Identification of risk genes in Chinese nonobstructive azoospermia patients based on whole-exome sequencing.

Nonobstructive azoospermia (NOA) is a severe condition in infertile men, and increasing numbers of causative genes have been identified during the last few decades. Although certain causative genes can explain the presence of NOA in some patients, a proportion of NOA patients remain to be addressed. This study aimed to investigate potential high-risk genes associated with spermatogenesis in idiopathic NOA patients by whole-exome sequencing. Whole-exome sequencing was performed in 46 male patients diagnosed with NOA. First, screening was performed for 119 genes known to be related to male infertility. Next, further screening was performed to determine potential high-risk causative genes for NOA by comparisons with 68 healthy male controls. Finally, risk genes with high/specific expression in the testes were selected and their expression fluctuations during spermatogenesis were graphed. The frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene pathogenic variant carriers was higher in the NOA patients compared with the healthy controls. Potential risk genes that may be causes of NOA were identified, including seven genes that were highly/specifically expressed in the testes. Four risk genes previously reported to be involved in spermatogenesis (MutS homolog 5 [MSH5], cilia- and flagella-associated protein 54 [CFAP54], MAP7 domain containing 3 [MAP7D3], and coiled-coil domain containing 33 [CCDC33]) and three novel risk genes (coiled-coil domain containing 168 [CCDC168], chromosome 16 open reading frame 96 [C16orf96], and serine protease 48 [PRSS48]) were identified to be highly or specifically expressed in the testes and significantly different in the 46 NOA patients compared with 68 healthy controls. This study on clinical NOA patients provides further evidence for the four previously reported risk genes. The present findings pave the way for further functional investigations and provide candidate risk genes for genetic diagnosis of NOA.

FAM9B serves as a novel meiosis-related protein localized in meiotic chromosome cores and is associated with human gametogenesis.

Meiosis is a complex process involving the expression and interaction of numerous genes in a series of highly orchestrated molecular events. Fam9b localized in Xp22.3 has been found to be expressed in testes. However, FAM9B expression, localization, and its role in meiosis have not been previously reported. In this study, FAM9B expression was evaluated in the human testes and ovaries by RT-PCR, qPCR, and western blotting. FAM9B was found in the nuclei of primary spermatocytes in testes and specifically localized in the synaptonemal complex (SC) region of spermatocytes. FAM9B was also evident in the follicle cell nuclei and diffusely dispersed in the granular cell cytoplasm. FAM9B was partly co-localized with SYCP3, which is essential for both formation and maintenance of lateral SC elements. In addition, FAM9B had a similar distribution pattern and co-localization as γH2AX, which is a novel biomarker for DNA double-strand breaks during meiosis. All results indicate that FAM9B is a novel meiosis-associated protein that is co-localized with SYCP3 and γH2AX and may play an important role in SC formation and DNA recombination during meiosis. These findings offer a new perspective for understanding the molecular mechanisms involved in meiosis of human gametogenesis.

A clinical trial on the consistency of bilateral testicular tissue histopathology and Johnsen score: single side or bilateral side biopsy?

To evaluate and compare left and right testicular tissue histopathology and Johnsen score, and to investigate the necessity for bilateral testicular biopsy. We recruited180 patients with non-obstructiveazoospermia (NOA) on testicular biopsy who had undergonetesticular sperm aspiration (TESA). Pathological sections of testicular tissue were diagnosed by specially-assigned doctors, who evaluated pathological findings, determined the Johnsen score and confirmed for the presence or absence of sperm. Sperm positive rates for left and right testicular histopathology were 55.0% and 51.7% respectively, and the proportion of Johnsen scores≥8 for left and right testes were 53.3% and 50.0%, respectively. Cohen kappa values revealed that the identification of sperm in bilateral testicular samples was not consistent and was related to random effects; Optimized cut-off value for bilateral testicular volume was 11ml (Johnsen score ≥8), and optimized cut-off values of E2 on left and right testes were 144.5pmol/L and 133.5 pmol/L (Johnsen score≤7). However, age, serum prolactin (PRL), follicle stimulating hormone (FSH), luteinizing hormone (LH) and total testosterone (TT) levels were not accurate predictors for the existence of testicular sperm. There was nostatistical significance between left and right testicular histopathology in terms of sperm positive rates or Johnsen score; the Johnsen score were caused entirely by random effects and a score from one side could not represent the other side. Therefore, we recommend that both testes need to undergo surgery when NOA patients undergo testicular biopsy or sperm retrieval.

