Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.     

Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.     

Genomic organisation and chromosomal assignment of ODF2 (outer dense fiber 2), encoding the main component of sperm tail outer dense fibers and a centrosomal scaffold protein

ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals.     

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa

Antisperm antibodies (ASAs) have been implicated in some instances of infertility. To characterize sperm antigens relevant to immunologic and immunocontraceptive development, SPAG2 (sperm-associated antigen 2) was identified by screening a human testis cDNA library with human sera positive for ASAs. Subsequently, two isoforms, SPAG2–1 and SPAG2–2, were identified in testis and placenta libraries, respectively. In the current study, Southern analysis of human genomic DNA with a probe common to the two SPAG2 isoforms indicated a single SPAG2 gene; therefore, alternative splicing is a likely mechanism for production of variant mRNAs. In situ hybridization of human testis sections demonstrated the expression of SPAG2 in primary spermatocytes, with decreased or arrested expression in postmeiotic cells. Immunofluorescence of Triton X-100–extracted spermatozoa with an anti-SPAG2 peptide antiserum indicate that SPAG2 is an intracellular component of the sperm flagellum. Electron microscopy refined this localization to the outer dense fibers (ODFs), structural filaments associated with the mammalian sperm axoneme. The ODFs have been reported to be composed of keratinlike intermediate filament proteins. However, SPAG2 does not exhibit the molecular characteristics of such proteins, nor does SPAG2 demonstrate sequence homology with previously characterized ODF proteins. Therefore, SPAG2 represents a novel protein of human sperm ODFs. Characterization of SPAG2 will further our understanding of ODF function in normal sperm motility and of flagellar abnormalities that lead to male infertility. Mol. Reprod. Dev. 50:284–293, 1998. © 1998 Wiley-Liss, Inc.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers

The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function.     

Isolation and characterization of rat sperm tail outer dense fibres and comparison with rabbit and human spermatozoa using a polyclonal antiserum

Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.     

Characterization of Spetex‐1, a new component of satellite fibrils associated with outer dense fibers in the middle piece of rodent sperm flagella

Spetex‐1, which has been isolated by differential display as a haploid spermatid‐specific gene, encodes a protein with two coiled‐coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri‐axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre‐embedding immunoelectron microscopy clearly demonstrated that Spetex‐1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex‐1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex‐1. We identified putative Spetex‐1 orthologs in many animal species, and both cysteine residues and coiled‐coil motifs were well conserved in mammalian orthologs of Spetex‐1. When Spetex‐1 was co‐transfected into COS‐7 cells with myc‐tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co‐localized in various sizes of aggregates in the cells. These data suggested that Spetex‐1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co‐operation with Tektin4. Mol. Reprod. Dev. 77: 363–372, 2010. © 2010 Wiley‐Liss, Inc.     

Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa.

We tested the effect of extracellular adenosine 5'-triphosphate (ATPo) on the activation of human spermatozoa. ATPo, in a concentration range from 50 microM to 5 mM, induced the acrosome reaction, which, at the optimal concentration of 2.5 mM, was maximal (30-35% of spermatozoa activated) within 60 min of the addition of the nucleotide. At the end of this incubation in the presence of ATPo no decrease in cell motility and viability was observed. Among other purine/pyrimidine nucleotides only the ATP analogue adeny-5'-lyl imidodiphosphate was effective (70% of ATP); a weak (10% of ATP) effect was also observed with CTP and the ATP analogues adenosine 5'-(beta gamma-methylene)triphosphate and adenosine 5'-O-(thiotriphosphate). ATPo did not cause Ca2+ release from intracellular stores, nor it caused Ca2+ influx from the extracellular milieu; on the contrary, it caused a clear, albeit slow, plasma membrane depolarization. ATPo-activated spermatozoa showed a nearly 100% success rate in the standard hamster egg fertilization test. Our results describe a new effect of ATPo in human spermatozoa with relevant potential applications in fertility studies.     

Polypeptide composition of rat sperm outer dense fibers. A simple procedure to isolate the fibrillar complex

Treatment of caput or cauda epididymal rat sperm with a low concentration (0.05%) of the cationic detergent cetyltrimethylammonium bromide and 30 mM 2-mercaptoethanol solubilized most of the sperm structures except for the sperm head and the outer dense fiber-connecting piece complex. The latter were purified, and about 10% of these complexes are formed by nine fibers attached to the connecting piece. Of these fibers, two are shorter than the other seven and presumably correspond to fibers 3 and 8 (Fawcett, D.W. (1975) Dev. Biol. 44, 394-436). Electron microscopy confirmed the purity of the isolated outer dense fibers and revealed their characteristic irregular cross-sectional shape. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed six major polypeptides (Mr = 87,000, 30,400, 26,000, 18,400, 13,000, and 11,500) with a high content of serine, aspartic and glutamic acids, proline, cysteine, leucine, and tyrosine. Furthermore, several lines of evidence indicate a close structural relationship between the components of 30,400 and 26,000 Da. The six major components of the fibers are phosphorylated at serine residues. These results indicate that the major components of rat sperm outer dense fibers are a unique family of phosphoproteins.     

Association of kinesin light chain with outer dense fibers in a microtubule-independent fashion

Conventional kinesin I motor molecules are heterotetramers consisting of two kinesin light chains (KLCs) and two kinesin heavy chains. The interaction between the heavy and light chains is mediated by the KLC heptad repeat (HR), a leucine zipper-like motif. Kinesins bind to microtubules and are involved in various cellular functions, including transport and cell division. We recently isolated a novel KLC gene, klc3. klc3 is the only known KLC expressed in post-meiotic male germ cells. A monoclonal anti-KLC3 antibody was developed that, in immunoelectron microscopy, detects KLC3 protein associated with outer dense fibers (ODFs), unique structural components of sperm tails. No significant binding of KLC3 with microtubules was observed with this monoclonal antibody. In vitro experiments showed that KLC3-ODF binding occurred in the absence of kinesin heavy chains or microtubules and required the KLC3 HR. ODF1, a major ODF protein, was identified as the KLC3 binding partner. The ODF1 leucine zipper and the KLC3 HR mediated the interaction. These results identify and characterize a novel interaction between a KLC and a non-microtubule macromolecular structure and suggest that KLC3 could play a microtubule-independent role during formation of sperm tails.     

Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum

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.     

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Increased calcium-ion influx is a component of capacitation of spermatozoa.

Capacitation (modifications required for gamete fusion) is produced by incubating guinea-pig spermatozoa in vitro in a chemically defined medium. It is shown that during such incubation a net uptake of Ca2+ by the sperm occurs in two distinguishable phases. An initial loose association of Ca2+, possibly to surface sites, is unaffected by agents (Mg2+, inhibitors of mitochondiral function) that prevent or delay the exocytotic spermatozoal acrosome reaction. The time course of a secondary Ca2+ uptake parallels or slightly precedes the time course of the acrosome reaction. This parallelism is maintained during a variety of treatments that either expedite (local anaesthetics, ionophore A23187, Triton X-100) or delay (Mg2+, low external Ca2+) the acrosome reaction. We conclude that the secondary Ca2+ influx described herein apparently serves to link alterations of the spermatozoal membrane to subsequent contractile and secretory components of the capacitation sequence.