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// Yen-Lin Chen 1, 2, 3 , Yi-Ting Tsai 1 , Ting-Ting Chao 4 , Yi-No Wu 5 , Meng-Chuan Chen 6 , Ying-Hung Lin 3 , Chun-Hou Liao 3, 7 , Shang-Shing P. Chou 2 and Han-Sun Chiang 3, 7 1 Department of Pathology, Cardinal Tien Hospital, New Taipei, Taiwan 2 Department of Chemistry, Fu-Jen Catholic University, New Taipei, Taiwan 3 Graduate Institute of Biomedical and Pharmaceutical Science, Fu-Jen Catholic University, New Taipei, Taiwan 4 Medical Research Center, Cardinal Tien Hospital, New Taipei, Taiwan 5 School of Medicine, Fu-Jen Catholic University, New Taipei, Taiwan 6 Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan 7 Division of Urology, Department of Surgery, Cardinal Tien Hospital, New Taipei, Taiwan Correspondence to: Han-Sun Chiang, email: 05382@mail.fju.edu.tw Keywords: cavernous nerve; Schwann cell; CIP2A; DAPK; GAS6/AXL Received: July 22, 2017 Accepted: October 28, 2017 Published: January 05, 2018 ABSTRACT Purpose: Impotence is one of the major complications occurring in prostate cancer patients after radical prostectomy (RP). Self-repair of the injured nerve has been observed in animal models and in patients after RP. However, the downstream signalling is not well documented. Here, we found that the DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. Materials and Methods: The 3 groups were a sham group, a 14-day post-bilateral cavernous nerve injury (BCNI) group and a 28-day post-BCNI group. Erectile function was assessed and immunohistochemistry was performed. The rat Schwann cell RSC96 line was chosen for gene knockdown, cell viability, western blot, immunofluorescence and co-immunoprecipitation assays. Results: The intracavernosal pressure was low on the 14 th day after BCNI and partially increased by the 28 th day. GAS6 and p-AXL expression gradually increased in the cavernous nerve after BCNI. RSC96 cells incubated with a GAS6 ligand showed increased levels of p-ERK1/2 and p-AKT. Moreover, DAPK and CIP2A.p-AXL and p-DAPK and CIP2A complexes were identified by both immunoblotting and co-immunoprecipitation. Conclusion: The DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. CIP2A inhibits PP2A activity, which results in p-DAPK(S308) maintenance and promotes Schwann cell proliferation. CIP2A is a potential target for the treatment of nerve injury after RP.

The main objective of this study was to evaluate the potential genetic effects of SEPT14 on male infertility through sequencing the SEPT14 coding region. To address this research gap, 254 men with sperm abnormalities and 116 normozoospermic men were recruited, and the whole-coding regions of SEPT14 were sequenced. Two heterozygous mutations, p.Ala123Thr (3/254 vs. 0/116) and p.Ile333Thr (3/254 vs. 0/116), were identified in these cases. A high percentage of defective sperm heads was found in sperm with mutated SEPT14. Both mutations are highly evolutionarily conserved among vertebrates. The results of a fine morphological and chromatin structural analysis indicated severely malformed sperm heads with abnormal chromatin packaging through transmission electron microscopy and Toluidine blue staining. Compared with controls, high DNA fragmentation was demonstrated in sperm from cases carrying SEPT14 mutations using the comet assay. In addition, these two mutations in SEPT14 affected its polymerization ability in vitro. These data revels that the two SEPT14 missense mutations impaired sperm head morphology and induced DNA damage. Our study suggests that genetic variant of SEPT14 is one of the effects for human sperm formation and male fertility.

