Epididymal protein ASF is a D-galactose-specific lectin with apoptotic effect on human breast cancer cell line MCF7.

Isolated caprine epididymal plasma glycoprotein "anti sticking factor" (ASF) interacts with caudal sperm surface in a D-galactose dependent manner. ASF acts as a Ca(2+) dependent soluble lectin principally activated in acidic pH. As a D-galactose specific lectin, it has a specific affinity for fibronectin as well as fibronectin receptor, i.e. integrins α5ß3 and α5ß1. By virtue of this particular property, it hampers the in vitro adhesion of the adherent breast cancer cell MCF7 with fibronectin. The effective anti-adhesive concentration of ASF promotes p53 dependent apoptosis in MCF7, which was established by Hoechst 33342 staining, DNA fragmentation assay, FITC tagged Annexin-V flowcytometry and western blot analysis. We suggest that ASF inhibits fibronectin-integrin interactions by binding with them and induces adhesion dependent apoptosis on adherent MCF7.

Role of forward-motility-stimulating factor as an extracellular activator of soluble adenylyl cyclase.

Forward-motility-stimulating factor (FMSF) is a protein, originally purified from bubaline serum, that promotes progressive motility of mature spermatozoa. FMSF binds to sperm surface receptors and activates transmembrane adenylyl cyclase (tmAC), causing a rise in intracellular cyclic AMP level ([cAMP]i) and subsequent activation of a protein kinase A/tyrosine kinase-mediated pathway that enhances forward motility. This article further evaluates how FMSF works in the caprine system, particularly identifying the stimulatory effect of this glycoprotein on soluble adenylyl cyclase (sAC). Elevated [cAMP]i, initially resulting from FMSF-dependent activation of tmAC, was associated with the release of Ca(2+) from an intracellular calcium store in the sperm head, likely via an inositol triphosphate-sensitive calcium ion channel. This peak Ca(2+) concentration of ∼125-175 nM was capable of stimulating sAC in vitro in a calmodulin-independent manner, thereby triggering more cAMP production. Our model proposes that a positive-feedback loop mediated by cAMP and Ca(2+) is established in FMSF-stimulated sperm, with cAMP playing the role of a chemical messenger at multiple steps, resulting in the observed progressive motility. Thus, FSMF stimulates a novel signaling cascade that synergistically activate both tmAC and sAC to achieve forward sperm motility.

Role of epididymal anti sticking factor in sperm capacitation.

Sperm capacitation depends on several features like hormones, ions, intracellular signaling, sperm associated molecules, etc. Anti sticking factor (ASF) is a novel sperm surface associated glycoprotein isolated from epididymal plasma. Function of ASF in vivo has not been revealed yet. The current study is an attempt to highlight the surface localization of ASF and corresponding biochemical changes that occurs in sperm cells during in vitro capacitation. In the presence of 1 nM ASF, percentage of bicarbonate and BSA induced capacitated cells in modified Tyrode medium (7.2) decreased from 72.45% to 16.25% as per Merocyanine 540 (M540)/DAPI stained flowcytometric analysis. Indirect immunocytostaining and western blot analysis shows that the amount of sperm surface bound residual ASF decline during in vitro capacitation. ASF at its effective concentrations notably reduced the bicarbonate and BSA induced cholesterol efflux. These data help in concluding ASF as a majorly responsible molecule that maintains caprine sperm membrane integrity by inhibiting cholesterol efflux. As the capacitation process, progress at in vitro condition, ASF is found to be released from the sperm surface and cell moved from non-capacitated to the capacitated state.

Extracellular regulation of sperm transmembrane adenylyl cyclase by a forward motility stimulating protein.

Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation of progressive motility by a physiologic extracellular factor in a mammalian species.

Occurrence of novel Cu(2+)-dependent sialic acid-specific lectin, on the outer surface of mature caprine spermatozoa.

