RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

Expression and localization of the deubiquitinating enzyme mUBPy in wobbler mouse testis during spermiogenesis.

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired.

Structure of msj-1 gene in mice and humans: a possible role in the regulation of male reproduction.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj-1 gene and searched for putative msj-1-like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions (Ha and Hb) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster (ugt1a@). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha. Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

MUBPy is a novel centrosome-associated protein and interacts with gamma-tubulin.

mUBPy is a deubiquitinating enzyme expressed preferentially in male germ cells and neurons. Recently, mUBPy has been shown to be involved in the down-regulation of growth factor receptors. In mouse spermatozoa mUBPy interacts with the sperm-specific molecular chaperone MSJ-1 and associates with the proteasome. The ubiquitin/proteasome system plays a key role during spermatogenesis to yield functional spermatozoa. Immunoelectron microscopy has been here used to localize both mUBPy and MSJ-1 in mouse spermatozoa. mUBPy and MSJ-1 label the cytoplasmic side of the acrosomal membrane and the centrosome, two sperm structures fundamental for a successful fertilization. In vitro protein interaction assay reveals that mUBPy is able to bind gamma-tubulin, a centrosomal protein marker. This protein interaction has been confirmed in vivo by double protein immunolabelling in spermatogenic cells. Upon the grounds of these findings and in the light of recent acquisition on the centrosome biology, we suggest that mUBPy could have a key role during mouse fertilization and propose mUBPy as a novel centrosomal component.

CAMP activates Rap1 in differentiating mouse male germ cells: a new signaling pathway mediated by the cAMP-activated exchange factor Epac?

Rap1, a Ras-like G-protein, is implicated in the signaling of various cellular processes as morphogenesis, differentiation, cell adhesion and spreading, and maintenance of T cell anergy and B cell activation. The effectors that mediate Rap1 signaling have not yet been definitely identified, with the exception of B-Raf which, however, is restricted to neuronal tissues and a small subset of other cell types, including in particular male germ cells. We previously showed that in mouse spermatids Rap1 could interact with B-Raf giving rise to a signaling complex. Here we investigated about the possible molecules which "switch on" Rap1 finding that cAMP could in vivo activate endogenous Rap1. Spermatid-enriched cell cultures stimulated with 8-(4-chlorophenylthio)-cyclic AMP yielded higher levels of GTP-bound Rap1 than unstimulated cells. Since cAMP-induced Rap1 activation is actually retained to occur through Epac, we checked whether this recently discovered Rap1 exchange factor is expressed in male germ cells. Our findings indicate that Epac is present in spermatogenic cells and exhibits a preferential subplasmalemmal localization, although it shows also an intracellular location, more or less pronounced depending on the type of spermatogenic cell examined. Taken together, our data show that cAMP activates Rap1 in differentiating male germ cells which express the cAMP sensor Epac, thus suggesting that this activation might occur directly through Epac.

Early defect in the expression of mouse sperm DNAJ 1, a member of the DNAJ/heat shock protein 40 chaperone protein family, in the spinal cord of the wobbler mouse, a murine model of motoneuronal degeneration.

Prevention of protein misfolding is ensured by chaperone proteins, including the heat shock proteins (HSP) of the DNAJ/HSP40 family. Detection of abnormal protein aggregates in various neurodegenerative diseases has led to the proposal that altered chaperone activity contributes to neurodegeneration. Msj-1, a DNAJ/HSP40 protein located around the spermatozoa acrosome, was recently found to be down-regulated in the testis of wobbler mutant mice. Wobbler is an unidentified recessive mutation which triggers progressive motoneuron degeneration with abnormal intracellular protein accumulations, and defective spermatozoa maturation. Here, we examined Msj-1 expression in the spinal cord of the mutants and their controls. Msj-1 transcripts were amplified by reverse transcription-polymerase chain reaction from mutant and wild-type spinal cord RNA. Sequencing of Msj-1 coding region revealed no change in the mutant. In contrast, decreased Msj-1 mRNA levels were observed in five to six-week-old wobbler mice spinal cord, when motoneuron degeneration is at its apex, as compared to controls. A similar decrease was observed in two-week-old wobbler spinal cord, when the number of motoneurons is still unaltered, indicating that the decreased mRNA content is intrinsic to the mutant and not simply related to the loss of cells expressing Msj-1. Assays of Msj-1 protein levels yielded similar results. Immunofluorescent labeling revealed numerous Msj-1-ir motoneurons in five-week-old control spinal cord while no signal was observed in age-matched wobbler. Our results show, therefore, that Msj-1 expression is down-regulated in both organs affected by the wobbler mutation, the CNS and the testis, and that this defect precedes the first histological signs of motoneuron degeneration. These results provide the first example of an association between transcriptional repression of a chaperone protein and a neurodegenerative process.