Male infertility is observed in approximately 50% of all couples with infertility. Intracytoplasmic sperm injection (ICSI), a conventional artificial reproductive technique for treating male infertility, may fail because of a severe low sperm count, immotile sperm, immature sperm, and sperm with structural defects and DNA damage. Our previous studies have revealed that mutations in the septin (SEPT)-coding gene SEPT12 cause teratozoospermia and severe oligozoospermia. These spermatozoa exhibit morphological defects in the head and tail, premature chromosomal condensation, and nuclear damage. Sperm from Sept12 knockout mice also cause the developmental arrest of preimplantation embryos generated through in vitro fertilization and ICSI. Furthermore, we found that SEPT12 interacts with SPAG4, a spermatid nuclear membrane protein that is also named SUN4. Loss of the Spag4 allele in mice also disrupts the integration nuclear envelope and reveals sperm head defects. However, whether SEPT12 affects SPAG4 during mammalian spermiogenesis remains unclear. We thus conducted this study to explore this question. First, we found that SPAG4 and SEPT12 exhibited similar localizations in the postacrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. Second, SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. Third, SEPT12 expression also altered the localizations of nuclear membrane proteins: LAMINA/C in the cells. This effect was specifically due to the expression of SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11. Based on these results, we suggest that SEPT12 is among the moderators of SPAG4/LAMIN complexes and is involved in the morphological formation of sperm during mammalian spermiogenesis.

Background.The pathophysiology of Taiwanese congenital bilateral absence of the vas deferens (CBAVD) is different from that in Caucasians. In particular, major cystic fibrosis transmembrane conductance regulator (CFTR) mutations and cystic fibrosis are absent in the former. Instead, deficiency in solute carrier family 9 sodium/hydrogen exchanger isoform 3 (SLC9A3) may play a role by generating obstructive azoospermia and degraded epithelial structure in the reproductive tract.Objectives.The objective of the study was to test whether SLC9A3 variants cause Taiwanese CBAVD.Materials and Methods.Six-month-oldSlc9a3−/−male mice were used to evaluate the effect of long-term SLC9A3 loss on the reproductive system. A case-control cohort of 29 men with CBAVD and 32 fertile men were genotyped for SLC9A3 variants.Results.SLC9A3 was expressed and localized in the apical border of the epithelium of human vas deferens and glandular epithelium of the seminal vesicle. SLC9A3 deficiency specifically induces atrophy of vas deferens and unfolding of seminal vesicle mucosa in mice. Loss of SLC9A3 increased the incidence of CBAVD in humans from 3.1% to 37.9% (p < 0.001). Up to 75.9% of CBAVD patients carry at least one variant in either SLC9A3 or CFTR.Discussion.Our findings build upon previous data associated with CBAVD pathogenesis. Here, we now report for the first time an association between CBAVD and loss ofSLC9A3and propose that specific defects in the reproductive duct due toSLC9A3variants drive CBAVD development.Conclusion.The data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3 function in reproduction.

Approximately 2–15% of couples experience infertility, and around half of these cases are attributed to male infertility. We previously identified TBC1D21 as a sterility-related RabGAP gene derived from infertile men. However, the in vivo function of TBC1D21 in male fertility remains unclear. Here, we show that loss of Tbc1d21 in mice resulted in male infertility, characterized by defects in sperm tail structure and diminished sperm motility. The mitochondria of the sperm-tail had an abnormal irregular arrangement, abnormal diameter, and structural defects. Moreover, the axoneme structure of sperm tails was severely disturbed. Several TBC1D21 interactors were selected via proteomic analysis and functional grouping. Two of the candidate interactors, a subunit protein of translocase in the outer membrane of mitochondria (TOMM20) and an inner arm component of the sperm tail axoneme (Dynein Heavy chain 7, DNAH7), confirmed in vivo physical co-localization with TBC1D21. In addition, TOMM20 and DNAH7 detached and dispersed outside the axoneme in Tbc1d21-deficient sperm, instead of aligning with the axoneme. From a clinical perspective, the transcript levels of TBC1D21 in sperm from teratozoospermia cases were significantly reduced when compared with those in normozoospermia. We concluded that TBC1D21 is critical for mitochondrial and axoneme development of mammalian sperm., Author summary Male sterility affects one in seven couples, and around half of these are the result of male factor disorders. The common conditions of male infertility cases include reduced sperm motility, low sperm account, and spermatozoa with abnormal morphology. We previously identified TBC1D21 as a sterility-related RabGAP gene from infertile men. However, the in vivo function of TBC1D21 in male fertility remains untested. Here, we created Tbc1d21 knockout mice. The knockout males were completely sterile, and had sperm-tail and mitochondria defects. Two interactors of TBC1D21, TOMM20, and DNAH7, may account for the irregular and defective patterns observed in Tbc1d21-/- mouse sperm tails. From a clinical perspective, TBC1D21 transcripts are reduced in sperm from patients exhibiting teratozoospermia. Our results indicate that TBC1D21 plays a critical role in the formation of intact mitochondria, and structure of mammalian sperm.