Ran-binding protein M is associated with human spermatogenesis and oogenesis.

The aim of the present study was to explore the underlying mechanism and diagnostic potential of Ran­binding protein M (RanBPM) in human spermatogenesis and oogenesis. RanBPM expression in human testis and ovaries was analysed using polymerase chain reaction (PCR) and western blotting, and immunofluorescence was performed on testis and ovary tissue sections during different developmental stages of spermatogenesis and oogenesis using RanBPM antibodies. Interactions with a variety of functional proteins were also investigated. RanBPM mRNA and protein expression levels were determined by PCR and western blotting in the tissue sections. Results revealed that the mRNA expression levels were highest in the testis followed by the ovary. The RanBPM protein was predominantly localized in the nucleus of germ cells, and the expression levels were highest in pachytene spermatocytes and cells surrounding spermatids in testis tissue. In ovary cells, RanBPM was localized in the nucleus and cytoplasm. In conclusion, the results suggested that RanBPM may have multiple roles in the regulation of germ cell proliferation during human spermatogenesis and oogenesis. This research may provide a novel insight into the underlying molecular mechanism of RanBPM and may have implications for the clinical diagnosis and treatment of human infertility.

Role of tripartite motif protein 27 as a gametogenesis-related protein in human germ cells.

BACKGROUND: The distribution and functional integrity of members of the tripartite motif (TRIM) protein family are essential for cell proliferation, development and apoptosis, and TRIM proteins have been linked to various cancers. To explore the diagnostic potential and mechanisms of TRIM27 in human spermatogenesis and oogenesis, we analyzed its localization pattern and putative roles in human testes and ovaries. METHODS: TRIM27 mRNA and protein levels in human testes and ovaries were investigated using RT-PCR and western blotting, respectively. TRIM27 was abundantly transcribed in human testes and ovaries, particularly during the early stages of spermatogenesis, and localized in the nuclei of primary spermatocytes. Immunofluorescence also revealed a diffuse distribution in the cytoplasm of round spermatids, and the protein was abundant in ovary tissue during various stages of oogenesis development. RESULTS: TRIM27 mRNA and protein was abundantly transcribed in male and female human germ cells by RT-PCR and western blotting in the human testes followed by the ovary. Immunohistochemical results revealed TRIM27 protein was abundant in the sex body of primary spermatocytes undergoing meiotic prophase during the first cycle of spermatogenesis. Moreover, Trim27 was diffusely localized in the cytoplasm of spermatids and round spermatids. Furthermore, TRIM27 was localized to both the nucleus and cytoplasm of human ovary cells. CONCLUSIONS: TRIM27 as a gametogenesis-related protein could play multiple roles in the regulation of sex body formation and germ cell proliferation during spermatogenesis and oogenesis. The identification and characterization of TRIM27 enhances our understanding of the molecular mechanisms underpinning its functions, and provides insight into its potential role in the pathogenesis of germ cell differentiation and infertility.

Trim27 interacts with Slx2, is associated with meiotic processes during spermatogenesis.

