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1.
Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria.  相似文献   

2.
Outer dense fibers (ODFs) are located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail. We have identified and describe here a human gene that is homologous to the Mst(3)CGP gene family of Drosophila melanogaster and encodes an ODF protein of 241 amino acids. The transcribed region has a size of ?lkb and contains two exons of 416 bp and 406 bp, respectively, not including the 3′ untranslated region. The gene is expressed in testis but not in human spleen, kidney, or brain and resembles in this respect the expression of the Drosophila Mst(3)CGP gene family in the male germline. An antiserum raised against a synthetic peptide derived from the N-terminus of the encoded sequence identified a protein of ? 32 kDa in an extract of human sperm flagella. By Southern-blot analyses and in situ hybridization, the ODF gene was localized to band q22 of chromosome 8. The isolation of a human gene encoding a sperm tail protein may provide the ability to identify and investigate, on the molecular level, possible reasons for human male infertility that are dependent on flagellar disturbances. © 1993 Wiley-Liss, Inc.  相似文献   

3.
Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non‐motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down‐regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α‐tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α‐tubulin and protect the acetylation level of α‐tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α‐tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo‐expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.  相似文献   

4.
5.
Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.  相似文献   

6.
The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.  相似文献   

7.
Infertility among couples of reproductive age is a perplexing condition when the cause is indeterminate. These cases are classified as unexplained infertility. In a subset of subjects, antisperm antibodies with sperm agglutinating and/or immobilizing activities have been detected in the blood or fluids of the reproductive tract. These cases are designated as immunologic infertility although a cause and effect relationship of the antibodies to infertility has not been established. In this review, seven target sperm antigens to antibodies associated with infertility and their encoding genes are described. The antisperm antibodies (ASAs) examined were obtained from infertile women or were monoclonal antibodies (mAb) raised against human sperm proteins. All the ASAs studied possessed potent sperm agglutinating and/or immobilizing activities. The target antigens were isolated from human and other mammalian sperm, and the encoding genes identified. The seven antigens are YWK-II, BE-20, rSMP-B, BS-63 (nucleoporin-related), BS-17 (calpastatin), HED-2 (zyxin), and 75- kDa. Each antigen is a distinct and separate entity and is produced by different cells of the reproductive tract, (e.g., germ cells, epididymal epithelial cells, and Sertoli cells). No single predominant target component has been found to interact with the ASAs. It is proposed that immunologic infertility is the consequence of the combined actions of multiple ASAs in immobilizing and/or agglutinating spermatozoa, blocking spermegg interaction, preventing implantation, and/or arresting embryo development.  相似文献   

8.
In addition to their role in sperm maturation, recent evidence has indicated that epididymal proteins have a role in male reproductive tract innate immunity. Herein we demonstrate that human and macaque epididymal protein isoforms in the SPAG (sperm associated antigen) 11 family, full length SPAG11C, K and L exhibit potent antibacterial activity against E. coli. Analysis of activities of the N- and C-terminal domains revealed that the human N-terminal peptide is bactericidal, while the C-terminal domains that contain the defensin-like 6 cysteine array in SPAG11C and partial arrays in SPAG11K and SPAG11L, lack antibacterial activity. The N-terminal peptide does not appear to contain all the determinants of activity since full-length human SPAG11C is more active than the isolated N-terminal peptide and since sulfhydryl reduction and alkylation, which would affect primarily the C-terminal peptides, completely abolished activities of the whole proteins. These results suggest that the structure conferred by the disulfide bonds in human SPAG11C contributes to the antibacterial activity of the whole molecule. The activities of the N-terminal peptide and of full length human SPAG11C were somewhat reduced in increasing NaCl concentrations. In contrast, the antibacterial activities of full length macaque SPAG11C, K and L were unaffected by the presence of NaCl suggesting a mechanism in the macaque that is less dependent upon electrostatic interactions. SPAG11C, K and L disrupted E. coli membranes but had no effect on erythrocyte membranes. Inhibition of E. coli RNA, DNA and protein synthesis by nonlethal concentrations of SPAG11 isoforms indicated an additional mechanism of bacterial killing. Abbreviation: SPAG11, sperm associated antigen 11; CFUs, colony forming units; NPN, N-phenyl-1-napthylamine; diSC3-5, 3,5-dipropylthiadicarbocyanine iodide; IAA, iodoacetamide; BME, β-mercaptoethanol  相似文献   

