Lateral diffusion of a human sperm-head antigen during incubation in a capacitation medium and induction of the acrosome reaction in vitro

An integral component of human spermatozoa, a glycoprotein of Mr 143,000 (two subunits of Mr 76,000 and 67,000) was recognized by the a-HS 1A.1 monoclonal antibody. The antigen was localized on the plasma membrane over the sperm head, as demonstrated by transmission electron microscopy. The antigen-antibody binding on gametes during changes in their functional state was followed by an indirect immunofluorescence assay of live human spermatozoa. In freshly ejaculated spermatozoa the antibody binding pattern revealed a patchwork quilt-like topography of the plasma membrane over the acrosome; the percentage of positive cells varied from 20 to 78% with a mean of 50% (n = 82). Incubation in a capacitation medium could increase this percentage up to 98%, revealing new epitopes in an energy-dependent and temperature-independent manner; concomitantly, a part of the antigen migrated in energy-independent and temperature-dependent manner and accumulated in a ring over the postacrosome. When an acrosome reaction was induced in vitro in the presence of Ca2+ with either A23187, ionomycin or human follicular fluid, the HS 1A.1 antigen migrated until immobilization in a well defined pattern around the equatorial segment (single band) or around the equatorial and postacrosomal segments (2 or, seldom, 3 bands). The new antigen localization resulted from a lateral diffusion of pre-existing molecules, occurred in only a few minutes, did not require energy and was temperature-dependent. At the same time, the well outlined large patch burst into a multitude of small spots before vanishing. this veil-like labelling was often observed in spermatozoa kept in the seminal plasma or treated with a metabolic poison. The HS 1A.1 antigen localization reflects surface changes induced by the incubation in a capacitation medium and the acrosome reaction. Apart from the regional heterogeneity of the plasma membrane of a single cell, as noted above, there were differences in the plasma membrane changes in individual spermatozoa from the same ejaculate as well as in semen samples from different donors. The new antibody binding pattern was often alike in successive ejaculates of the same donor. In patients consulting for infertility the percentage of positive cells was often low and migration of the antigen was slight or absent.     

Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.     

Monoclonal antibodies to bull sperm surface antigens

Four monoclonal antibodies were generated during the present investigation. Mice were immunized with washed ejaculated bull spermatozoa. Spleen cells from the immunized mice were fused with myeloma cells (SP2/0) and four different hybridoma clones were obtained, producing specific monoclonal antibodies. These antibodies were designated as SP₂A₅, SP₁A₂, SP₁C₄ and SP₁C₆ respectively. All belonged to the IgG sub-class 1. The specificity of these monoclonal antibodies was tested using both ELISA (enzyme-linked immunosorbent assay) and indirect immunofluorescence staining techniques. Quantitative estimation of antibody-antigen reaction was done by optical density measurements. Ejaculated bull spermatozoa were always reactive for each ELISA procedure. Other test cells including spleen, testicular, ovarian, uterine and pancreatic cells from both bull and rabbit were non-reactive. Bull testicular spermatozoa were also non-reactive. However, seminal fluid (without sperm) was reactive. All four monoclonal antibodies were reactive to the midpiece of the ejaculated bull spermatozoa. In addition, SP₂A₅ was also reactive to the acrosomal area and SP₁A₂ was reactive to the acrosomal and the post-acrosomal area. Cytoplasmic droplets of ejaculated spermatozoa from bull and human were also possibly reactive to one antibody (SP₁C₆). The results clearly suggest the heterogeneous nature of the sperm cell plasma membrane and precise molecular alteration in the plasma membrane components (antigens) as the sperm cells differentiate and mature during their transit through the epididymis.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Sperm antibodies in vasectomized men and their effects on fertilization

Sera (vbs, n = 25) and seminal plasma (vsp, n = 21) from vasectomized men (n = 25) were analyzed for cross-reaction with lithium diiodosalicylate (LIS)-solubilized human sperm extract, protamine, and fertilization antigen (FA-1) with an enzyme-linked immunosorbent assay (ELISA). Among the vbs tested, 44% reacted with human sperm extract, 28% reacted with protamine, and 44% reacted with FA-1 for at least one class of antibodies (IgG, IgA, or IgM). In contrast to the sera, the seminal plasma showed minimal reactions. Neither the vbs nor vsp were found to contain immune complexes, indicating that the antibodies were present in free form. Vasectomized sera that reacted with FA-1 showed a significant (p less than 0.0001) inhibition of human sperm penetration of zona-free hamster ova. The immunoabsorption of FA-1-positive sera with purified FA-1 significantly increased the penetration rates. Affinity-purified human immunoglobulins reactive with FA-1 and not those reactive with protamine reduced sperm penetration rates. Thus, antibodies in vbs reactive with FA-1 are relevant to infertility, causing an inhibition of fertilization. These data will have clinical relevance for diagnosis and treatment of infertility after successful vasovasostomy.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.     