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu(2+)) ion (300 µM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 µM concentration. However, asialofetuin is not capable of inhibiting this Cu(2+)-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu(2+) ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu(2+) dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu(2+)-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.

Receptor expression is essential for forward motility in the course of sperm cell maturation.

Forward motility stimulating factor (FMSF) is a glycoprotein previously purified from buffalo blood serum that promotes progressive motility of caprine caudal spermatozoa. We have prepared a functionally active covalent conjugate of this factor with horseradish-peroxidase (HRP) to obtain an idea of its binding efficacy on maturing spermatozoa. Receptor-assay was performed using FMSF-HRP conjugate in saturating conditions to bind with spermatozoa isolated from different epididymal segments. Activity and binding profile of the motility stimulating factor coincided, suggesting both these parameters come into play only partially when spermatozoa reach the maturation state in the distal-corpus region and largely in caudal part (around 24% and 80% binding and 10% and 79% forward motility, respectively). Spermatozoa from caput up to mid-corpus regions neither displayed any substantial binding with FMSF nor exhibited significant induction in forward motility. Study of cell surface-bound FMSF on maturing spermatozoa in physiological milieu demonstrated their presence on anterior spermhead and suggests a nearly similar pattern of occurrence. Flow-cytometric analysis also implies analogous presence of this receptor. The factor was also immunodetected in uterine fluids of cattle species. This study displays a maturation-dependent expression of FMSF-receptor and consequential stimulation of forward motility that may be crucial for its journey to meet the ovum.

Copper: a biphasic regulator of caprine sperm forward progression.

Copper is essential for spermatogenesis and its presence has been demonstrated in male and female reproductive fluids in several mammalian species. However, little is known about the physiological significance of this trace element in the regulation of forward progression of mammalian sperm cells which is essential for sperm fertility potential in vivo. The purpose of this investigation was to determine the physiological role of the bivalent copper ion (Cu(2+)) on mammalian sperm forward motility using a chemically-defined medium and caprine cauda epididymal sperm model. Sperm forward motility was significantly enhanced by Cu(2+) in a dose-dependent manner; maximal activation (approx 20%) was noted at the 5 µM level of the metal. Above 10 µM Cu(2+) sperm motility decreased, showing that Cu(2+) exerts a biphasic regulation on sperm motility. These findings have been confirmed using a spectrophotometric motility assay, an objective method of motility analysis. At lower concentrations (up to 5 µM), copper enhanced sperm membrane lipid peroxidation as well as the level of intra-sperm cyclic adenosine mono phosphate (c-AMP), but at a higher level it caused marked inhibition of both of the biochemical parameters. The observed correlation of Cu(2+)-dependent biphasic modulation of sperm membrane lipid peroxidation and intrasperm c-AMP with sperm forward motility is consistent with the view that Cu(2+) regulation of sperm motility is mediated by membrane lipid peroxidation, which in turn modulates the level of intra-sperm c-AMP, a well-known activator of sperm motility.

A novel membrane protein-specific serine/threonine kinase: tissue distribution and role in sperm maturation.

Our recent studies have described for the first time the purification of an ectoprotein kinase to apparent homogeneity using caprine sperm as the model. Purified ectokinase (CIK) is a novel membrane protein-specific kinase that phosphorylates serine and threonine residues of ectophosphoproteins. This study, using ELISA based on ecto-CIK antibody demonstrates that ecto-CIK level is remarkably higher in the sperm membrane than in the cytosol. The epididymal sperm maturational event as well as sperm vertical velocity is associated with a significant increase in the ecto-CIK level. Ecto-CIK, the membrane protein-specific kinase, is also present in all the tissues tested and is predominantly localized in the cell membrane. Ubiquitous localization of the novel kinase on the mammalian cell membrane suggests that the kinase may play pivotal role in gamete as well as somatic cell regulation by modulating membrane biology through serine/threonine phosphorylation of specific membrane proteins located in the ectodomains.