M. Mollova

MONOCLONAL ANTIBODIES TO INTRA-ACROSOMAL PROTEINS INHIBIT GAMETE BINDING IN VITRO

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.

Changes in immunochemical localisation of acrosomal and sperm proteins in boar spermatozoa during capacitation and induced acrosome reaction

Abstract Three monoclonal antibodies that stained acrosin of 55, 53, 45 and 38 kDa (ACR.2), acrosomal protein of 28 kDa (ACR.4) and sperm protein of 17 kDa (ACR.3) were used for immunostaining and immunoblotting analysis of boar spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Two proteins (acrosin and the 28 kDa protein) showed similar changes in the acrosome during capacitation and induced acrosome reaction. For detection of these proteins, immunoelectron microscopy was also used. The immunofluorescence test revealed that most spermatozoa lost acrosin and the 28 kDa protein after incubation with ionophore A 23187. The lost proteins were detected in the culture medium by immunoblotting. During capacitation and induced acrosome reaction, sperm protein of 17 kDa (ACR.3) showed a different pattern of labelling. The changes in relocation of specific proteins during capacitation and acrosome reaction support a concept of physiological preparation of spermatozoa for sperm-egg interaction.

Sertoli Cell Quiescence - New Insights

It is currently accepted that the Sertoli cells are proliferatively active only during the embryogenesis and early fetal development, seizing to divide after puberty, when the spermatogenic niche is prepared, and they become terminally differentiated. So far, only seasonal breeders from mammals have been reported as having season‐dependent variations in adult Sertoli cells number and proliferation activity. In this review, we will try to shed light on testis somatic cell plasticity and discuss new evidence for some unique proliferative features Sertoli cells harbor.

Isolation and biological characterization of boar sperm capacitation-related antigen

Mollova M, Djarkova T, Ivanova M, Stamenova M, Kyurkchiev S. Isolation and biological characterization of boar sperm capacitation‐related antigen. AJRI 1999; 42:254–262

Identification and characterization of human acrosomal antigen defined by a monoclonal antibody with blocking effect on in vitro fertilization

A monoclonal anti-human sperm antibody (Mab 1A1) has been produced by fusion of myeloma cells with splenocytes from a BALB/c mouse immunized with in vitro capacitated human spermatozoa. Immunofluorescence studies with Mab 1A1 show that it recognizes an antigen(s) (Ag 1A1) which is located in the acrosome of human spermatozoa. As shown by Western blotting experiments, 1A1 antigen represents a family of proteins with Mr ranging from 20 kDa to 34 kDa. Immunofluorescence observations on epitope exposure and location suggest that during in vitro capacitation of human spermatozoa, Mab 1A1 epitope-bearing molecules are concentrated in regularly arranged granules in the acrosome. After long-term incubation the epitope is exposed on the apical acrosome surface exhibiting a spot-like arrangement. The 1A1 epitope is widely distributed among mammalian species: boar, ram, mouse and rat acrosome is intensively stained by Mab 1A1. The antibody inhibits in vitro fertilization mainly by blocking sperm attachment to and penetration through the zona pellucida when included in the medium for the in vitro fertilization of mouse, porcine and human oocytes.

A possible biological role of boar sperm diaphorase investigated by a specific monoclonal antibody

Abstract A monoclonal antibody (Mab 5D8) against soluble NADH diaphorase from boar spermatozoa was used to investigate the possible biological role of this enzyme in the process of fertilization. The localization of the sperm diaphorase was confirmed by the indirect immunoperoxidase technique which showed that both fixed and in vitro capacitated spermatozoa were positively stained in the region of the mitochondrial sheath after treatment with Mab 5D8. Cytochemical staining for the specific enzyme showed that Mab 5D8 inhibited the diaphorase activity of live capacitated spermatozoa. Data on sperm motility indicate the potency of Mab 5D8 to significantly decrease the hyperactivated sperm population when present in the capacitation medium. After treatment with Mab 5D8, these spermatozoa showed reduced penetrating activity, but no changes were observed in their binding activity in an in vitro fertilization (IVF) system. When Mab 5D8 was added to the fertilization medium, there was considerable inhibition of sperm binding and even more marked suppression of sperm penetration through the zona pellucida. These findings demonstrate the essential biological role of sperm diaphorase during fertilization.

Monoclonal Antibodies Reacting Against Capacitated but not With Freshly Ejaculated Boar Spermatozoa

PROBLEM: The involvement of individual sperm proteins in differentiation of antigenically specific and functionally defined regions on sperm membrane has not yet been completely elucidated.