Isoform-Level Gene Expression Profiles of Human Y Chromosome

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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 Diba Ahmadi Rastegar, Mehdi Sharifi Tabar, Mehdi Alikhani, Pouria Parsamatin, Fazel Sahraneshin Samani, Marjan Sabbaghian, Mohammad Ali Sedighi Gilani, Ali Mohammad Ahadi, Anahita Mohseni Meybodi, Abbas Piryaei, Naser Ansari-Pour, Hamid Gourabi, Hossein Baharvand, and Ghasem Hosseini Salekdeh J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00520 • Publication Date (Web): 10 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Proteome Research

åIsoform Level Gene Expression Profile of Human Y Chromosome Azoospermia Factor Genes and their X Paralogues in the Testicular Tissue of Non-obstructive Azoospermia Patients Diba Ahmadi Rastegar1#, Mehdi Sharifi Tabar1, Mehdi Alikhani1, Pouria Parsamatin1, Fazel Sahraneshin Samani2, Marjan Sabbaghian3, Mohammad Ali Sedighi Gilani3, Ali Mohammad Ahadi4, Anahita Mohseni Meybodi5, Abbas Piryaei2, Naser Ansari-Pour6, Hamid Gourabi5, Hossein Baharvand7,8, and Ghasem Hosseini Salekdeh1,9* 1. Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 2. Stem Cells and Developmental Biology Group of Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3. Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 4. Department of Genetics, Faculty of Science, Shahrekord University, Shahrekord, Iran. 5. Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 6. Faculty of New Sciences and Technology, University of Tehran, Tehran, Iran 7. Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 8. Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran 9. Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran

*Corresponding authors: Ghasem Hosseini Salekdeh, Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Tel: +98 21 22306485, Fax: +98 21 23562507, Email: [email protected]

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Abstract 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 MA 2 ACS Paragon Plus Environment

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patients 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.

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



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1. Introduction The human Y chromosome proteome project (Y-HPP) aims to map and annotate all proteincoding genes located on the Y chromosome (Y for short hereafter)1-3. It has been well documented that the human Y plays fundamental roles in brain function, spermatogenesis and other male-specific disorders4, 5. Dysfunction of Y genes is thus responsible for a broad range of male-related disorders, among which male infertility is the most prominent.6 This is because infertility is considered as a worldwide health problem that affects approximately 15% of couples, of which half is male-related7. Defects in spermatogenesis are thought to be caused by various acquired or congenital factors where 15-30% of the latter cases are attributable to genetic factors8. From the 1970s, special attention has been paid to the Y after an association between deletions on the long arm (Yq) and spermatogenic failure6, 9. These deleted regions were named as azoospermia factor (AZF) regions accordingly and subsequent analyses identified three intervals (AZFa, b, c) each of which contains critical genes for efficient spermatogenesis10-12. The AZFa interval, spanning 792 kb, maps to proximal Yq and contains two functional genes, DDX3Y and USP9Y. Both of the genes have functional X paralogues, namely DDX3X and USP9X13. DDX3Y (the DEAD [Asp–Glu–Ala–Asp] box polypeptide 3, Y-linked gene formerly known as DBY) is a member of the DEAD-box protein family of RNA helicases expressed in testicular tissue, predominantly spermatogonial cells14, 15. USP9Y (a Y-linked gene encoding the ubiquitin-specific peptidase 9) is the second AZFa gene which encodes a protein with a Cterminal ubiquitin hydrolase domain16. The AZFb interval spans a total of 6.23 Mb and encompasses nine genes, namely XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1 and PRY17. Among these, only


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RBMY1A1, HSFY1, EIF1AY and PRY have been investigated with respect to their expression, deletion and mutation18-20. The AZFc region spans 3.5 Mb and maps to distal Yq. This region is largely constituted by arrays of repeating DNA blocks named amplicons. This makes the sequence prone to structural variation21, 22 and genes in this region are thus found in multiple copies23. Three protein-coding gene families located in this interval are BPY2, CDY1 and DAZ1. Altogether, AZFa, AZFb and AZFc microdeletions are thought to lead to non-obstructive azoospermia (NOA) in men. Clinical management of men with NOA undergoing treatment is of high importance since testicular sperm extraction (TESE) from NOA patients along with successful intracytoplasmic sperm injection (ICSI) is currently the mainstream approach in reproductive medical centers24-26. Therefore, prediction of sperm presence in the testicular biopsy of men with NOA will assist in the management of NOA patients26, 27. Reports show that 3765% of the men with NOA lack sperm in their testicular biopsy and there is no reliable preoperative clinical parameter or biomarker to predict sperm presence 27. Men in whom sperm are not retrieved may needlessly face the risk of psychological distress and financial cost. Furthermore, female partners of men with NOA who fail sperm retrieval may undergo unnecessary ovarian stimulation and the couple bearing further financial expenses. The availability of a genetic marker to predict the presence of retrievable testicular sperm in men with NOA seems to be promising.

