Inhibition of the antibody responses to rat blood transfusion antigens in mice by boar seminal immunosuppressive fraction.

PROBLEM: Immunosuppressive fraction of boar seminal vesicle fluid (ISF) was tested to muffle primary and secondary antibody responses to xenotranfusions. Contemporaneously, heparin non-binding fraction of seminal plasma (H- fraction), presumed to be identical to ISF, was used to support the results. METHOD: To study their similarity, ISF and H- fraction were analyzed by high-performance liquid chromatography and the separated proteins by N-terminal sequencing. In sera of mice treated with ISF or H- fraction, the productions of antibodies against rat erythrocytes and blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The productions of IgM, IgA, and IgG subclasses were followed by sandwich ELISA. RESULTS: ISF and H- fraction were proved to be equal complexes of porcine seminal plasma (PSP) proteins PSP I and PSP II. Both inhibited antibody responses to rat erythrocytes and serum and the concentrations of IgM, IgG, IgG1 and IgG2 after the first transfusion with a long-lasting effect. Both suppressed the secondary antibody production if applied before the second transfusion. IgA and IgG3 stayed uninfluenced. ISF and H- fraction had an equal immunosuppressive effect. CONCLUSIONS: ISF was characterized biochemically, found to be identical to H- fraction, and determined to be powerful in overcoming unwanted exaggerated antibody responses to xenotransfusion.

Immunosuppressive effect induced by intraperitoneal and rectal administration of boar seminal immunosuppressive factor.

The immunosuppressive component was isolated from boar seminal vesicle secretion and administered i.p. or rectally to male mice. By means of the immunofluorescent method, the seminal immunosuppressive component was found on the membranes of 50-70% of white blood cells of treated mice the first day after i.p. and the third day after rectal administration. The immunosuppressive component was observed on the membranes of 10-20% of white cells even at the 17th day after treatment. Intraperitoneal or rectal administration of the immunosuppressive component led to a decrease in the white cell concentration in blood of treated mice. These findings indicate that rectal deposition of semen may compromise some aspects of the immune system and may be an important cofactor in the development of viral or bacterial infections among homosexual men.

Effect of intra-rectal administration of boar seminal immunosuppressive fraction on mouse lymphocytes.

The repeated deposition of an immunosuppressive fraction isolated from boar vesicular gland secretion into the rectum of healthy male and female mice reduced responses of lymphocytes to mitogens in vitro. Rectal deposition of the immunosuppressive component also led to a decrease in the activity of plaque-forming cells. These findings indicate that repeated rectal deposition of semen may compromise some aspects of the immune system and may be an important cofactor in the development of viral or bacterial infections among homosexual men.

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.

Proteins and glycosaminoglycans in the intercellular matrix of the human cumulus-oophorus and their effect on conversion of proacrosin to acrosin.

Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction.

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration.

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.

Activation of proacrosin by a locally produced component of human follicular fluid.

Components of human follicular fluid were separated on Sepharose 6B columns and the effects of different fractions on the conversion of pig proacrosin to acrosin were examined. A high-molecular-weight fraction (Mr greater than 3,000,000) of follicular fluid was a potent stimulator of this reaction. The proacrosin converting activity was absent in the corresponding fraction of blood serum. The acceleration of proacrosin activation was dependent on the concentration of material with proacrosin converting activity. The results indicate that human follicular fluid contains a high-molecular-weight component of local origin which is capable of accelerating proacrosin in a dose-dependent manner.

The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF.

Cumulus cells surrounding pre-ovulatory human oocytes were found to secrete a variety of proteins which became firmly associated with the cumulus intercellular material. Antibodies raised against human cumuli oophori completely blocked fertilization in vitro by impairing the sperm-zona pellucida interaction. A group of glycoproteins of high mol. wt were identified as the main cumulus cell secretory products. These proteins showed a marked affinity for human spermatozoa and were potent stimulators of the conversion of human and boar proacrosin into acrosin and of human sperm acrosome reaction. Another fraction of proteins of human cumulus intercellular matrix with an apparent mol. wt of approximately 25,000 daltons was also found to stimulate significantly the acrosome reaction of human spermatozoa, although this fraction had no proacrosin-converting activity. These results indicate that proteins secreted by pre-ovulatory human cumulus cells have an indispensable role in the development of human sperm fertilizing ability. This effect seems to be realized by a concerted action of different types of cumulus-derived proteins just prior to and during the sperm-zona pellucida interaction. Disorders of cumulus cell secretory activity may account for some cases of idiopathic infertility and repeated IVF failures.