Congenital bilateral absence of vas deferens (CBAVD) is a special entity in obstructive azoospermia. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are involved in Taiwanese CBAVD but most heterozygous 5T variant. The solute carrier family 9 isoform 3 (SLC9A3) is the Na+/H+ exchanger, which interacts with CFTR and regulates the Ca2+ homeostasis. Loss of SLC9A3 decreases CFTR protein and causes obstructive azoospermia in mice. It also causes mal-reabsorption by the efferent tubules, which leads to the obstructive phenomenon and eventually results in testicular atrophy. In 6-month old SLC9A3 deficiency mice, the atrophy of their vas deferens and seminal vesicles become more prominent. Decreases of CFTR expression in the reproductive organ in the SLC9A3 deficient (−/−) mice prove the interaction between CFTR and SLC9A3 in the reproductive tract. Most of Taiwanese CBAVD have at least one variant of SLC9A3 deletion and CFTR IVS8-5T, which co-contribute to Taiwanese CBAVD. The report indicates SLC9A3 deficiency can reverse the pathological changes in the gastrointestinal tract of CF mice. Further research can explore the definite mechanism of SLC9A3 and its role interacting with CFTR in different organ systems, which can contribute to novel treatment for the patients with cystic fibrosis and CBAVD. Keywords: Congenital bilateral absence of vas deferens, Cystic fibrosis transmembrane conductance regulator, Solute carrier family 9 isoform 3

Solute carrier family 9 isoform 3 (SLC9A3), a Na⁺/H⁺ exchanger, regulates the transepithelial absorption of Na⁺ and water and is primarily expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens. Loss of the Slc9a3 allele in mice enhances intestinal fluid and causes diarrhoea as a consequence of diminished Na⁺ and HCO₃- absorption. Hence, the loss also causes male infertility and reveals the abnormal dilated lumen of the rete testis and calcification in efferent ductules. However, whether loss of Slc9a3 alleles also disrupts mammalian spermatogenesis remains unknown. First, through immunoblotting, we determined that SLC9A3 is highly expressed in the murine testis compared with the small intestine, epididymis, and vas deferens. During murine spermatogenesis, SLC9A3 is specifically expressed in the acrosome region of round, elongating, and elongated spermatids through immunostaining. Furthermore, SLC9A3 signals are enriched in the acrosome of mature sperm isolated from the vas deferens. In Slc9a3 knockout (KO) mice, compared with the same-aged controls, the number of spermatids on the testicular section of the mice progressively worsened in mice aged 20, 35, and 60 days. Sperm isolated from the epididymis of Slc9a3 KO mice revealed severe acrosomal defects. Our data indicated that SLC9A3 has a vital role in acrosomal formation during spermiogenesis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na+/H+ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3–/–epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations., Author summary Cystic fibrosis (CF) is the most common inherited life-threatening disease in Caucasians. The most well-known cause of CF is a genetic defect in CFTR, an apical membrane chloride and bicarbonate channel. The symptoms of CF include defects in the respiratory, digestive, and male reproductive systems. Most male patients with CF are infertile due to congenital bilateral absence of the vas deferens (CBAVD), which leads to obstructive azoospermia. Nevertheless, Taiwanese patients with CBAVD do not carry the common mutations of CFTR found in Caucasians. We have identified a potential candidate, SLC9A3, of which a single copy is lost in Taiwanese patients with CBAVD. In addition to the previously reported role of SLC9A3 in the digestive system and efferent ductules, we now report that the SLC9A3 deficiency causes obstructive azoospermia and impairs the epithelial structure of the reproductive tract. Loss of SLC9A3 also leads to dramatic reduced expression of CFTR in the reproductive tract. We suggest that the interplay between SLC9A3 and CFTR is responsible for CF-related infertility. Thus, we have characterized a potential critical player in the pathogenesis of CBAVD and provide a new diagnostic candidate for Asian patients with CBAVD.

Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12) is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identified as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1). NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12–NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the filament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck of wild-type sperm. These results indicate that SEPT12–NDC1 complexes are involved in mammalian spermiogenesis.

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

There is an unmet need for treatment of erectile dysfunction resulting from radical prostatectomy and cavernous nerve (CN) injury. Given the neuroprotective properties of docosahexaenoic acid (DHA), we investigated its effect on penile functional and structural recovery in a rat model of bilateral cavernous nerve injury. Rats were subject to CN injury and received intraperitoneal administration of either vehicle or a DHA nanoemulsion (nano-DHA) at 10, 50, or 250 μg/kg. Functional testing and histological analyses were performed at 28 days post-injury. The maximum intracavernosal pressure (ICP) and other measures of erectile function were significantly higher in the nano-DHA groups than in the vehicle group (p p

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm.

Endothelial progenitor cells (EPCs) are bone marrow-derived endothelial cells capable of circulating, proliferating, and differentiating into mature endothelial cells. Circulating EPCs can be directly recruited to some extent at sites of injury, and their administration could accelerate repair or endothelialization of the damaged tissue. We investigated the effects of intracavernous injections of EPCs into the corpora cavernosa of rats with erectile dysfunction (ED) caused by bilateral cavernous nerve (CN) injury. Overall, 24 male Sprague-Dawley rats were randomized into three groups: sham surgery, vehicle-only, or EPC treatment. Rats in the EPC treatment and vehicle-only groups were subjected to bilateral CN injury before injection of EPCs or vehicle, respectively, into the corpora cavernosa. Four weeks after surgery, erectile function was assessed by measuring maximum intracavernosal pressure (ICP), change in ICP, area under the ICP curve, and ratio of change in ICP and mean arterial pressure (MAP; ΔICP/MAP). Penile tissue was histomorphometrically analyzed for the expression of neural nitric oxide synthase (nNOS), neurofilament-1 (NF-1), von Willebrand factor (vWF), endothelial NOS (eNOS), and smooth muscle cell content. Maximum ICP and all other functional parameters of erectile function were significantly reduced in the vehicle-only group vs. the sham and EPC treatment groups (all p

Male infertility has become a worldwide health problem, but the etiologies of most cases are still unknown. SEPT12, a GTP-binding protein, is involved in male fertility. Two SEPT12 mutations (SEPT12(T89M) and SEPT12(D197N)) have been identified in infertile men who have a defective sperm annulus with a bent tail. The function of SEPT12 in the sperm annulus is still unclear. Here, we found that SEPT12 formed a filamentous structure with SEPT7, SEPT 6, SEPT2 and SEPT4 at the sperm annulus. The SEPT12-based septin core complex was assembled as octameric filaments comprising the SEPT proteins 12-7-6-2-2-6-7-12 or 12-7-6-4-4-6-7-12. In addition, the GTP-binding domain of SEPT12 was crucial for its interaction with SEPT7, and the N- and C-termini of SEPT12 were required for the interaction of SEPT12 with itself to polymerize octamers into filaments. Mutant mice carrying the SEPT12(D197N) mutation, which disrupts SEPT12 filament formation, showed a disorganized sperm annulus, bent tail, reduced motility and loss of the SEPT ring structure at the sperm annulus. These phenotypes were also observed in an infertile man carrying SEPT12(D197N). Taken together, our results demonstrate the molecular architecture of SEPT12 filaments at the sperm annulus, their mechanical support of sperm motility, and their correlation with male infertility.