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25(Mm), expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor.

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

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

A novel rap1/B-Raf/14-3-3 theta protein complex is formed in vivo during the morphogenetic differentiation of postmeiotic male germ cells.

The 14-3-3 family of proteins is expressed in a broad range of organisms and tissues. Based on data essentially obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways, in particular those involving the activity of the Raf family protein kinases. The 14-3-3 theta mouse isoform is expressed almost exclusively in testis and brain. In an effort to understand the function of 14-3-3 theta in testis, we sought to identify endogenous proteins that interact with 14-3-3 theta in spermatogenic cells. A recombinant 14-3-3 theta fusion protein was used in Far Western and GST pulldown assays. Here we report that 14-3-3 theta interacts in vivo and in vitro with 93- to 95-kDa B-Raf, originally described as specific of neural tissues and never reported in male germ cells. Moreover, in mouse spermatids, i.e., the haploid cytodifferentiating cells, a so far unidentified protein complex formed by endogenous Rap1/B-Raf/14-3-3 theta can be coimmunoprecipitated. The intracellular localization of endogenous 14-3-3 theta, B-Raf, and Rap1 was analyzed in distinct spermatogenic cell types and a peculiar codistribution of the three proteins was immunorevealed in differentiating spermatids. Together, these data demonstrate that a protein complex formed by endogenous Rap1, 93- to 95-kDa B-Raf, and 14-3-3 theta exists in vivo and the finding that this has been detected in cytodifferentiating, not dividing cells, strengthens the hypothesis for a role of Rap1/B-Raf-mediated signaling in cell morphogenesis and differentiation.

Two-dimensional distortion of space representation in unilateral neglect: perceptual and response-related factors.

The paper reports the results of an experiment in which left-neglect patients were required to point at the location they judged vertically to correspond (within the frame of the visual stimulus display they were given) with a cue that was variably located along a left-right axis lying proximally or distally with respect to the left-right axis over which they had to give their response. Patients were found to make rightward errors as in a similar, single-case study. The significant positive correlation between those errors and the degree of response bias on a manual-response version of the Milner Landmark Task suggests that rightward pointing errors made by left-neglect patients in conditions such as those set in the present experiment are due to a dysfunction selectively affecting an output-related component of space representation.

Signaling events during male germ cell differentiation: bases and perspectives.

