Proteomic profiling of TBC1 domain family member 21-null sperms reveals the critical roles of TEKT 1 in their tail defects.

BACKGROUND: Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown. RESULTS: Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21-/- sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures. CONCLUSIONS: These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation.

Deleterious genetic changes in AGTPBP1 result in teratozoospermia with sperm head and flagella defects.

Approximately 10%-15% of couples worldwide are infertile, and male factors account for approximately half of these cases. Teratozoospermia is a major cause of male infertility. Although various mutations have been identified in teratozoospermia, these can vary among ethnic groups. In this study, we performed whole-exome sequencing to identify genetic changes potentially causative of teratozoospermia. Out of seven genes identified, one, ATP/GTP Binding Protein 1 (AGTPBP1), was characterized, and three missense changes were identified in two patients (Affected A: p.Glu423Asp and p.Pro631Leu; Affected B: p.Arg811His). In those two cases, severe sperm head and tail defects were observed. Moreover, AGTPBP1 localization showed a fragmented pattern compared to control participants, with specific localization in the neck and annulus regions. Using murine models, we found that AGTPBP1 is localized in the manchette structure, which is essential for sperm structure formation. Additionally, in Agtpbp1-null mice, we observed sperm head and tail defects similar to those in sperm from AGTPBP1-mutated cases, along with abnormal polyglutamylation tubulin and decreasing △-2 tubulin levels. In this study, we established a link between genetic changes in AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation.

Novel five nucleotide deletion in dysferlin leads to autosomal recessive limb-girdle muscular dystrophy.

Muscular dystrophy (MD) is a genetic disorder that causes progressive muscle weakness and degeneration. Limb-girdle muscular dystrophy (LGMD) is a type of MD that mainly causes muscle atrophy within the shoulder and pelvic girdles. LGMD is classified into autosomal dominant (LGMD-D) and autosomal recessive (LGMD-R) inheritance patterns. Mutations in the Dysferlin gene (DYSF) are common causes of LGMD-R. However, genetic screening of DYSF mutations is rare in Taiwan. Herein, we identified a novel c.2867_2871del ACCAG deletion and a previously reported c.937+1G>A mutation in DYSF from a Taiwanese family with LGMD. The primary symptoms of both siblings were difficulty climbing stairs, walking on the toes, and gradually worsening weakness in the proximal muscles and increased creatine kinase level. Through pedigree analysis and sequencing, two siblings from this family were found to have compound heterozygous DYSF mutations (c. 937+1G>A and c. 2867_2871del ACCAG) within the separated alleles. These mutations induced early stop codons; if translated, truncated DYSF proteins will be expressed. Or, the mRNA products of these two mutations will merit the nonsense-mediated decay, might result in no dysferlin protein expressed. To our knowledge, this is the first report of a novel c.2867_2871del ACCAG deletion in DYSF. Further research is required to examine the effects of the novel DYSF mutation in Taiwanese patients with LGMD.

Decreased Klotho Expression Causes Accelerated Decline of Male Fecundity through Oxidative Injury in Murine Testis.

Oxidative stress is the etiology for 30-80% of male patients affected by infertility, which is a major health problem worldwide. Klotho protein is an aging suppressor that functions as a humoral factor modulating various cellular processes including antioxidation and anti-inflammation, and its dysregulation leads to human pathologies. Male mice lacking Klotho are sterile, and decreased Klotho levels in the serum are observed in men suffering from infertility with lower sperm counts. However, the mechanism by which Klotho maintains healthy male fertility remains unclear. Klotho haplodeficiency (Kl+/-) accelerates fertility reduction by impairing sperm quality and spermatogenesis in Kl+/- mice. Testicular proteomic analysis revealed that loss of Klotho predominantly disturbed oxidation and the glutathione-related pathway. We further focused on the glutathione-S-transferase (GST) family which counteracts oxidative stress in most cell types and closely relates with fertility. Several GST proteins, including GSTP1, GSTO2, and GSTK1, were significantly downregulated, which subsequently resulted in increased levels of the lipid peroxidation product 4-hydroxynonenal and apoptosis in murine testis with low or no expression of Klotho. Taken together, the loss of one Kl allele accelerates male fecundity loss because diminished antioxidant capability induces oxidative injury in mice. This is the first study that highlights a connection between Klotho and GST proteins.

Localization Patterns of RAB3C Are Associated with Murine and Human Sperm Formation.

Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to several critical cellular processes such as cytokinesis, cytoskeletal remodeling, and vesicle transportation. In our previous study, we found that SEPT14 mutations resulted in teratozoospermia with >87% sperm morphological defects. SEPT14 interactors were also identified through proteomic assays, and one of the peptides was mapped to RAB3B and RAB3C. Most studies on the RAB3 family have focused on RAB3A, which regulates the exocytosis of neurotransmitters and acrosome reactions. However, the general expression and patterns of the RAB3 family members during human spermatogenesis, and the association between RAB3 and teratozoospermia owing to a SEPT14 mutation, are largely unknown. Materials and Methods: Human sperm and murine male germ cells were collected in this study and immunofluorescence analysis was applied on the collected sperm. Results: In this study, we observed that the RAB3C transcripts were more abundant than those of RAB3A, 3B, and 3D in human testicular tissues. During human spermatogenesis, the RAB3C protein is mainly enriched in elongated spermatids, and RAB3B is undetectable. In mature human spermatozoa, RAB3C is concentrated in the postacrosomal region, neck, and midpiece. The RAB3C signals were delocalized within human spermatozoa harboring the SEPT14 mutation, and the decreased signals were accompanied by a defective head and tail, compared with the healthy controls. To determine whether RAB3C is involved in the morphological formation of the head and tail of the sperm, we separated murine testicular tissue and isolated elongated spermatids for further study. We found that RAB3C is particularly expressed in the manchette structure, which assists sperm head shaping at the spermatid head, and is also localized at the sperm tail. Conclusions: Based on these results, we suggest that the localization of RAB3C proteins in murine and human sperm is associated with SEPT14 mutation-induced morphological defects in sperm.

Preparation of Catechin Nanoemulsion from Oolong Tea Leaf Waste and Its Inhibition of Prostate Cancer Cells DU-145 and Tumors in Mice.

Anti-cancer activity of catechin nanoemulsions prepared from Oolong tea leaf waste was studied on prostate cancer cells DU-145 and DU-145-induced tumors in mice. Catechin nanoemulsions composed of lecithin, Tween-80 and water in an appropriate proportion was prepared with high stability, particle size of 11.3 nm, zeta potential of -67.2 mV and encapsulation efficiency of 83.4%. Catechin nanoemulsions were more effective than extracts in inhibiting DU-145 cell growth, with the IC50 being 13.52 and 214.6 µg/mL, respectively, after 48 h incubation. Furthermore, both catechin nanoemulsions and extracts could raise caspase-8, caspase-9 and caspase-3 activities for DU-145 cell apoptosis, arresting the cell cycle at S and G2/M phases. Compared to control, catechin nanoemulsion at 20 µg/mL and paclitaxel at 10 µg/mL were the most effective in reducing tumor volume by 41.3% and 52.5% and tumor weight by 77.5% and 90.6% in mice, respectively, through a decrease in EGF and VEGF levels in serum.

ACTN4 Mediates SEPT14 Mutation-Induced Sperm Head Defects.

Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerized SEPTs participate in the modulation of various cellular processes, such as cytokinesis, cell polarity, and membrane dynamics, through their interactions with microtubules, actin, and other cellular components. The main objective of this study was to dissect the molecular pathological mechanism of SEPT14 mutation-induced sperm head defects. To identify SEPT14 interactors, co-immunoprecipitation (co-IP) and nano-liquid chromatography-mass spectrometry/mass spectrometry were applied. Immunostaining showed that SEPT14 was significantly localized to the manchette structure. The SEPT14 interactors were identified and classified as (1) SEPT-, (2) microtubule-, (3) actin-, and (4) sperm structure-related proteins. One interactor, ACTN4, an actin-holding protein, was selected for further study. Co-IP experiments showed that SEPT14 interacts with ACTN4 in a male germ cell line. SEPT14 also co-localized with ACTN4 in the perinuclear and manchette regions of the sperm head in early elongating spermatids. In the cell model, mutated SEPT14 disturbed the localization pattern of ACTN4. In a clinical aspect, sperm with mutant SEPT14, SEPT14A123T (p.Ala123Thr), and SEPT14I333T (p.Ile333Thr), have mislocalized and fragmented ACTN4 signals. Sperm head defects in donors with SEPT14 mutations are caused by disruption of the functions of ACTN4 and actin during sperm head formation.

Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice.

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.

SEPT14 Mutations and Teratozoospermia: Genetic Effects on Sperm Head Morphology and DNA Integrity.

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.

SLC9A3 Affects Vas Deferens Development and Associates with Taiwanese Congenital Bilateral Absence of the Vas Deferens.

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-old Slc9a3 -/-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 of SLC9A3 and propose that specific defects in the reproductive duct due to SLC9A3 variants drive CBAVD development. CONCLUSION: The data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3 function in reproduction.

Testis-Specific SEPT12 Expression Affects SUN Protein Localization and is Involved in Mammalian Spermiogenesis.

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.

