Michal Simon

Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods

Artificial insemination with frozen-thawed spermatozoa is commonly used in cattle breeding. A simple and fast procedure is needed for routine evaluation of the acrosomal status of frozen-thawed bovine sperm. Therefore, the purpose of this study was to test two staining procedures used to determine the viability and integrity of acrosome of frozen-thawed bovine spermatozoa. Double staining and Hoechst/FITC-Pisum sativum agglutinin (FITC-PSA) labelling were tested for evaluating the viability and acrosome reaction induced by calcium ionophore of bull spermatozoa. In our experiments no significant differences were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%). The viability of sperm stained by the double staining method was 67.17%, and a higher portion of viable sperm (82.67%) was observed by staining with the Hoechst procedure (P < 0.01). On the basis of the results obtained it is concluded that both methods can be used for detecting the acrosome reaction of frozen-thawed bovine spermatozoa.

Ruminant cluster CD41/CD61

After analysis of flowcytometry data on bovine and ovine cells and immunoprecipitation studies, a cluster of seven MAbs-CAPP2 (3W-232), IL-Al64 (M430), IL-Al66 (3W-226), IVA30 (3W-069), IVA31 (3W-468), IVA38 (3W-349) and IVA125 (3W528)-was shown to detect bovine and ovine CD41/Cd61 (also known as allS Mateo et al., 1996a). MAb Co-35E4, not submitted to the workshop, also precipitated the platelet integrin and cross-reacted with several species (Pintado et al., 1995).

Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies

A panel of 18 monoclonal antibodies (mAbs) defined by the third workshop as specific for platelets, clustered in three preliminary groups: PC7, PC13 and PC27. These mAbs were further analysed by immunoprecipitation using extracts of iodinated and biotinylated peripheral blood mononucleated cells (PBMC) and platelets. We could confirm the existence of mAbs with specificities to WC9 (in PC7) and CD41/61 (in PC13). Two mAbs formed a new cluster, WC13, which may be homologous to human CD31 (in PC27). The influence of EDTA and thrombin on the expression of the different antigens on the platelet membrane was assessed by flow cytometry (FCM) analysis, as well as cross-reactivity with platelets from different species.

Analysis of the expression of platelet antigens CD9 and CD41/61 in transgenic rabbits with the integrated human blood clotting factor VIII gene construct.

The objective of this research was to study the expression of cell membrane molecules CD9 and CD41/61 of transgenic rabbit with integrated human factor VIII (rhFVIII) gene construct. The expressions of these molecules have been monitored during two lactations of transgenic rabbits and simultaneously compared with the expression of the same molecules of non-transgenic rabbits. The immunochemical analysis by indirect immunofluorescence, ELISA and indirect immunoperoxidase staining of blood cells and udder tissues show that the insertion of the WAP-hFVIII gene construct into the rabbit genome, do not influence the expression of cell membrane antigens CD9 and CD41/61 on the blood platelets, polymorphonuclear blood cells, milk somatic cells and mammary gland tissues.

Two monoclonal antibodies from the platelet panel recognize sheep plasma fibrinogen

Among the monoclonal antibodies (mAbs) submitted to the third Workshop, two mAbs, IVA120 (3W-323) and IVA198 (3W-290), could be identified to recognize the sheep fibrinogen molecule. The apparent molecular weight of the immunoprecipitated 48-60 kDa cell surface protein under reducing conditions suggested this antigen could be the fibrinogen molecule. ELISA and immunoblotting assays, performed with commercially available sheep plasma fibrinogen, confirmed that these two mAbs recognize two different epitopes present on the sheep fibrinogen molecule.

Characterization of tetraspanin protein CD81 in mouse spermatozoa and bovine gametes.

Sperm-egg interaction and fusion represent a key moment of fertilization. In mammals, it is not possible without the interaction of the tetraspanin superfamily proteins including CD81. A detailed immunohistochemical localization of CD81 was monitored in bovine oocytes during different maturation stages, as well as during early embryogenesis. In addition, characterization of CD81 was carried out in bovine and mouse sperm. In bovine eggs, CD81 was detected on the plasma membrane of the germinal vesicle, metaphase I and metaphase II oocytes. During fertilization, accumulation of CD81 molecules in the perivitelline space of fertilized oocytes, which appeared as vesicles associated with plasma membrane, was observed. In majority of bull-ejaculated sperm and caput, corpus and cauda epididymal sperm, as well as mouse cauda epididymal sperm, CD81 was found on the plasma membrane covering the apical acrosome. Although the process of capacitation did not influence the localization of CD81, it was lost from the surface of the acrosome-reacted spermatozoa in bull, in contrast to mouse sperm where there was a relocalization of the CD81 protein during acrosome reaction across the equatorial segment and later over the whole sperm head. The presented results highlight conservative unifying aspects of CD81 expression between cattle and mouse, together with mouse-specific traits in sperm CD81 behaviour, which emphasizes certain species-specific mechanisms of fertilization to be considered.

Comparative fluorescence analysis of the bovine sperm using IVA-520 (anti-CD46 antibody) and lectins: probable localisation of CD46 on bovine sperm membrane.

Membrane cofactor protein (CD46) is complement regulatory protein with probable function in the reproduction process. Expression of CD46 on human, mice, rat and guinea pig spermatozoa is restricted to the inner acrosomal membrane. In spite of the presence of anti-sperm antibodies and other potential complement activating agents in follicular fluid, CD46 is not expressed on the plasma membrane of spermatozoa as the other complement regulatory proteins (DAF and CD59) in human. Using dual immunofluorescence labelling with mAb IVA-520 (anti-bovine CD46) and various lectins with different binding pattern or monoclonal antibody ACR.4, targeted against intra-acrosomal protein, we excluded the expression of CD46 on the inner acrosomal membrane as well as in the acrosomal content but, we suggested the localization of this molecule on the outer acrosomal membrane and possibly on the plasma membrane of bovine sperm.

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract

The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%). This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.

Immunohistochemical reactivity of anti-platelet monoclonal antibodies

Ten mAbs of preliminary clusters PC13 and PC27 with specificity for bovine platelets were studied by immunohistochemistry. Cryostat sections of bovine lymph node, spleen, thymus, small intestine, liver, kidney and smears of bone marrow cells were used. Five mAbs (CAPP2, IVA30, IVA125, IL-A164 and IL-A166) assigned to cluster PC13 (CD41/CD61) stained platelets and non-lymphocytic cells of various tissues. Our data confirm the presence of two specificities in PC27: three mAbs (IVA120, IVA197 and IVA198) specific for fibrinogen strongly reacted with the endothelial and reticular tissues whereas the other two mAbs Co-3D1D4 and Buf13 (WC13) were negative.