ABSTARCT Formation of the XY body is believed to prevent recombination between X and Y chromosomes during meiosis. We recently demonstrated that SYCP3-like X-linked 2 (Slx2) could be involved in synaptonemal complex formation as well as XY body maintenance during meiosis. In order to further investigate the role and composition of XY body protein complexes in meiotic processes and spermatogenesis, a yeast 2-hybrid screening was performed, and the tripartite motif protein 27(Trim27) was found to interact with Slx2 and co-localized in the XY body. Trim27 has a tripartite motif (TRIM) consisting of a RING finger, B-box and coiled-coil domains, and is a transcriptional regulator that is expressed in various tumor cell lines. In this study, we showed that Slx2 and Trim27 were highly expressed in meiosis of mouse testis. And the Slx2/Trim27 interaction was confirmed in vivo by co-immunoprecipitation and mammalian 2-hybrid interaction assays. Moreover, cytoimmuno localization experiments revealed that Slx2/Trim27 was co-localized to the XY body of spermatocytes during meiosis, and immunohistochemical results revealed co-localization of Trim27 and γ-H2AX in the XY body of primary spermatocytes in the mouse testis. Trim27 may therefore be a transcriptional regulation protein connecting Slx2 and γ-H2AX, thereby promoting the formation of a more potent XY body protein complex in meiotic processes and spermatogenesis. In conclusion, Trim27 connecting Slx2 may regulate meiotic processes in multiple ways by influencing XY body formation and germ cell proliferation during spermatogenesis.

The prevalence of erectile dysfunction among subjects with late-onset hypogonadism: a population-based study in China.

INTRODUCTION: The concurrence of chronic diseases and some well-defined risk factors significantly impacts the prevalence of erectile dysfunction (ED). AIM: To determine whether late-onset hypogonadism (LOH) impacts the prevalence of ED using investigation reproductive health data of middle-aged and aging males in China. METHODS: The reproductive health status of 1498 males, aged 40-69 years, was evaluated using questionnaires of LOH based on the Androgen Deficiency in Aging Males (ADAM) and Aging Male Symptoms scale (AMS), as well as the International Index of Erectile Function-5 (IIEF-5) assessment. The 10th percentile of serum total testosterone (TT) and calculated free testosterone (cFT) levels of controls were set as cut-off levels of AD. The main outcome measures were used to assess the prevalence of LOH and ED according to different subject characteristics. RESULTS: Of the 1472 subjects who completed the questionnaires who supplied hormone measurements, the prevalence of self-reported ED and identified by the IIEF-5 assessment were 11.28% and 77.85%, respectively. The IIEF-5 assessment revealed a prevalence of ED of 55.34%, 88.20%, and 91.77%, respectively, among those aged 40-49, 50-59, and 60-69 years. AD rates of ED subjects were 13.73% and 40.69% according to the TT and cFT cut-off levels. The prevalence of ED among subjects positive for LOH (ADAM+ and AMS+) were 88.81% and 95.80%, respectively. The prevalence of ED among the AD subjects (TT and cFT cut-off levels) with LOH (ADAM+ and AMS+) were 86.67%/81.82%. And the prevalence of ED among clinical LOH subjects (ADAM+ and AMS+) were 89.51%/98.48%. CONCLUSIONS: We found that middle-aged and aging Chinese males were at a relatively high risk of ED. The prevalence of ED among subjects with LOH symptoms was greater than in all recruited subjects. The effect of LOH on the prevalence of ED far outweighed the risk of decreased testosterone levels.

Effect of sildenafil on erectile dysfunction and improvement in the quality of sexual life in China: a multi-center study.

BACKGROUND: Erectile dysfunction (ED) is a common medical condition in middle-aged and elderly men; however, large-scale and multi-center epidemiologic studies about the treatment effects on ED in China are lacking. OBJECTIVE: To elucidate the efficacy and safety of a phosphodiesterase type 5 inhibitor (PDE5-i) in the treatment of men with ED in China. METHODS: Patients clinically diagnosed with ED from 53 andrology centers in 15 metropolitan areas in China who were willing to undergo treatment for ED were enrolled in the study. Each participant received 4 weeks of unique PDE5-i treatment, and completed the following forms (International Index of Erectile Function score 5 [IIEF-5], the Erection Hardness Score [EHS], Self-Esteem and Relationship [SEAR], and SF-36 of Health Related Quality of Life). Pre-and post-treatment data were compared using descriptive analysis. RESULTS: A total of 1956 ED patients were included in this study; 1922 patients provided valid questionnaires for analysis. Four weeks of sildenafil treatment was considered effective and safe. Specifically, the IIEF-5 sores (11.30 ± 3.7 vs. 20.02 ± 5.1, P < 0.05), EHS levels (99.1% patients increases to level 3 or 4), and the SEAR scores (32.5 vs. 55.1, P < 0.05) were significantly improved compared to baseline. Sildenafil therapy also significantly improved the satisfaction, enjoyment, and frequency of sexual attempts and sexual activity, as well as physical vigor and mental health scores. CONCLUSION: The present study provides direct evidence regarding the efficacy and safety of sildenafil therapy in a large sample of Chinese men with ED, thus verifying that sildenafil improved the symptoms and quality of sexual life.

