Construction of a Catsper1 DNA Vaccine and Its Antifertility Effect on Male Mice

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.     

Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse

Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.     

RNF151, a testis-specific RING finger protein, interacts with dysbindin

RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII-IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation.     

Up-regulation of CatSper genes family by selenium

Background  

CatSper1-4 are a unique family of sperm cation channels, which are exclusively expressed in the testis and play an important role in sperm motility and male fertility. Despite their vital role in male fertility, almost nothing is known about the factors regulating their expression. Here, we investigated the effects of selenium (Se) on the expression of CatSper genes and sperm parameters in aging versus young male mice.     

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins. © 1995 Wiley-Liss, Inc.     

Zonadhesin: characterization, localization, and zona pellucida binding.

Zonadhesin is a multiple-domain transmembrane protein that is believed to function as a sperm-zona pellucida binding protein. In this study we sequenced zonadhesin from rabbit testis and analyzed its processing, expression, localization, and zona pellucida binding. We show that the precursor protein occurs exclusively in the testis and that proteolytic processing results in the formation of three fragments: p43 (D1 domain), p97 (D2-D4 domains), and p58 (D4 domain-C-terminal). In mature spermatozoa the p43 and p97 fragments exist as disulfide-bonded dimers. During spermatogenesis, synthesis of zonadhesin mRNA chiefly occurs in primary spermatocytes, whereas the protein is abundant in both Sertoli cells and spermatids. In spermatozoa the protein is localized exclusively to the anterior acrosome but is not available for binding antibody on live spermatozoa. Once the acrosome reaction is induced, zonadhesin is lost from the spermatozoon, but remains with the acrosomal shroud. We show that recombinant D4 domain can bind zona pellucida, and we propose that zonadhesin functions after the acrosome reaction has been initiated to bind the acrosomal shroud to the zona pellucida.     

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.     

Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis

Kelch-like 10 (KLHL10) is a member of the BTB (Bric-a-brac, Tramtrack, and Broad-Complex)-kelch protein superfamily essential for spermiogenesis and male fertility. In a search for KLHL10-interacting proteins using a yeast two-hybrid assay, we identified Cullin3 (CUL3) as one of multiple KLHL10-interacting partners. Yeast cotransformation assays revealed that CUL3 bound the BTB/POZ domain of KLHL10. Northern blot and quantitative RT-PCR analyses demonstrated that Cul3 mRNA was preferentially expressed in the testis. In situ hybridization analysis localized Cul3 mRNA to spermatids in the adult testis. CUL3 protein was detected in elongating and elongated spermatids (steps 10-16) by immunofluorescent microscopy. The expression pattern of CUL3 resembles KLHL10. CUL3 was coimmunoprecipated with KLHL10, and KLHL10 was also detected in the CUL3 immunoprecipitants using testis lysates. These findings suggest that KLHL10, like other BTB/kelch proteins, interacts with CUL3 to form a CUL3-based ubiquitin E3 ligase that functions specifically in the testis to mediate protein ubiquitination during spermiogenesis.     

MSJ-1, a mouse testis-specific DnaJ protein, is highly expressed in haploid male germ cells and interacts with the testis-specific heat shock protein Hsp70-2

The MSJ-1 gene encodes a murine DnaJ homologue that is expressed specifically in adult testis. DnaJ proteins act as cochaperones of Hsp70 proteins in promoting diverse cellular functions. In this study we used recombinant MSJ-1 proteins to produce MSJ-1 antiserum and to carry out in vitro binding assays. In a wide immunoscreening of mouse tissues, affinity-purified MSJ-1 antibodies recognize a unique protein of 30 kDa in male germ cells only. MSJ-1 is able to interact with the testis-specific Hsp70-2 protein and can be coimmunoprecipitated with Hsp70-2 from spermatogenic cells; binding of these two chaperones is consistent with the presence of a third component, which is so far unknown. MSJ-1 is weakly detected in early round spermatids, and its protein content increases in cytodifferentiating spermatids where it colocalizes with the developing acrosome and their postnuclear region. Hsp70-2, which is known to be highly expressed in meiotic cells, shows a subcellular localization in late differentiating spermatids that overlaps that of MSJ-1. MSJ-1 is also maintained in testicular and epididymal spermatozoa, where it sharply demarcates into two distinct cell areas; the outer surface of the acrosomal vesicle, and the centrosomal area. On the whole, our findings are consistent with a role for MSJ-1 in acrosome formation and centrosome adjustment during spermatid development, whereas its presence in mature spermatozoa suggests a special function during fertilization, shortly afterward, or both.     

Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Spatio-developmental distribution of the prion-like protein doppel in Mammalian testis: a comparative analysis focusing on its presence in the acrosome of spermatids

The first prion-like protein doppel, officially designed as prion protein dublet, does not seem to be needed for prion disease progression, whereas its physiological function seems to be related to male fertility. Its expression is primarily detected in the male genital tract, and Prnd-inactivated male mice are sterile. We investigated the location of Doppel in the testis of various species of mammal to determine its physiological function. Doppel is expressed early during ontogenesis, and is found in both germ cells and Sertoli cells in mice, rats, boars, and humans. Doppel is permanently expressed in the Sertoli cells but at different levels according to species. Its expression in testicular germ cells was primarily detected in spermatids, with a transient presence in the acrosome. These data suggest that Doppel may play a physiological role in acrosome biogenesis and may be of use in studies of patients suffering from idiopathic infertility.     

Molecular Cloning and Putative Functions of KIFC1 for Acrosome Formation and Nuclear Reshaping during Spermiogenesis in Schlegel's Japanese Gecko Gekko japonicus

Spermiogenesis, occurring in the male testis, is a complicated and highly-ordered developmental process resulting in the production of fertile mature sperm. In Gekko japonicus, this process occurs in 7 steps during which the spermatids undergo dramatic changes in the cytoskeleton and nucleus. Here, we cloned and sequenced the cDNA of the mammalian KIFC1 homologue in the testis of G. japonicus. The 2 344 bp full-length cDNA sequence contained a 191 bp 5'-untranslated region, a 134 bp 3'-untranslated region and a 2 019 bp open reading frame encoding a protein of 672 amino acids. Tissue expression analysis revealed the highest expression of kifc1 mRNA was in the testis. Fluorescence in situ hybridization revealed that the kifc1 mRNA signal was hardly detected in step 1 spermatids but became concentrated at the acrosome of step 2 spermatids and abundant in the nucleus of step 5 spermatids where the nucleus then undergoes dramatic elongation and compression. The kifc1 mRNA signal then gradually disappears in mature sperm. This expression of KIFC1 at specific stages of spermiogenesis in G. japonicus implies its important role in the major cytological transformations such as acrosome biogenesis and nucleus morphogenesis.     

精索静脉曲张对青春期大鼠睾丸中VEGF和Flt-1蛋白表达的影响

目的研究血管内皮生长因子(VEGF)及其受体Flt-1蛋白在实验性左侧精索静脉曲张(ELV)大鼠睾丸中的表达和定位,探讨它们在精索静脉曲张(VC)致男性不育中的作用。方法建立青春期大鼠ELV模型,采用免疫组化法检测VEGF及Flt-1在ELV4周、8周组及相应对照组大鼠睾丸中的表达变化。结果 VEGF和Flt-1蛋白在大鼠睾丸中定位具有细胞特异性。VEGF蛋白表达于生精细胞、精子细胞发育中的顶体、Sertoli和Leydig细胞胞质内;Flt-1表达于精子细胞发育中的顶体及Leydig细胞胞质中。ELV4周组睾丸中VEGF蛋白的表达显著增加(P<0.01),8周时其表达量下降(P<0.01);ELV4周组与8周组睾丸中Flt-1蛋白的表达均比相应对照组下降(P<0.01),ELV8周组比4周组显著减少(P<0.01)。结论 ELV可影响青春期大鼠睾丸中VEGF和Flt-1蛋白的表达量,可能会影响精子的发生、发育,因而该变化可能是VC引起男性不育的原因之一。     

Identification of SAMT family proteins as substrates of MARCH11 in mouse spermatids

MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN–MVB pathway. However, the physiological substrate of MARCH11 has not been identified. In this study, we have identified and characterized SAMT1, a member of a novel 4-transmembrane protein family, which consists of four members. Samt1 mRNA and its expression product were found to be specific to the testis and were first detected in germ cells 25 days after birth in mice. Immunohistochemical analysis further revealed that SAMT1 was specifically expressed in haploid spermatids during the cap and acrosome phases. Confocal microscopic analysis showed that SAMT1 co-localized with MARCH11 as well as with fucose-containing glycoproteins, another TGN/MVB marker, and LAPM2, a late endosome/lysosome marker. Furthermore, we found that MARCH11 could increase the ubiquitination of SAMT1 and enhance its lysosomal delivery and degradation in an E3 ligase activity-dependent manner. In addition, the C-terminal region of SAMT1 was indispensable for its ubiquitination and proper localization. The other member proteins of the SAMT family also showed similar expression profile, intracellular localization, and biochemical properties, including ubiquitination by MARCH11. These results suggest that SAMT family proteins are physiological substrates of MARCH11 and are delivered to lysosomes through the TGN–MVB pathway by a ubiquitin-dependent sorting system in mouse spermatids.     

Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.