Biomedical subjects
Publications and source records attributed to E L Golubeva.
Generation of free radicals in ejaculate samples from infertile patients was evaluated using chemiluminescent technique. The presence of antisperm antibodies in samples increased the possibility of damages to spermatozoon plasma membranes due to excessive generation of free radicals.
Conflicting opinions about the effects of antisperm antibodies on fertilization can be due to inadequacy of experimental approaches in evaluating the antisperm immunity. Detection of antisperm antibodies bound to the surface of live spermatozoa can be associated with aggregation of surface antigen-antibody complexes followed by metabolic activation of spermatozoa and acrosomal reaction, which impair cell resistance. New concept of antisperm immunity and its influence on reproduction can be formulated only after comprehensive studies of the mechanisms of spermatozoon response to binding of antisperm antibodies. Further improvement of quantitative assays of antisperm antibodies and evaluation of their effect on spermatozoon function should be aimed at selection of experimental conditions preventing changes in spermatozoa coated with antisperm antibodies during in vitro manipulations.
Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective method for the quantitative detection of antisperm antibodies (ASA). But the cross-linking of cell surface antigen (Ag) with bivalent antibodies and/or antigen-antibody (Ag-Ab) complexes with second antibodies may induce the reorganization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationship between aggregation of Ag-Ab complexes on the sperm surface and the results of indirect FCM test. Swim-up spermatozoa of normozoospermic men were incubated with ASA-positive sera from infertile patients and with second antibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum under different conditions and then analysed by FCM and fluorescence microscopy. It was shown that low temperature, cytochalasin B, excess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag-Ab complexes and dramatically increase ASA quantity determined on the sperm surface. However, inhibition of aggregation on the live sperm surface was observed only in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag-Ab complex shedding from the sperm surface during experimental manipulation limits the use of indirect FCM test for quantitative ASA determination.
Acrosome status in human spermatozoa from 20 normozoospermic men was evaluated by flow cytometry following the induction of the acrosome reaction with the ionophore A23187. Dual fluorescence staining of methanol fixed spermatozoa incubated with and without (control) the ionophore A23187 was performed with probes which targeted the outer acrosomal membrane (OAM) (rhodamine-labelled Arachis hypogaea agglutinin) or constituents of the acrosomal vesicle (fluorescein-labelled Pisum sativum agglutinin). Flow cytometry analysis revealed two major subpopulations of cells: acrosome-intact and acrosome-reacted spermatozoa after induction of the acrosome reaction. The intensity of green and red fluorescence in acrosome-reacted spermatozoa was significantly lower than that of the acrosome-intact control spermatozoa (P < 0.0001). The intensity of green fluorescence in the acrosome-intact subpopulation of spermatozoa was significantly higher than that of the control population (P < 0.002). Exposure of spermatozoa to the ionophore A23187 resulted in reliable enhancement of the number of spermatozoa with very high intensity of green and/or red fluorescence compared with the control (P < 0.03). An inverse correlation between the number of acrosome-reacted spermatozoa and spermatozoa with a very high intensity of green and/or red fluorescence was demonstrated (r = -0.631, P < 0.01). This method provides an objective and efficient procedure for quantitative estimation of the acrosomal status of human spermatozoa.
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Memories about scientific activities of P. K. Anokhin cover "Gorbi" period of his work, pre-war work in VIEM in (All-Union Institute of Experimental Medicine) Moscow, renew of researches the war (1944-1952), stress the losses, caused by scientific session, devoted to problems of I. P. Pavlov physiological doctrine in 1950. Indicated roots and ways of forming of functional system theory and system genesis conception. Overview of scientific works, belong to academics, headed by P. K. Anokhin.