Identification and Characterization of Xlr5c as a Novel Nuclear Localization Protein in Mouse Germ Cells.

BACKGROUND: Spermatogenesis is the complex process by which diploid stem cells generate haploid germ cells in gamete production. Members of the Xlr (X-chromosome linked, lymphocyte regulated) superfamily play essential roles in spermatogenesis. The expression, localization and role in spermatogenesis of one such member, Xlr5c, has not been reported previously. METHODOLOGY/PRINCIPAL FINDINGS: Xlr5c mRNA and protein levels in murine testes and other tissues were investigated using RT-PCR and Western blotting. Xlr5c was abundantly transcribed in mouse testes, particularly during the early stages of spermatogenesis and throughout prophase I in the nuclei of spermatocytes. Xlr5c was specifically localized at synaptonemal complexes(SCs) region in preleptotene and pachytene spermatocytes, as was the homologous Xlr protein Sycp3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Xlr5c was abundantly transcribed in germ cells, localized at SCs region, where it may play a potential role during the early stages of spermatogenesis. Identification and characterization of this novel testis protein may offer a new perspective for understanding of the molecular mechanisms involved in germ cell differentiation.

miRNA-mediated regulation of heat shock proteins in human ejaculated spermatozoa.

BACKGROUND/AIM: Heat shock proteins (HSPs) are expressed in human spermatozoa and play a role in sperm function. MicroRNAs (miRNAs) regulate gene expression, and the possible involvement of miRNAs in the regulation of HSP gene expression in sperm was investigated in this study. MATERIALS AND METHODS: miRNAs differentially expressed in 8 copies of an oligoasthenozoospermic semen group (OA) were identified by comparison with a normal male semen control group (NC) using microarray technology. Potential targets of HSP proteins among the differentially expressed miRNAs were further investigated. Results: HSP40, HSP60, HSP70, and HSP90 were all found to be expressed in human ejaculated spermatozoa. A total of 32 miRNAs showed significant differences in expression between the OA and NC groups. Ten of these miRNAs encoded potential targets of HSPs. CONCLUSION: These results show that specific miRNAs are expressed in human ejaculated spermatozoa. These miRNAs appear to be involved in regulating the expression of HSP40, HSP70, and HSP90, and this in turn affects sperm function.

Correlation between body mass index and semen quality in male infertility patients.

BACKGROUND/AIM: The question of whether body mass index (BMI) affects semen quality and male fertility is controversial. The purpose of this research was to determine whether there is a correlation between BMI and semen analysis parameters. MATERIALS AND METHODS: A total of 617 male infertility patients were recruited and separated into 3 groups according to BMI values as follows: normal weight group (n = 334), overweight group (n = 220), and obese group (n = 63). Height and weight were measured and a routine semen analysis was performed for all patients. RESULTS: Significant differences existed in BMI, age, and sperm motility (progressive motility) among the 3 groups. BMI and abstinence period were negatively correlated with sperm motility (P < 0.05 and P < 0.01), although they did not correlate with semen volume, total sperm number, concentration, and rate of sperm with normal morphology (P > 0.05). Abstinence, BMI, and age had a linear correlation with sperm motility (P < 0.01) in that order of influence. CONCLUSION: Sperm motility, an important semen parameter with respect to male fertility, is reduced in men with increased BMI, and BMI is one of the risk factors that influence semen quality.