9.
Cdk5 and p35 are integral components of the sperm tail outer dense fibers (ODFs), which contribute to the distinct morphology and function of the sperm tail. In this study, we sought to characterize and investigate the significance of Cdk5/p35 association with ODFs. We show that ODF2 interacts with Cdk5 and p35 but not with the Cdk5/p35 heterodimer. By using deletion mutants, the ODF2 binding region in p35 was mapped to residues 122 to 198. This overlaps the Cdk5 binding region in p35, explaining the inability of ODF2 to bind to the Cdk5/p35 complex. In vitro phosphorylation assay showed that although Cdk5/p35 does not phosphorylate ODF2, it phosphorylates ODF1. Mass spectrometry revealed that Cdk5/p35 specifically phosphorylates Ser193 in the ODF1 C-terminal region containing the Cys-X-Pro motif, the interaction site for the novel RING finger protein, ODF1 interacting protein (OIP1), a candidate E3 ubiquitin ligase, that also localizes in the sperm tail. Cdk5 phosphorylation of ODF1 Ser193 results in enhanced ODF1-OIP1 interaction. These findings suggest that Cdk5 may be important in promoting ODF1 degradation, and potentially, the detachment and fragmentation of the sperm tail following fertilization.  相似文献   

10.
Serum from an infertile male with high-titer anti-sperm antibodies was used to identify a novel human sperm antigen by screening of a testis expression library. The clone, initially designated Repro-SA-1 (HUGO-approved symbol SPAG6), was found to encode a sequence highly enriched in testis. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in prostate, ovary, spleen, thymus, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, liver, muscle, kidney, and pancreas. The gene was mapped to chromosome 10p11.2-p12. Antibodies raised against SPAG6 sequences localized the protein to the tails of permeabilized human sperm. Both the Chlamydomonas protein and SPAG6 contain eight contiguous armadillo repeats, which place them in a family of proteins known to mediate protein-protein interactions. The cloning of the human homologue of the Chlamydomonas PF16 locus provides a new avenue to explore the role of the axoneme central pair in human sperm function.  相似文献   

11.
12.
13.
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. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.  相似文献   

14.
Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger.  相似文献   

15.
Declining fertility rates in both human and animals is a cause for concern. While many of the infertility cases are due to known causes, idiopathic infertility is reported in 30% of the infertile couples. In such cases, 18% of the infertile males carry antisperm antibodies (ASAs). Such data are lacking in livestock, wherein 20–30% of the animals are being culled due to low fertility. In males, the blood–testis barrier (BTB) and biomolecules in the semen provide an immuno‐tolerant microenvironment for spermatozoa as they traverse the immunologic milieu of both the male and female reproductive tracts. For example, insults from environmental contaminants, infections and inflammatory conditions are likely to impact the immune privilege state of the testis and fertility. The female mucosal immune system can recognize allogenic spermatozoa‐specific proteins affecting sperm kinematics and sperm‐zona binding leading to immune infertility. Elucidating the functions and pathways of the immune regulatory molecules associated with fertilization are prerequisites for understanding their impact on fertility. An insight into biomolecules associated with spermatozoal immune tolerance may generate inputs to develop diagnostic tools and modulate fertility. High‐throughput sequencing technologies coupled with bioinformatics analyses provides a path forward to define the array of molecules influencing pregnancy outcome. This review discusses the seminal immune regulatory molecules from their origin in the testis until they traverse the uterine environment enabling fertilization and embryonic development. Well‐designed experiments and the identification of biomarkers may provide a pathway to understand the finer details of reproductive immunology that will afford personalized therapies.  相似文献   

16.
17.
The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.  相似文献   

18.
Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.  相似文献   

19.
20.
A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.  相似文献   

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