In all species, reproductive function depends on the ability of the individual to produce functional differentiated gametes. Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into mature haploid spermatozoa. Thus from a genetic point of view, spermatogenesis can be divided into two phases, namely the diploid and haploid phase. Indeed, this complex differentiation process is still more intriguing since primary spermatocytes, if genetically diploid, are functionally tetraploid, while elongating spermatids, the germ cells undergoing the most dramatic morphological changes, if genetically haploid, become functionally anucleate due to ongoing condensation of chromatin resulting in an inactive nuclear DNA. This multi-step differentiative pathway is dependent on a specific environment provided by the anatomical and cellular relationships that take place in the testis and more specifically within the seminiferous tubules. Already, early anatomists (mind comes to Enrico Sertoli and Gustaf Retzius) were fascinated by the mixed cellular composition of the testis correctly deciphered as a whole of interacting and interdependent cell types despite the fact these belong to two well-established and different cell lineages, i.e, the somatic and germinal line. Since their time (the XIX century) up to-day a conspicuous bulk of experimental work and a relative massive bibliographic documentation have been provided. From this it stands out : a) a sophisticated role played by the cyclic hormonal control elicited by the hypothalamic-pituitary axis; b) the structural membrane specializations of Sertoli-germ cell communications; c) the existence and action of a paracrine and autocrine testicular regulative secretion; d) a regulation of germ cell gene expression, highly specialized both at transcriptional, posttranscriptional, and translational level; e) an active participation of the haploid genome in the final steps of cell differentiation. Each of these points has been the matter of several more and less recent reviews to which the present author hands back in the course of this note. However all these points, although topics of separate and extensive treatises, are conceptually jointed by a 'leit-motiv', that is, the intracellular transduction of an exogenous signal evoking a specific stimulatory/inhibitory, proliferative/differentiative event. The spirit with which the present author interpreted this minireview was to recall some points to which to draw attention having as a scenario the complex process of male germ cell differentiation in mammals.

MSJ-1, a new member of the DNAJ family of proteins, is a male germ cell-specific gene product.

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein-protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages and in situ hybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.

Molecular cloning and developmental pattern of expression of MSJ-1, a new male germ cell-specific DNAJ homologue.

A cDNA encoding a new member of the DnaJ protein family has been isolated by screening a mouse testicular expression library. The predicted protein, named MSJ-1, is 242 amino acid residues-long, containing the fingerprinting J domain in the NH2 terminus. A wide tissutal Northern blot analysis reveals that MSJ-1 is expressed only in the testis, while in situ hybridization analyses demonstrate that the mRNA is first transcribed in spermatids. The antiserum developed against a MSJ-1/GST fusion protein recognizes a protein of 30 kDa in germ cell protein extracts.

Molecular cloning and tissue-specific expression of the mouse homologue of the rat brain 14-3-3 theta protein: characterization of its cellular and developmental pattern of expression in the male germ line.

The highly conserved 14-3-3 family of proteins, originally reported as brain-specific and then found in various somatic cells and oocytes, interacts with several important signal transduction kinases so that actually the 14-3-3 protein are considered as modulators of multiple signal transduction pathways. Here we show that a 14-3-3 protein is also expressed in the male germ cells, thus extending the protein cellular distribution to a cell line never reported to express 14-3-3 proteins. Screening of a mouse spermatogenic cells lambda gt11 cDNA library with affinity-purified polyclonal antibodies to the tyrosine kinase SP42 allowed the isolation of several positive clones. Sequencing of a positive cDNA clone revealed a 735-nucleotide open reading frame encoding a protein of 245 amino acids (27,778 Da). The predicted protein was found to be identical to the most recently discovered 14-3-3 isoform, the theta subtype from a rat brain. Here we demonstrate that 14-3-3 theta mRNA is highly expressed in testis and brain only. Western immunoblot analyses confirm the Northern blot data. Developmental Northern and Western blot analyses are consistent with an expression and translation of the 14-3-3 theta gene throughout spermatogenesis. However, analysis of RNA from purified populations of spermatogenic cells at different developmental stages and immunohistochemistry on adult testis sections reveal that within the testis the 14-3-3 theta gene products are most abundant in meiotic prophase spermatocytes, and, above all, in differentiating spermatids. Both testicular and epididymal spermatozoa are negative. The present study is the first report on the presence and molecular characterization of the 14-3-3 theta gene product in the male germ line. Our observations suggest that this specific member of the 14-3-3 protein family could play distinct modulatory roles in the complex development of the mammalian male germ cell lineage.

sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species.