BACKGROUND: DAZ gene family is crucial for human spermatogenesis that requires the precise co-ordination of cell cycle events. CDC25A is recognized as the downstream substrate of DAZ gene family and is thought to function on the M-phase regulation of cell cycles. We investigated the expression profiles of CDC25A in the testes of infertile men and evaluated the relationship between CDC25A levels and testicular phenotype, clinical hormonal parameters and sperm retrieval results. METHODS: The protein and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemistry and quantitative real-time-PCR. CDC25A in human spermatozoa was investigated by western blotting and immunofluorescence staining. RESULTS: The CDC25A protein was expressed mainly in spermatocyte, spermatid and spermatozoa. CDC25A transcript levels were significantly decreased (P = 0.0009) in patients with spermatogenic failure, especially in men with meiotic arrest and Sertoli cell-only syndrome. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval (P = 0.005). CONCLUSIONS: Decreased CDC25A is associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies are necessary to explore the functional roles of CDC25A in human spermatozoa.

Objective To determine the expression profiles of the DAZ gene family in men with and without spermatogenic failure. Design Prospective case study. Setting University-based reproductive clinics and genetics laboratory. Patient(s) Thirty-four infertile men presenting with azoospermia. Intervention(s) The mRNA transcript concentrations of the DAZ family genes (BOULE, DAZL, DAZ) and the housekeeping GAPDH gene in the testes of azoospermic men were examined by quantitative competitive–reverse transcription–polymerase chain reaction (QC-RT-PCR). The steady-state concentrations of mRNA encoding for each gene in each testicular sample were normalized by the amounts of GAPDH. Main outcome measure(s) Transcript ratios (gene/GAPDH) of BOULE, DAZL, and DAZ. Result(s) The transcript ratios for BOULE, DAZL, and DAZ were significantly decreased in tissues with spermatogenic failure (hypospermatogenesis, maturation arrest, and Sertoli cell–only). However, the ratios of BOULE/DAZL and DAZ/DAZL did not reveal any significant difference in all tissues. Three patients with DAZ deletion possess lower transcripts of BOULE and DAZL. Conclusion(s) All members of the DAZ gene family play important roles in human spermatogenesis. Decreased concentrations of DAZ family members in men with spermatogenic failure may be due to the secondary effect of germ cell loss, and transcriptional control of BOULE, DAZL, and DAZ are not altered in the various degrees of spermatogenic failure. Although the sample size is limited, no compensatory increase of DAZL or BOULE transcription was found in men with DAZ deletion.