The GPI-anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.     

The detection of the herpes simplex virus 1 and 2 antigens in clinical specimens by using monoclonal antibodies

The possibility of using monoclonal antibodies (McAb), obtained earlier, for the detection of herpes simplex virus (HSV) in clinical specimens taken from sick and infected persons was studied. The examination of 90 persons revealed that the mixture of McAb 4A and 2C could effectively detect the presence of HSV antigen in the indirect immunofluorescence assay (IFA) directly in cells contained in cytological preparations (smears, scrapes, impressions) obtained from different organs of patients. The search of optimum combinations of McAb for the detection of HSV antigens by the method of the solid-phase enzyme immunoassay (EIA) was carried out. This study, made on purified HSV used as an experimental model, revealed that the maximum sensitivity could be achieved with the use of two McAb (4f6 and 7c4) out of three McAb (4f6, 7c4 and 3d10). The approbation of both variants of EIA on clinical specimens taken from 99 patients (blood clots, seminal fluid, scrapes of cervical canal cells, peripheral blood lymphocytes) showed that the addition of McAb 3d10 made it possible to detect 8 more positive specimens. 754 specimens from 337 patients were studied with the use of McAb-based EIA, and in 204 of these patients (61%) HSV antigen was detected. The results obtained with the use of our McAb were compared with the data obtained with certified commercial test systems. The coincidence of the EIA data with those obtained with the use of the Murex Wellcozyme HSV test system (UK) was registered in 75% of cases (in 15 out of 20 cases). The coincidence of the IFA data with those obtained with the use of the Sanofi test system (France) was observed in all 19 cases (100%).     

Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma

Indirect immunofluorescence studies with polyclonal antibodies show that caltrin binds to the plasma membrane over the acrosome and principal tail regions of bovine spermatozoa but not to the postacrosomal area or the midpiece. Calcium influx into bovine epididymal spermatozoa maintained in a simple salt medium containing DL-beta-hydroxybutyrate is prevented by caltrin freshly prepared from bovine seminal plasma through a procedure employing only gel permeation columns. Older preparations, on the other hand, enhance calcium uptake into these cells. Caltrin freshly prepared through a purification scheme that includes a cation exchanger only induces enhancement of calcium uptake into bovine epididymal spermatozoa maintained under identical conditions. It is postulated that early during sperm transit through the female reproductive tract, caltrin bound to the sperm plasma membrane protects the sperm cells from calcium influx. As the cells enter the oviduct where meeting with the egg could take place, factors present in the surrounding milieu may cause caltrin to change from an inhibitor to an enhancer of calcium uptake. The acrosome reaction and possibly hyperactivation, two components of capacitation that require calcium influx as an initial event, then take place.     

Cyclic nucleotide phosphodiesterases in human spermatozoa and seminal fluid: Presence of an active PDE10A in human spermatozoa

BackgroundCyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for sperm functions such as capacitation, motility and acrosome reaction. It is well known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. The present study was undertaken to characterize cAMP-PDE activity in human semen.MethodscAMP-PDE activity was measured in human sperm and seminal plasma using family specific PDE inhibitors. Three sperm fractionation methods were applied to assess cAMP-PDE activity in spermatozoa. Western blots were used to validate the presence of specific family in sperm and seminal plasma.ResultsUsing three sperm fractionation methods, we demonstrated that in human sperm, the major cAMP-PDE activity is papaverine-sensitive and thus ascribed to PDE10. In seminal plasma, total cAMP-PDE activity was 1.14 ± 0.39 fmol of cAMP hydrolyzed per minute per μg of protein. Using specific inhibitors, we showed that the major cAMP-PDE activity found in human seminal plasma is ascribed to PDE4 and PDE11. Western blot analysis, immunoprecipitation with a specific monoclonal antibody, and mass spectrometry confirmed the presence of PDE10 in human spermatozoa.ConclusionThis study provides the first demonstration of the presence of functional PDE10 in human spermatozoa and functional PDE4 and PDE11 in human seminal plasma.General significanceSince the contribution of cyclic nucleotides in several sperm functions is well known, the finding that PDE10 is an active enzyme in human spermatozoa is novel and may lead to new insight into fertility.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.     