sp42, a tyrosine kinase of 42 kDa originally found in ejaculated boar spermatozoa, is so far the only tyrosine protein kinase to have been purified from mature male germ cells. We have developed and characterized rabbit polyclonal antibodies specifically directed against the boar sperm enzyme, which has been here purified to homogeneity. Anti-sp42 serum and sp42 affinity-purified antibodies work very well in western blot, immunoprecipitation and immunocytochemistry, and do not inhibit sp42 catalytic activity. Immunoblotting analyses reveal the presence of sp42 both in maturing boar epididymal (caput, corpus and cauda segment) spermatozoa and in testicular spermatogenic cells, thus establishing that the protein is effectively expressed in the germ cells and is not a sperm-associated protein secreted by the epididymal epithelium or male accessory glands. This finding is further strengthened by the fact that sp42 is not glycosylated, since different lectins fail to bind to sp42 and treatment of sp42 with different deglycosylation enzymes does not result in a reduction of the molecular mass of sp42. When different boar tissues are immunoscreened in western blot analysis, the results are all sp42-negative. The extension of the study to other mammalian species (human, mouse and rat) demonstrates that proteins immunologically related to boar sp42, which share the same molecular mass and tyrosine kinase activity, are both expressed in spermatogenic cells and maintained in mature sperm cells. Intriguingly, when a wide spectrum of somatic mouse and rat tissues is probed with sp42-antiserum, no tissue presents anti-sp42 immunoreactivity. Immunocytochemistry shows that in boar spermatozoa sp42 is confined to the tail mid-piece, while by immunohistochemistry carried out on sections of adult rat testis the appearance time of the kinase appears to be consistent with a post-meiotic synthesis in haploid spermatids. Altogether, these results demonstrate that boar sp42 is a new male germ cell-specific gene product, with highly conserved tissue expression extended to other mammalian species, and suggest a possible role played by the cytoplasmic tyrosine kinase in the cell signalling network specific to haploid male germ cells.

Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: its potential for tyrosine as well as serine phosphorylation towards microtubule-associated protein 2 and histone H 2B.

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 microM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149-154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M(r) 42,000, is stimulated by Mg2+ > Mn2+ but not by Ca2+, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa.

Tyrosine protein kinase in boar spermatozoa: identification and partial characterization.

A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase--the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases.

Evidence for Ca(2+)-mediated F-actin/phospholipid binding of human sperm calpactin II.

We have previously reported the presence in human spermatozoa of calpactin II, a calcium-binding component of the membrane skeleton (Berruti, Exper. Cell Res. 179, 374, 1988). Reported here are studies which show the ability of sperm calpactin II to interact with filamentous actin and acidic phospholipids in a Ca(2+)-dependent fashion. At high Ca2+ concentrations (greater than 1 mM) sperm calpactin II binds to actin filament and it is resolubilized by EGTA. Liposome binding experiments reveal that sperm calpactin II does associate with phospholipidic vesicles at micromolar free Ca2+ levels. These interaction properties together with the cellular distribution of the protein revealed by immunofluorescence analysis in Ca2+ and ionophore A23187-treated human spermatozoa allow to hypothesize a physiological role for calpactin II in sperm Ca(2+)-mediated events.

Evidence for a calsequestrin-like calcium-binding protein in human spermatozoa.

Human spermatozoa were investigated for the presence of protein(s) recognized by antibodies against calsequestrin, the high capacity, moderate affinity Ca2(+)-binding protein, originally described in striated muscle fibers. Western immunoblots of detergent-soluble sperm extracts probed with polyclonal antibodies raised against human skeletal muscle calsequestrin identified a strongly cross-reactive protein. This protein resembles muscle calsequestrin in many respects. In fact, its migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is pH dependent, its apparent molecular mass being 64 kDa in alkaline SDS-PAGE and 44 kDa in neutral SDS-PAGE; its isoelectric point is acidic (4.6); it is metachromatically stained blue by the carboxycyanine dye, Stains-All; it is a Ca2(+)-binding protein (45Ca blot overlay). Indirect immunofluorescence experiments showed that the immunoreactive protein has an intracellular localization confined to the tail mid-piece. From these findings we conclude that human sperm cells express a protein structurally and antigenically related to skeletal muscle calsequestrin; a basis for a novel interpretation of Ca2(+)-mediated events in spermatozoa is thus provided.