Objectives To investigate the transcriptional levels of four azoospermia factor genes in the testis of azoospermic men and to investigate the association between transcriptional levels and the results of sperm retrieval. Methods Thirty-eight azoospermic men with normal karyotype and without Y chromosome gene deletions were enrolled. The amounts of USP9Y (ubiquitin specific protease 9, Y chromosome), DBY (dead box on the Y), RBMY1 (RNA-binding motif on the Y, 1), and DAZ (deleted in azoospermia) transcripts were examined by quantitative competitive-reverse transcriptase-polymerase chain reaction. The steady-state concentrations of mRNA encoding for each gene in each testicular sample were normalized by the amounts of a housekeeping gene (glyceraldehyde phosphate dehydrogenase [GAPDH]). Differences in the transcript ratios (gene transcript/GAPDH transcript) among patients with different testicular histologic findings (normal spermatogenesis, hypospermatogenesis, maturation arrest, Sertoli cell-only syndrome) were analyzed. For each gene, the association between the transcript ratios and the results of sperm retrieval was evaluated. Results No statistically significant difference was found in the transcript ratios of USP9Y and DBY among the four histologic patient groups (P = 0.33 and P = 0.21, respectively). In contrast, statistically significant decreases were found in the transcript ratios of RBMY1 and DAZ in patients with spermatogenic failure (P = 0.0002 and P = 0.002, respectively). The transcript ratios of USP9Y and DBY were not associated with the results of sperm retrieval, and the transcript ratios of RBMY1 and DAZ revealed a positive association with successful sperm retrieval. Conclusions The decreased transcriptional levels of RBMY1 and DAZ in patients with spermatogenic failure may reflect the generalized loss of germ cells. The transcriptional levels of RBMY1 and DAZ may have the potential of becoming useful parameters in the prediction of successful sperm retrieval.

Objective To address phenotype/genotype correlation in a man with i(Y)(p10). Design Case report. Setting University-based reproductive genetics laboratory. Patient(s) A 27-year-old azoospermic man with i(Y)(p10), relatively normal stature, and testicular Sertoli-cell-only syndrome. Intervention(s) Testicular biopsy, cytogenetic study, Y-chromosome deletion mapping analysis, fluorescence in situ hybridization (FISH). Main outcome measure(s) Expression of Y-chromosome genes. Result(s) We have identified one azoospermic man with i(Y)(p10) of 312 Taiwanese men presenting with a severe spermatogenic defect. Y-chromosome deletion mapping analysis confirmed deletions of all Yq sequences, including a putative growth controlling gene. Fluorescence in situ hybridization (FISH) analysis showed duplication of Yp material. The patient had normal stature considering midparental height. He also had no germ cells in the testicular tissue (Sertoli-cell-only syndrome) resulting from the loss of azoospermia factor in Yq. Conclusion(s) Among structural rearrangements of the Y-chromosome, the isochromosome of Yp occurs very rarely. This case is the first reported case of an isochromosome Yp with a detailed description of testicular histology and body height.

Asthenozoospermia is a major cause of male infertility. However, the molecular mechanisms underlying sperm-motility defects remain largely unknown in the majority of cases. In our previous study, we applied a proteomic approach to identify unknown proteins that were downregulated in spermatozoa with low motility compared to spermatozoa with good motility. Several sperm motility- related proteins have been identified. In this study, 3-hydroxyisobutyrate dehydrogenase (HIBADH), one of the proteins identified using the proteomic tools, is further characterized.Reverse-transcription polymerase chain reactions (RT-PCR), western blotting, and immunofluorescence assays (IFA) were preformed to investigate the expression pattern. The enzymatic activity of HIBADH was evaluated in sperm with good (50 %), moderate (50 %) and lower motility (20 %).Using RT-PCR, we found that transcripts of HIBADH are enriched in the cerebellum, heart, skeletal muscle, uterus, placenta, and testes of male humans. In western blotting, it is expressed in the placenta, testes, and spermatozoa. During spermiogenesis, HIBADH is located at the mid-piece (a specialized development from the mitochondria) of elongating, elongated, and mature sperm. The enzymatic activity of HIBADH in sperm with moderate and lower motility were significantly reduced compared with good motility (P0.0001 and P0.05, respectively).Our study indicated that HIBADH is involved in the mitochondrial function of spermatozoa, and maintains sperm motility. It may serve as a sperm-motility marker.

It is estimated that 10-15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12(+/+)/Septin12(+/-) chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm

Septins (full name: Septin; symbol name: SEPT) belong to a family of polymerizing GTP binding proteins that are required for many cellular functions, including membrane compartmentalization, vesicle trafficking, mitosis and cytoskeletal remodeling. Two of the 14 family members in the mammalian species, Septin12 and 14 are expressed specifically in the testis. In the mouse, knockout of Septin4 and Septin12 leads to male sterility with distinctive sperm pathology (defective annulus or bent neck). In humans, sperm with abnormal expression pattern of SEPT4, 7 and 12 are more prevalent in infertile men. How septin filament is assembled /dissembled and how the SEPT-related complex regulates spermatogenesis still await further investigation.