Recently, it was shown that the longevity of many Y-linked genes is because of selection to maintain expression, in males, of dosage-sensitive, widely expressed X–Y gene pairs at levels comparable to their autosomal ancestors3. We thus hypothesized that the expression pattern of



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AZF region genes and their alternative transcript variants, as well as their X- homologues, may assist to predict sperm presence.

There is growing evidence that almost all multi-exon human genes have more than one mRNA isoform28. During alternative splicing, the coding and noncoding regions of a single gene are rearranged to generate several mRNA, yielding distinct protein isoforms with differing biological functions. Notably, altered expression of specific isoforms for numerous genes is linked with disease formation and its prognosis29, 30. We hypothesized that isoform level expression profiles would be better than gene level expression profiles at discriminating between fertile and infertile testicular tissue. Therefore, in the first step we conducted a comprehensive expression profiling of alternative transcript variants of all AZF genes and their paralogues in the testicular tissue of sertoli cell only syndrome (SCO) and maturation arrest (MA) patients and identified differentially expressed transcripts.

We then analyzed these transcripts in MA samples with successful sperm retrieval and those lacking any sperm cell. Our results indicate that not only individual AZF transcript variants play a vital role in spermatogenesis but the X-linked homologues and the autosomal ones, seem also to be involved in germ cell development and male fertility. More importantly, we show that certain transcripts can predict sperm presence in MA patients.

2. Materials and Methods 2.1. Subjects Among the azoospermic patients who referred to the Royan Infertility Clinic (Tehran, Iran) from the year 2012 to 2014, 104 azoospermic men aged 26-45 who had underwent TESE were 6 ACS Paragon Plus Environment

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selected for this study. The target group was selected through a comprehensive andrological examination including medical history, semen and hormonal analysis (FSH, LH and testosterone) at the Department of Andrology of Royan Institute. Men with Y-chromosome microdeletions or an abnormal karyotype were excluded, resulting in a total of 92 patients in the case group. Twelve normospermic males who had undergone testicular biopsy were also recruited as the control group. This study was approved by the Ethics Committee of Royan Institute. Informed written consent was taken from all participants.

2.2. Testicular tissue evaluation and analysis Testicular tissue specimens were collected and instantly frozen in liquid nitrogen and stored at 80°C for RNA and protein extraction. Based on histological and cytological evaluations, the most advanced spermatogenic cell identified, determined the subtype of the specimen. Accordingly, testicular biopsies were classified into complete spermatogenesis (n=12), complete maturation arrest at the spermatocyte stage (n=28) and Sertoli cell only syndrome (n=40).

2.3. Maturation arrest samples with successful sperm retrieval Twenty-four testicular biopsy specimens from 12 maturation arrest patients with successful sperm retrieval (MA+) and 12 maturation arrest patients with failed sperm retrieval (MA-) were samples to find predictive genetic markers. All testicular samples were obtained from TESE procedures in an attempt to obtain sperm for ICSI.

2.4. Chromosome-specific primer designing 7 ACS Paragon Plus Environment


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The human Y and X chromosomes encode paralogues with highly similar alternative splicing transcript variants. Designing variant-specific primers would thus be challenging. To distinguish between the two, we designed highly specific primers using the Vector NTI software (Lifescience, USA). Primers were designed at the exon-intron boundaries for most of the transcripts to avoid any amplification due to possible genomic contamination (see Supplementary Table1). The specificity of the primer pairs were then checked using Basic Local Alignment Search Tool31 (BLAST) to verify the accuracy of PCR amplifications, PCR products were analyzed by melting curve analysis, gel electrophoresis and DNA sequencing.

2.5. RNA isolation & Quantitative RT-PCR Total RNA (1-5 µg) was isolated using the TRIzol (TRIzoL Reagent, Invitrogen Life Technologies) method. The resultant RNA was reverse transcribed into cDNA and then diluted up to 25 ng/µl for quantitative real-time PCR (qPCR). The expression of candidate genes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (as an internal control) was evaluated using qPCR and SYBR Green I chemistry. The samples were tested in triplicate on Rotor-Gene 6000TM instrument (Corbett Life Science, Australia). The cycling conditions were an initial step at 95°C for 10 min followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s.

