authors.affiliation: Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Alikhani
authors.affiliation: Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.
authors.forename: Pouria
authors.initials: P
authors.lastname: Parsamatin
authors.forename: Fazel
authors.initials: F
authors.lastname: Sahraneshin Samani
authors.forename: Marjan
authors.initials: M
authors.lastname: Sabbaghian
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Sadighi Gilani
authors.affiliation: Department of Genetics, Faculty of Science, Shahrekord University , Shahrekord, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Mohammad Ahadi
authors.forename: Anahita
authors.initials: A
authors.lastname: Mohseni Meybodi
authors.affiliation: Anatomy and Cell Biology Department, School of Medicine, Shahid Beheshti University of Medical Sciences , Tehran, Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Piryaei
authors.affiliation: Faculty of New Sciences and Technology, University of Tehran , Tehran, Iran.
authors.forename: Naser
authors.initials: N
authors.lastname: Ansari-Pour
authors.forename: Hamid
authors.initials: H
authors.lastname: Gourabi
authors.forename: Hossein
authors.initials: H
authors.lastname: Baharvand
authors.affiliation: Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.|Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran , Karaj, Iran.
authors.forename: Ghasem Hosseini
authors.initials: GH
authors.lastname: Salekdeh
mesh_terms: D000328:Adult; D053713:Azoospermia / Q000235:genetics* / Q000473:pathology; D041321:Chromosomes, Human, X; D041322:Chromosomes, Human, Y; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008875:Middle Aged; D013737:Testis / Q000473:pathology*
keywords: AZF; HPP; Human Proteome Project; TESE; alternative transcript variants; azoospermia; genetic molecular markers; human Y chromosome; sperm retrieval