Synthesis and application of peptide immunogens related to the sperm tail protein tpx-1, a member of the CRISP superfamily of proteins.

The synthesis of peptides containing 0, 1 and 2 cysteine residues related to the human sperm tail protein, tpx-1, is described. These synthetic peptides, following conjugation to keyhole limpet hemocyanin modified with maleimidobenzoic acid N-hydroxysuccinimide ester, were used as immunogens to generate polyclonal antibodies in female New Zealand white rabbits. The binding characteristics of the derived antipeptide sera were evaluated using indirect and competitive ELISA procedures. Western immunoblot experiments also confirmed that these synthetic peptide immunogens are able to generate high-titer polyclonal antibodies capable of cross-reacting with the mature tpx-1 protein present in crude rat sperm tail/testis preparations as well as in outer dense fiber preparations. Consequently, these synthetic peptides represent promising candidates for investigations into the role of tpx-1 in the immunoregulation of sperm function in the rat and other mammalian models, with the derived antisera also providing an avenue to explore possible sites of expression of tpx-1 proteins in other tissues.     

Antibodies to rat sperm tail polypeptides recognize Sertoli cell secretory proteins

We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail.     

Human outer dense fiber gene, ODF2, localizes to chromosome 9q34

We have isolated the human homolog of the rat Odf2 gene. In rat, Odf2, the 84-kDa major outer dense fiber protein, interacts strongly and specifically with Odf1, the 27-kDa major outer dense fiber protein. The interaction is mediated by leucine zippers during ODF assembly along the sperm axoneme. We compared homology and genomic structure to rat and mouse Odf2 genes. Using fluorescence in situ hybridization, we mapped the human Odf2 gene (ODF2) to chromosome 9q34.     

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.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

A sperm cytoskeletal protein TSA70 is a novel phosphorylated member of cenexin/odf2 family

Using post-vasectomy monoclonal antibody we recently identified a testis specific sperm auto-antigen called TSA70 which is post-meiotically expressed and plays a role in sperm motility and capacitation-acrosome reaction. In the present study, we report its cytoskeletal nature based on its resistance to various high ionic salt solutions. TSA70 is developmentally regulated and appears postpubertally. The two protein spots identified by 2D WB namely TSA1-pI=5.821, MW=77.050 and TSA3-pI=6.173, MW=75.519 showed sequence homology to Cenexin/odf2 indicating that two are isoforms of the same protein. The immunoreactivity of TSA70 with anti-Cenexin antibody substantiates its homology with Cenexin/odf2. In silico analysis revealed the presence of two leucine zippers in TSA70 and also predicted potential phosphorylation sites at serine, threonine, and tyrosine residues. The phosphorylated status of TSA70 was further confirmed by immunoblot analysis. The differential cellular expression suggests that TSA70 is a novel member of Cenexin/odf2 family that exhibits functional divergence.     

Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.     

Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing-thawing

A sublethal environmental stress, high-hydrostatic pressure (HHP) was reported to significantly improve the motility, viability and fertility parameters of frozen bull and boar semen. However, the mechanism of how HHP treatment improves survival rates at sperm cryopreservation remains unclear. The purpose of this study was to evaluate the effect of HHP treatment of fresh boar semen on the protein profile of boar sperm before and after freezing. Fresh, extended semen of eight boars was split, one part was treated with 200, 300 or 400bar for 90min using a custom made pressuring device before the start of the semen freezing procedure, and the other part was prepared without HHP treatment. After thawing, samples were checked for motility. The effect of HHP treatment on the post-thaw motility of frozen semen was significant (P=0.02). Post-thaw motility of each treatment groups increased compared to control (46% vs. 52%, 56% and 56%; control vs. 200bar, 300bar and 400bar treatments). Samples for protein analysis were collected from the 300bar treatment group before HHP treatment at room temperature (25+/-3 degrees C), at 5 degrees C of the cooling process and after thawing with or without HHP treatment. The sperm were lysed using a urea-pyranoside-dithiothreitol buffer to extract their proteins for protein analysis. Approximately 800microg total proteins were assayed by two-dimensional gel electrophoresis and stained with colloidal Coomassie blue. The levels of 125 protein spots were quantified. The results revealed that the levels of 7 protein spots differed significantly among treatments. The identities of various protein constituents were identified by mass spectrometry and database searching. Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor were identified as HHP response proteins being significantly higher in HHP-treated samples. Testis-specific glyceraldehyde 3-phosphate dehydrogenase, outer dense fiber of sperm tails 2 isoform 10, cytosolic 5'-nucleotidase 1B, and quinone oxidoreductase represented the cooling and freezing related proteins. The differing levels of these identified proteins could be valuable for further exploring the protective mechanism of the HHP treatment in frozen-thawed porcine sperm.     

Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa

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.     

Possible function of caudal nuclear pocket: degradation of nucleoproteins by ubiquitin-proteasome system in rat spermatids and human sperm.

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Antigenic homology between rat sperm tail polypeptides and Sertoli cell secretory proteins

A high performance liquid chromatographic procedure has been used for the purification of rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein to determine their role during spermatogenesis. These two proteins display binding affinity for each other and appear antigenically related. We have observed that: 1. S70 and S45-S35 heterodimeric protein coelute during purification, 2. polyclonal antiserum raised against protein S70 recognizes common antigenic determinants in polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein, and 3. a monoclonal antibody that recognizes polypeptide S35 but does not crossreact with either protein S70 or polypeptide S45, immunoprecipitates the S70/S45-S35 heterodimeric protein complex. In immunofluorescent experiments, antisera raised against protein S70 and polypeptide components of S45-S35 heterodimeric protein immunoreact with two major sperm intracellular structures: the acrosome and periaxonemal outer dense fibers of sperm tail. Immunoreactivity was not detected on the sperm plasma membrane surface of unfixed, living sperm. Outer dense fibers extracted from sperm tails by a combined treatment with cetylthrimethylammonium bromide and 2-mercaptoethanol, yielded a characteristic polypeptide pattern. In immunoblotting experiments sperm tail polypeptides were recognized by polyclonal antisera raised against Sertoli cell secretory proteins. We conclude that Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein are antigenically related to each other and to keratin-like polypeptides from sperm tail.     

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1,000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation. © 1996 Wiley-Liss, Inc.     

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.     

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.     

Proteomic profiling of accessory structures from the mouse sperm flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.