RESUMO
BACKGROUND: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. RESULTS: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. CONCLUSIONS: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mamíferos/metabolismo , Metaloproteases/metabolismo , Oócitos/metabolismo , Folículo Ovariano/fisiologia , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Cricetinae , Primers do DNA/genética , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Mamíferos/crescimento & desenvolvimento , Camundongos , Dados de Sequência Molecular , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Coelhos , Ratos , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
Assuntos
Fertilização , Isoantígenos/metabolismo , Metaloproteases/metabolismo , Oócitos/metabolismo , Proteínas de Plasma Seminal/metabolismo , Acrossomo/metabolismo , Processamento Alternativo , Animais , Ligação Competitiva , Far-Western Blotting , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Feminino , Imunoprecipitação , Isoantígenos/genética , Masculino , Metaloproteases/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Gravidez , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Plasma Seminal/genética , Interações Espermatozoide-Óvulo , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-HíbridoRESUMO
PURPOSE: Compliance with post-vasectomy semen analysis could be improved with the availability of a simple, rapid and accurate home test. SpermCheck Vasectomy, a highly sensitive lateral flow immunochromatographic diagnostic device, was designed to detect extreme oligospermia or azoospermia in men after vasectomy. We report the results of clinical and consumer testing of SpermCheck. MATERIALS AND METHODS: A prospective, noncomparative observational study assessed the ability of SpermCheck Vasectomy to predict post-vasectomy sperm counts obtained using a hemacytometer procedure based on standard World Health Organization methodology. Consumer studies evaluated ease of use. RESULTS: A cohort of 144 post-vasectomy semen samples was tested in the clinical trial. SpermCheck was 96% accurate in predicting whether sperm counts were greater or less than a threshold of 250,000 sperm per ml, a level associated with little or no risk of pregnancy. Sensitivity was 93% (95% CI 79% to 98%) and specificity was 97% (91% to 99%). The positive predictive value of the test was 93% (79% to 98%), and most importantly the negative predictive value was 97% (91% to 99%). The test gave a positive result 100% of the time at sperm concentrations of 385,000/ml or greater. Consumer studies with 109 lay volunteers showed that SpermCheck was easy to use. Volunteers obtained the correct or expected test result in every case and the correct response rate on a 20 question survey about the test was 97%. CONCLUSIONS: SpermCheck Vasectomy, a simple and reliable immunodiagnostic test that can provide evidence of vasectomy success or failure, offers a useful alternative to improve compliance with post-vasectomy sperm monitoring. It is currently the only Food and Drug Administration approved test for this purpose.
Assuntos
Comportamento do Consumidor , Testes Imunológicos/instrumentação , Kit de Reagentes para Diagnóstico , Contagem de Espermatozoides/instrumentação , Vasectomia , Adolescente , Adulto , Idoso , Desenho de Equipamento , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.
Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Glicoproteínas/imunologia , Infertilidade Masculina/imunologia , Polissacarídeos/metabolismo , Espermatozoides/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Antígeno CD52 , Sequência de Carboidratos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Masculino , Dados de Sequência Molecular , Mucinas/química , Mucinas/metabolismo , Polissacarídeos/imunologia , Sêmen/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
To identify novel sperm alloantigens relevant to immune infertility, sera from infertile men containing antisperm antibodies (ASA) were employed on 2-D immunoblots of human sperm proteins. An immunoreactive protein spot (MW: 44 kDa, pI: 4.5) was microsequenced and the related cDNA was cloned yielding a 309 amino acid sequence corresponding to a gene currently annotated in Genbank as TSGA2 homolog (mouse) to signify 'testis specific gene A2'. In Genbank the protein deduced from this gene is currently named human meichroacidin, an orthologue of meichroacidin previously identified in mouse spermatocytes. Human TSGA2 mapped to chromosome 21q22.3. Human meichroacidin (hMCA) contained a single potential tyrosine phosphorylation site and five casein kinase phosporylation motifs. The N-terminus contained a Membrane Occupation Recognition Nexus (MORN) motif found in the lipid kinase-phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family and junctophilins. However hMCA lacked the characteristic kinase homology domain of PIP5K. Northern blot analysis revealed 1.5 kb hMCA transcripts in testis and trachea with lower levels in thyroid and spinal cord. A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated occurrence of the mRNA messages in all the ciliated tissues tested with highest levels of messages in testis and trachea. Western blot analysis showed the presence of hMCA protein in brain, thyroid and trachea at the identical mass, 44 kDa, as in human testis. However, this immunoreactive pattern differed from that of sperm in which a 38 kDa form was also evident suggesting that hMCA undergoes proteolytic processing. In human testis, hMCA localized to the tails of developing spermatids and did not localize to the nucleus of either spermatocytes or spermatids. EM immunocytochemistry localized hMCA within the radial spokes of the axonemal complex of the sperm flagellum, and immunofluorescence studies revealed h-meichroacidin in the cilia of epithelial cells in the trachea and ependyma. Bioinformatic identification of orthologues of meichroacidin in several lower organisms including ciliates and flagellates suggest the protein plays a role in flagellar motility across phyla. We propose the term radial spoke protein 44 as an accurate designation, preferable to human meichroacidin because it denotes the restricted localization of the protein to the radial spokes of the axonemes of both sperm and cilia. Further, since the human gene is expressed in brain, thyroid, trachea and lung in addition to testis, we suggest that the gene name be changed from TSGA2 [testis specific gene A2] to radial spoke protein 44 [RSP44].