The role of SLC9A3 in Taiwanese patients with congenital bilateral absence of vas deferens (CBAVD).

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.

TBC1D21 Potentially Interacts with and Regulates Rap1 during Murine Spermatogenesis.

Few papers have focused on small guanosine triphosphate (GTP)-binding proteins and their regulation during spermatogenesis. TBC1D21 genes (also known as male germ cell RAB GTPase-activating protein MGCRABGAP) are related to sterility, as determined through cDNA microarray testing of human testicular tissues exhibiting spermatogenic defects. TBC1D21 is a protein specifically expressed in the testes that exhibits specific localizations of elongating and elongated spermatids during mammalian spermiogenesis. Furthermore, through co-immunoprecipitation (co-IP) and nano liquid chromatography⁻tandem mass spectrometry (nano LC⁻MS/MS), Rap1 has been recognized as a potential TBC1D21 interactor. This study determined the possible roles of Rap1 and TBC1D21 during mammalian spermiogenesis. First, the binding ability between Rap1 and TBC1D21 was verified using co-IP. Second, the stronger signals of Rap1 expressed in elongating and elongated murine spermatids extracted from testicular sections, namely spermatogonia, spermatocytes, and round spermatids, were compared. Third, Rap1 and TBC1D21 exhibited similar localizations at postacrosomal regions of spermatids and at the midpieces of mature sperms, through isolated male germ cells. Fourth, the results of an activating Rap1 pull-down assay indicated that TBC1D21 overexpression inactivates Rap1 activity in cell models. In conclusion, TBC1D21 may interact with and potentially regulate Rap1 during murine spermatogenesis.

CDC42 Negatively Regulates Testis-Specific SEPT12 Polymerization.

Septin (SEPT) genes encode well-preserved polymerizing GTP-binding cytoskeletal proteins. The cellular functions of SEPTs consist of mitosis, cytoskeletal remodeling, cell polarity, and vesicle trafficking through interactions with various types of cytoskeletons. We discovered that mutated SEPTIN12 in different codons resulted in teratozoospermia or oligozoospermia. In mouse models with a defective Septin12 allele, sperm morphology was abnormal, sperm count decreased, and sperms were immotile. However, the regulators of SEPT12 are completely unknown. Some studies have indicated that CDC42 negatively regulates the polymerization of SEPT2/6/7 complexes in mammalian cell lines. In this study, we investigated whether CDC42 modulates SEPT12 polymerization and is involved in the terminal differentiation of male germ cells. First, through scanning electron microscopy analysis, we determined that the loss of Septin12 caused defective sperm heads. This indicated that Septin12 is critical for the formation of sperm heads. Second, CDC42 and SEPT12 were similarly localized in the perinuclear regions of the manchette at the head of elongating spermatids, neck region of elongated spermatids, and midpiece of mature spermatozoa. Third, wild-type CDC42 and CDC42Q61L (a constitutive-acting-mutant) substantially repressed SEPT12 polymerization, but CDC42T17N (a dominant-negative-acting mutant) did not, as evident through ectopic expression analysis. We concluded that CDC42 negatively regulates SEPT12 polymerization and is involved in terminal structure formation of sperm heads.

DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

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 14th day after BCNI and partially increased by the 28th 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.

nNOS-positive minor-branches of the dorsal penile nerves is associated with erectile function in the bilateral cavernous injury model of rats.

The changes in neuronal nitric oxide synthases (nNOS) in the dorsal penile nerves (DPNs) are consistent with cavernous nerve (CN) injury in rat models. However, the anatomical relationship and morphological changes between the minor branches of the DPNs and the CNs after injury have never been clearly explored. There were forty 12 week old male Sprague-Dawley rats receiving bilateral cavernous nerve injury (BCNI). Erectile function of intracavernous pressure and mean arterial pressure were measured. The histology and ultrastructure with H&E stain, Masson's trichrome stain and immunohistochemical stains were applied on the examination of CNs and DPNs. We demonstrated communicating nerve branches between the DPNs and the CNs in rats. The greatest damage and lowest erectile function were seen in the 14th day and partially recovered in the 28th day after BCNI. The nNOS positive DPN minor branches' number was significantly correlated with erectile function. The sub-analysis of the number of nNOS positive DPN minor branches also matched with the time course of the erectile function after BCNI. We suggest the regeneration of the DPNs minor branches would ameliorate the erectile function in BCNI rats.

SLC9A3 Protein Is Critical for Acrosomal Formation in Postmeiotic Male Germ Cells.

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.

Loss of SLC9A3 decreases CFTR protein and causes obstructed azoospermia in mice.

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.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during 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.

SEPT12-NDC1 Complexes Are Required for Mammalian Spermiogenesis.

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.