Objective To test the diagnostic efficiency of a gene-specific, five-marker screening strategy for the detection of Y chromosome deletions. Design Prospective case study. Setting University genetics laboratory and reproductive clinics. Patient(s) Six hundred twenty-seven infertile men and 212 fertile men. Intervention(s) Peripheral blood samples were screened for Y chromosome deletions in a triple-blind fashion using three protocols: protocol I consisted of five gene-specific markers, including USP9Y, DBY, SMCY, RBM1 , and DAZ ; protocol II included 14 gene-specific markers; and protocol III consisted of six sequence-tagged sites (STSs) markers recommended by EAA/EMQN. Main Outcome Measure(s) Deletion status of Y chromosome genes or sequence-tagged sites. Result(s) Protocols I and II identified the same 41 infertile patients with Y deletions. Protocol III identified 38 infertile patients with Y deletions, and all 38 patients were also identified by protocols I and II. One patient with isolated USP9Y deletion and two patients with isolated DBY deletions, as detected by protocols I and II, could not be identified by protocol III. Conclusion(s) We observed mostly consistent results between our protocols and the EAA/EMQN protocol. This gene-specific, five-marker screening panel provides the same diagnostic efficiency as the EAA/EMQN protocol and may be considered an alternative to the EAA/EMQN protocol.

Background Testicular sperm retrieval can lead to paternity for azoospermic patients with spermatogenic failure. The human BOULE gene, a meiotic regulator of germ cells, is a gene whose altered expression may be associated with sterility. We determined the levels of BOULE transcripts in the testes of azoospermic patients, and evaluated the relationship between BOULE transcript levels and patients' testicular phenotypes, clinical parameters and sperm retrieval results. Methods and results BOULE transcript levels in the testes of 41 azoospermic patients were examined by quantitative competitive-reverse transcription-polymerase chain reaction. A significant decrease in BOULE transcript levels was detected in patients with spermatogenic failure, and BOULE transcript levels progressively decreased with increasing severity of testicular failure. BOULE transcript levels did not correlate with the serum hormone parameters measured. Significantly higher BOULE transcript levels were detected in 19 patients with successful sperm retrieval than in 12 patients with failed sperm retrieval. When using a cut-off value of 0.5 for BOULE transcript ratio to predict the success of sperm retrieval, both the sensitivity and specificity value were 100%. Conclusions We suggest the BOULE transcript plays an important role in human spermatogenesis and that the levels may predict the presence of testicular sperm in patients with spermatogenic failure.

Single-strand conformation polymorphism analysis of exon-containing genomic DNA segments of the deleted-in-azoospermia-like (DAZL) gene was performed in 160 infertile Taiwanese men presenting with severe oligozoospermia and nonobstructive azoospermia. An A--G transition at nucleotide 386 in exon 3 was identified. The mutation is located within the RNA-recognition motif (aa 32-117) domain of the DAZL protein and will lead to Thr54--Ala change (T54A) of DAZL protein. Analysis of cDNA from testicular tissue of infertile carriers showed absence of expression for the T54A allele, implying that the allele carrying T54A polymorphism is hardly, if ever, expressed. The frequencies of T54A allele in patients and the control group were 7.39% and 0.86%, respectively (P = 0.0003). The phenotypes varied significantly in cases with heterozygous T54A polymorphism, ranging from hypospermatogenesis and maturation arrest to Sertoli cell-only syndrome. A combination of DAZ gene deletion and T54A polymorphism did not worsen the phenotype. Our findings provide strong evidence for the role of the autosomal DAZL gene in human spermatogenesis.