2.6. Generation of rabbit polyclonal antibodies against Y chromosome proteins In order to provide high quality and quantity of antibodies for Y-HPP project, we generated homemade antibodies against most of the proteins from AZF regions according to standard protocols as described below.


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2.6.1. Antigen preparation Specific sequences of genes located in the AZFa, AZFb and AZFc regions of Y chromosome, where possible, were cloned on pET-28a(+) expression vector. Corresponding recombinant proteins were produced in E. coli Bl21. Recombinant proteins were extracted by sonication, lysed by a urea buffer (8 M urea and 0.1 M sodium phosphate, pH: 8.8) and purified by 6×HisNi-NTA chromatography as described before19. Amicon columns (Millipore, USA) were applied to desalt and concentrate the recombinant proteins.

2.6.2. Rabbit immunization and polyclonal antibody production Young female New Zealand white rabbits (Albino) were immunized by subcutaneously injection of 400 µg protein emulsified in Freund’s complete adjuvant. After a month, rabbits were boosted with 200 µg protein emulsified in Freund’s incomplete adjuvant. Rabbits were given two another boosters at three-week intervals followed by bleeding two weeks after last booster. Either antisera or protein G purified antibodies were used for antibody validation and immunodetections.

2.6.3. Confirmation of rabbit polyclonal antibodies Generated antibodies were tested by sequential experiments. First, antibody reactions against recombinant proteins (20ng) were tested by western blot technique. Then, antibodies were evaluated against a panel of proteins (40µg each) extracted from different tissue specimens by western blot. Finally, antibodies were assessed using IHC/IF (Supplementary Figure S1)



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2.7. Western blotting Concentration of extracted proteins were estimated by dye binding method 32. 40 µg protein from each sample was loaded on a 12% SDS-polyacrylamide gel. After gel electrophoresis, the proteins were blotted onto a PVDF membrane (Bio-Rad, USA) by a wet transblotting apparatus following manufacturer instructions (Bio-Rad, USA). The blotted membranes were incubated in TBST (Tris buffer saline with 0.1% Tween-20) containing 5% non-fat dried milk at room temperature for 1.5 h. The membranes were incubated with individual antibodies (antisera, 1:10000 for homemade antibodies and about 1:6000 for the commercial ones in blocking solution) for 6 h at 4◦C. Subsequently, the membranes were washed three times with TBST for 15 min, and then were incubated with peroxidase-labeled secondary antibodies for 1 hr at room temperature. After three times washings in TBST, blots were incubated with chemiluminescent peroxidase substrate (Sigma-Aldrich, PQ0201, Germany) in dark room and exposed to X-ray films (GE, 28906835)33. Commercial antibodies used in this study are as follows: DDX3Y (wh0008653m1), USP9Y (WH0008287M5), HSFY1 (sab1402645), RBMY1A1 (av40257), CYORF15A (hpa039741), CYORF15B (WH0084663M1), EIF1AY (sab1402445), BPY2 (sab1402645) and CDY (av48645) from Sigma-Aldrich (United States), RPS4Y2 (ab74709), DAZ (ab54605) and KDM5D (ab35492) from Abcam (United States), and XKRY (sc83952) and PRY (sc79704) from Santa Cruz Biotechnology (Germany). GAPDH (ab9483) was used as loading control.

2.8. Immunohistochemistry Slides were initially baked, deparaffinized in xylene and passed through graded alcohols. Antigen was then retrieved with Trypsin 0.5% (Gibco 27250018): CaCl2 1% (1:1) for 30 10 ACS Paragon Plus Environment

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minutes at 37°C. All further steps were carried out at room temperature in a hydrated chamber. Slides were pretreated with peroxidase block for 5 minutes to quench endogenous peroxidase activity and were then washed in PBST. Slides were blocked using normal goat serum (Dako, 250ul added to PBST) and subsequently incubated with homemade/commercial primary antibodies (diluted in PBST) overnight. Slides were then washed in PBST and treated with goat anti-mouse-HRP (Sigma-Aldrich, A0168) or goat anti-rabbit-HRP (Abcam, ab97051) for 30 minutes. After further washing, immunoperoxidase staining was developed using a 3,3’diaminobenzidine (DAB) chromogen (Sigma-Aldrich, D5905) for 5 min. Slides were finally dehydrated in graded alcohol and xylene, then mounted and coverslipped.

2.9. Statistical analysis Differences in transcripts expression among the three histological groups were analyzed by means of analysis of variance (ANOVA) using SPSS version 16, and post-hoc analyses was performed by Duncan’s test. Multiple comparisons between means of binary categories were performed using Student’s t-test. P values less than p

Isoform-Level Gene Expression Profiles of Human Y Chromosome

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