R. N. Peterson

Sertoli cell junctions: morphological and functional correlates

Publisher Summary Modern cell biology has shown that cellular recognition and adhesion are not always manifested by junctions of a classical type or by morphological entities recognizable by standard transmission electron microscopy techniques. This chapter describes several junctions in which experimental data are the primary evidence for suggesting a junctional relationship between cells. The cyclic nature of spermatogenesis and the continually changing morphology of the Sertoli cell provide a dynamic framework in which junctions are periodically formed and eliminated. Desmosomes are important in maintaining the integrity of the seminiferous epithelium, a function, which should not be underemphasized, because many germ cell types appear only loosely arranged in the seminiferous epithelium. Based on circumstantial evidence, it is reasonable to suspect that, as yet, unrecognized junctional forms are present in the relationship of the Sertoli cell and germ cells and will be revealed in the future through methodological advances.

Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.

Morphologic Characteristics of the Chemically Induced Acrosome Reaction in Human Spermatozoa

The morphologic changes accompanying the acrosome reaction in human spermatozoa, as it is induced by the antibiotic A23187 and calcium ions, are described. The reaction is shown to be similar to that observed in other species when the reaction occurs spontaneously or is induced by physiologic fluids. The reaction in human spermatozoa differs from the chemically induced reaction in other species in that plasma membrane microfilaments, prominent in the boar, and tubular-like elements prominent in boar, rabbit, and monkey sperm, are not observed. Motility remains high when human spermatozoa are treated with A23187 and calcium and it is possible that these agents may be useful in the study of certain causes of infertility.

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Abstract Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.

Development of the acrosome and alignment, elongation and entrenchment of spermatids in procarbazine-treated rats.

Intraperitoneally administered procarbazine caused, among other features previously reported (Russell et al., 1983), specific defects in the acrosome of cap phase spermatids of the rat seminiferous epithelium. The effect of procarbazine was to fragment and eventually cause resorption of the acrosomes of a small number of steps 5--9 spermatids. Although the acrosome was lost, close union of the leaflets of the nuclear envelope underlying the acrosomal sac was maintained as was the marginal fossa and acrosomal zonule. Spermatids at steps 8 and 9 of development, which had lost their acrosomes, showed nuclei which were eccentric within the cell--a feature which normally occurs at these steps of spermiogenesis in acrosome intact cells. Even without an acrosomal sac, the plasma membrane of these cells (in stage VIII) became orientated to the region of the nuclear membrane which would have underlaid the acrosome. Although abundant, Sertoli ectoplasmic specialization did not become aligned with the spermatid head. The spermatid failed to become orientated within the seminiferous epithelium and failed to enter the crypts within the Sertoli cell as usually occurs during the elongation process. Thus, the presence of an acrosome is not likely related to the formation of an eccentric nucleus or the alignment of the surface of the nucleus which would normally underlay the acrosome with the cells plasma membrane (internal alignment). The presence of an acrosome may be related to the alignment of the spermatid head with the ectoplasmic specialization, which in turn may influence the orientation and positioning of the late spermatids within the seminiferous epithelium (external alignment) and their position within recesses of the Sertoli cell. This study also suggests a role for the manchette in the process of elongation of the spermatid.

Use of a fluorescent dye to measure drug-induced changes in the membrane potential of boar spermatozoa.

Abstract The fluorescence emission intensity of the dye 3,3′ dipentyloxo-carbocyanine iodide equilibrated with washed boar spermatozoa and valinomycin or gramicidin varies with external potassium or sodium concentration in a manner indicating that dye fluorescence is related to the plasma membrane potential. The membrane potential in turn is shown to be dependent on energy metabolism. The drugs propranolol, lidocaine and diphendydramine, which possess local anesthetic-like properties, induce a rapid depolarization which can be reversed by valinomycin at low drug concentrations and a slower apparently energy dependent depolarization at higher drug concentrations that is not reversible. Low concentrations of these drugs decrease forward progression of sperm but have little effect on the percentage motile cells. Theophylline increases the frequency of contraction of drug-treated cells but not their forward progression, these findings are discussed in terms of the role of the sperm membrane in the control of motility.

Isolation of Major Proteins from Boar Sperm Plasma Membranes by Preparative Gel Electrophoresis and Localization of a Major Polypeptide Using Specific Monoclonal Antibodies

A preparative procedure using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is described for isolating major boar sperm plasma membrane polypeptides (PMPs) in soluble form. Proteins were first separated on 6 mm diameter gels using a pH gradient expanded in the acidic region. The second dimension used 6 mm thick, 10% acrylamide gels. Major proteins identified by Coomassie staining were excised and electroeluted. The procedure was applied to the isolation of a group of proteins in the molecular weight range 40K-50K which comprise a major fraction of the total integral membrane protein in these cells (groups 4 and 5). Yields of electrophoretically pure soluble polypeptides from these groups were between 0.3 mg - 0.5 mg from the processing of 16 gels per week. Electroeluted proteins were also used to elicit monoclonal antibodies to major proteins. Monoclonal antibodies to the major plasma membrane protein referenced as 4.85 were isolated and shown to be specific to this protein by transblotting procedures. This protein was primarily localized over the anterior portion of the principal segment of ejaculated sperm by indirect FITC fluorescence microscopy. The ability to isolate 60-100 mg of plasma membranes per week from the cauda epididymides of boars also permitted developing a procedure for the rapid fractionation of large amounts of detergent solubilized plasma membranes by isoelectric focusing in flatbeds of Biogel P200. For the first time, individual proteins of sperm surface proteins can be isolated in large enough amounts to begin detailed biochemical characterization, localization, and functional testing.