SLX2 interacting with BLOS2 is differentially expressed during mouse oocyte meiotic maturation.

Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation.

SYCP3-like X-linked 2 is expressed in meiotic germ cells and interacts with synaptonemal complex central element protein 2 and histone acetyltransferase TIP60.

Meiosis is the process by which diploid germ cells produce haploid gametes. A key event is the formation of the synaptonemal complex. In the pachytene stage, the unpaired regions of X and Y chromosomes form a specialized structure, the XY body, within which gene expression is mostly silenced. In the present study, we showed that SYCP3-like X-linked 2 (SLX2, 1700013H16Rik), a novel member of XLR (X-linked Lymphocyte-Regulated) family, was specifically expressed in meiotic germ cells. In the spermatocyte SLX2 was distributed in the nucleus of germ cells at the preleptotene, leptotene and zygotene stages and is then restricted to the XY body at the pachytene stage. This localization change is coincident with that of phosphorylated histone H2AX (γH2AX), a well-known component of the sex body. Through yeast two-hybrid screening and coimmunoprecipitation assays, we demonstrated that SLX2 interacts with synaptonemal complex central element protein 2 (SYCE2), an important component of synaptonemal complex, and histone acetyltransferase TIP60, which has been implicated in remodeling phospho-H2AX-containing nucleosomes at sites of DNA damage. These results suggest that SLX2 might be involved in DNA recombination, synaptonemal complex formation as well as sex body maintenance during meiosis.

Mouse Fem1b interacts with and induces ubiquitin-mediated degradation of Ankrd37.

Ankyrin repeat domain 37 (Ankrd37), a protein containing ankyrin repeats (ARs) and a putative nuclear localization signal (NLS), is highly conserved from zebrafish to humans. In mouse testes, Ankrd37 protein was initially present in the cytoplasm of elongating spermatids, and finally restricted to the nuclei of spermatozoa during spermatogenesis. Ankrd37 bound to feminization 1 homolog b (Fem1b) as indicated by yeast two-hybrid screening and co-immunoprecipitation assays. Ankrd37 facilitated the transport of Fem1b protein from cytoplasm to nuclei in co-transfected CHO cells. In addition, the protein level of Ankrd37 was decreased in a Fem1b dose-dependent manner as shown by the transfection experiments, and Ankrd37 was ubiquitinated in the presence of Fem1b. As the nematode Fem-1 has been shown to target its downstream effector TRA-1 for ubiquitin-mediated degradation, we report in the present study that mouse Fem1b targets Ankrd37 for degradation in the same manner.

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice.

BACKGROUND: Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. CONCLUSIONS/SIGNIFICANCE: SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.

Microisolation and microcloning of bovine X-chromosomes for identification of sorted buffalo (Bubalus bubalis) spermatozoa.

Flow-cytometry sorting technology has been successfully used to separate the X- and Y-chromosome bearing spermatozoa for production of sex-preselected buffalo. However, an independent technique should be employed to validate the sorting accuracy. In the present study, X-chromosomes of bovine were micro-dissected from the metaphase spreads by using glass needles. Then X-chromosomes were then amplified by PCR and labelled with Cy3-dUTP for use as a probe in hybridization of the unsorted and sorted buffalo spermatozoa -chromosome. The results revealed that 47.7% (594/1246) of the unsorted buffalo spermatozoa were positive for X- chromosome probe, which was conformed to the sex ratio in buffalo (X:Y spermatozoa=1:1); 9.6% (275/2869) of the Y-sorted buffalo spermatozoa and 86.1% (1529/1776) of the X-sorted buffalo spermatozoa showed strong X-chromosome FISH signals. Flow cytometer re-analysis revealed that the proportions of X- and Y-bearing spermatozoa in the sorted X and Y semen was 89.6% and 86.7%, respectively. There were no significant differences between results assayed by flow-cytometry re-analysis and by FISH in this study. In conclusion, FISH probe derived from bovine X- chromosomes could be used to verify the purity of X and Y sorted spermatozoa in buffalo.