A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, Oreochromis niloticus

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT-30) against a molecular weight (Mr) = 120,000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration-dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT-30, suggesting that the TAT-30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with TAT-30 demonstrated that the sperm immobilizing factor was more than Mr = 1,000,000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120,000-TAT-30 positive protein. Additionally, lectin blot analysis showed that the TAT-30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT-30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells.     

Trophoblast/leukocyte-common antigen is expressed by human testicular germ cells and appears on the surface of acrosome-reacted sperm

The murine monoclonal antibody H316 reacts with a cell-surface antigen of human trophoblast, leukocytes, certain epithelia, and several malignant cell types. We have found that the H316 antibody also recognizes an antigen synthesized by pre- and post-meiotic human testicular germ cells and is expressed in the acrosomal region of methanol-fixed testicular, epididymal, and ejaculated sperm. The antigen is poorly expressed on the surface of fresh ejaculated motile sperm, but is detectable on most viable sperm after a 6-h incubation in medium containing human serum albumin (HSA), or 60-min incubation with the calcium ionophore A23187 (both treatments induce sperm acrosomal changes termed capacitation and acrosome reaction). We found that antigen recognized by H316 is immunoprecipitated as a single, broad 50 kDa band from radiolabeled ionophore-treated sperm extracts and that preincubation of HSA-capacitated sperm with this antibody causes a moderate, but significant, inhibition of hamster egg penetration. These data indicate that the antigen recognized by the H316 monoclonal antibody is synthesized by testicular germ cells and is surface-expressed on capacitated/acrosome-reacted sperm populations. Its potential as a human sperm acrosome reaction marker, and possible biological role in sperm-egg or sperm-lymphocyte interactions, warrants further investigation.     

Characterization and activity of angiotensin-converting enzyme in Holstein semen

The study was designed to perform immunodetection in spermatozoa and seminal plasma, immunolocalization in spermatozoa, and evaluation of the enzymatic activity of angiotensin-converting enzyme (ACE) in the semen of Holstein bulls. We used ejaculates from five bulls as part of a regular collection of semen. The monoclonal anti-ACE antibody recognized a single protein band with 100 kDa in detergent extract prepared from sperm and in seminal plasma. ACE enzymatic activity in sperm was 43.7, 21.3, 45.6, 60.0, and 57.7 mU/mL in bulls 1, 2, 3, 4, and 5, respectively, and 0.3, 2.3, 3.0, 2.3, and 2.6 mU/mL in seminal plasma of the same bulls, respectively. The average percentages of sperm with acrosome reactions after treatment with heparin were 28.3%, 28.6%, 35.2%, 25.0%, and 32.3%, respectively. These values were higher than the percentages of acrosome reactions in controls and the captopril group (P<0.05), although no difference was seen between the captopril and control groups (P>0.05). After 4h of incubation, motility in the control group (32.9%) was significantly higher than that in the heparin (15.7%) and captopril (12.1%) groups. No difference was found in motility after the capacitation assay in the heparin and captopril groups (P>0.05). In conclusion, ACE was immunologically localized in the acrosome of the spermatozoa of Holstein bull, the specific enzymatic activity of ACE in detergent-extracted spermatozoa and seminal plasma was inhibited by captopril, and this ACE inhibitor reduced the percentage of sperm with progressive motility and acrosome reactions after capacitation in vitro.     

Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.     

Seminal CD38 Enhances Human Sperm Capacitation through Its Interaction with CD31

Human sperm have to undergo a maturational process called capacitation in the female reproductive tract. Capacitation confers upon the sperm an ability to gain hypermotility and undergo acrosome reaction. Previous studies have suggested that seminal plasma proteins induce the capacitation of sperm in the female reproductive tract for the successful fertilization of the oocyte. However, the function of seminal plasma proteins in capacitation remains largely unclear. To the end, we found that soluble CD38 (sCD38) in seminal plasma increases the capacitation of sperm via specific interactions between sCD38 and the CD31 on the sperm. Upon the association of sCD38 with CD31, tyrosine kinase Src phosphorylates CD31, a process blocked by Src inhibitors. Shc, SHP-2, Grb2, and SOS, as well as Src kinase were found to associate with the phosphorylated CD31. The sCD38-induced phosphorylation of CD31 initiates a cascade reaction through the phosphorylation of Erk1/2, which results in the acrosome reaction, and sperm hypermotility. These processes were prevented by Src, Ras and MEK inhibitors. Taken together, these data indicate that the sCD38 present in seminal plasma plays a critical role in the capacitation of sperm.