Assuntos
Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Isoantígenos/metabolismo , Cauda do Espermatozoide/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Western Blotting , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Epitélio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Infertilidade/sangue , Isoantígenos/imunologia , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Cauda do Espermatozoide/ultraestrutura , Medula Espinal/citologiaRESUMO
PURPOSE: Members of the SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome) family of cancer-testis antigens are promising targets for tumor immunotherapy because they are normally expressed exclusively during spermiogenesis on the adluminal side of the blood-testis barrier, an immune privileged compartment. EXPERIMENTAL DESIGN AND RESULTS: This study analyzed the human SPANX genomic organization, as well as SPAN-X mRNA and protein expression in somatic and cancer cells. The SPANX family consists of five genes, one of which is duplicated, all located in a gene cluster at Xq27.1. From the centromere, the arrangement of the five SPANX genes mapped on one contiguous sequence is SPANXB, -C, -A1, -A2, and -D. Reverse transcription-PCR analyses demonstrated expression of SPAN-X mRNA in melanoma and ovarian cell lines, and virtual Northern analysis established SPANX gene expression in numerous cancer cell lines. Immunoblot analysis using polyclonal antisera raised against recombinant SPAN-X confirmed the translation of SPAN-X proteins in melanoma and ovarian tumor cell lines. The immunoreactive proteins migrated between M(r) 15,000 and M(r) 20,000 similar to those observed in spermatozoa. Immunoperoxidase labeling of melanoma cells and tissue sections demonstrated SPAN-X protein localization in the nucleus, cytoplasm, or both. Ultrastructurally, in melanoma cells with nuclear SPAN-X, the protein was associated with the nuclear envelope, a localization similar to that observed in human spermatids and spermatozoa. Significantly, the incidence of SPAN-X-positive immunostaining was greatest in the more aggressive skin tumors, particularly in distant, nonlymphatic metastatic melanomas. CONCLUSIONS: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy.
Assuntos
Antígenos de Neoplasias/genética , Melanoma/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Humanos , Incidência , Masculino , Melanoma/patologia , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espermátides/metabolismo , Espermátides/patologia , Células Tumorais CultivadasRESUMO
We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.
Assuntos
Acrossomo/metabolismo , Glicosilfosfatidilinositóis/genética , Receptores de Superfície Celular/genética , Interações Espermatozoide-Óvulo/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Acrossomo/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos , Antígenos Ly/genética , Sequência de Bases , Western Blotting , Cricetinae , Feminino , Fertilização/fisiologia , Glicosilfosfatidilinositóis/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Família Multigênica , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.
Assuntos
Acrossomo/química , Isoantígenos/genética , Muramidase/química , Proteínas de Plasma Seminal/genética , Espermatozoides/enzimologia , Acetilglucosamina/metabolismo , Adulto , Anticorpos/química , Anticorpos/farmacologia , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Feminino , Humanos , Técnicas In Vitro , Isoantígenos/química , Isoantígenos/imunologia , Masculino , Microscopia Eletrônica , Peso Molecular , Oligossacarídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/imunologia , Capacitação Espermática , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/ultraestruturaRESUMO
Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.
Assuntos
Antígenos CD/análise , Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Glicoproteínas/análise , Glicoproteínas/imunologia , Modelos Animais , Espermatozoides/imunologia , Vacinas Anticoncepcionais , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Evolução Biológica , Western Blotting , Antígeno CD52 , Callithrix/imunologia , Anticoncepção Imunológica , Epididimo/imunologia , Epitopos/análise , Epitopos/química , Epitopos/imunologia , Imunofluorescência , Glicosilação , Humanos , Imuno-Histoquímica , Técnicas de Imunoadsorção , Macaca fascicularis/imunologia , Macaca nemestrina/imunologia , Macaca radiata/imunologia , Masculino , Pan troglodytes/imunologia , Papio/imunologia , Especificidade da Espécie , Espermatozoides/fisiologiaRESUMO
To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.
Assuntos
Acrossomo/imunologia , Isoantígenos/análise , Isoantígenos/imunologia , Proteínas de Plasma Seminal/imunologia , Testículo/imunologia , Acrossomo/fisiologia , Acrossomo/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Especificidade de Anticorpos , Antígenos , Sequência de Bases , Membrana Celular/química , Mapeamento Cromossômico , Cromossomos Humanos Par 6 , Clonagem Molecular , DNA Complementar/genética , Hormônios Esteroides Gonadais , Humanos , Soros Imunes/imunologia , Infertilidade Masculina/imunologia , Isoantígenos/química , Isoantígenos/genética , Ponto Isoelétrico , Masculino , Espectrometria de Massas , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Ratos , Proteínas Recombinantes/imunologia , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/ultraestruturaRESUMO
To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.