Rastegar, Diba Ahmadi Tabar, Mehdi Sharifi Alikhani, Mehdi Parsamatin, Pouria Sahraneshin Samani, Fazel Sabbaghian, Marjan Gilani, Mohammad Ali Sadighi Ahadi, Ali Mohammad Mohseni Meybodi, Anahita Piryaei, Abbas Ansari-Pour, Naser Gourabi, Hamid Baharvand, Hossein Salekdeh, Ghasem Hosseini 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 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 <i>(USP9Y</i>, <i>DDX3Y</i>, <i>XKRY</i>, <i>HSFY1</i>, <i>CYORF15A, CYORF15B, KDM5D, EIF1AY</i>, <i>RPS4Y2</i>, <i>RBMY1A1</i>, <i>PRY, BPY2, DAZ1</i>, and <i>CDY1</i>) 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 <i>HSFY1-1</i>, <i>HSFY1-3</i>, <i>BPY2-1</i>, <i>KDM5C2</i>, <i>RBMX2, </i>and <i>DAZL1</i> 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. WB;5D;5C;41 alternative transcripts;PRY;CDY;USP 9Y DDX 3Y XKRY;HSFY;SCOS;IHC;PCR;Human Y Chromosome Azoospermia Factor Genes;NOA;sperm retrieval;28 premiotic maturation arrest;expression;X Chromosome Paralogs;AZFc region genes;1A;RBMX;KDM;X chromosome homologue transcripts;CYORF;12 MA patients;Y chromosome genes;TESE;EIF;BPY;isoform level signature;testicular sperm extraction;RPS;15B;4Y;spermatogenesi;protein level;X chromosome counterparts;DAZL 1 transcripts;1AY;testis cell types;RBMY;azoospermic men;15A 2015-09-04
    https://acs.figshare.com/articles/journal_contribution/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/2135281
10.1021/acs.jproteome.5b00520.s001