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L D Russell

Publications and source records attributed to L D Russell.

At least 109 records · Page 6Linked to original sources
PubMed1986

Reconstruction of a type-B configuration monkey Sertoli cell: size, shape, and configurational and specialized cell-to-cell relationships.

A model of a stage-V monkey Sertoli cell was reconstructed from electron micrographs taken of semiserial sections. The configuration (type-B) was one in which spermatids were positioned near the lumen, their heads occupying shallow cylindrical recesses at the apical portion of the Sertoli cell. The cell volume was calculated to be 4,100 micron3, the surface area 2,400.68 micron2, and the surface-to-volume ratio 0.58:1. The reconstructed cell extended from the basal lamina to the tubular lumen and was generally of the tall columnar type although its surface contour was highly irregular. The dimensions of the cell [centripetal (68.46 micron), circumferential (18.40 micron), and longitudinal (21.63 micron)] were determined and cell surfaces designated. Relative and absolute surface areas of the reconstructed cell which faced other Sertoli cells, germ cells, basal lamina, and tubular lumen were calculated. Junctions and surface specializations were enumerated, catalogued, and depicted on diagrams of the cell surface. Where appropriate, type-A rat and type-B monkey Sertoli cells were compared and discussed. Morphometry was utilized to analyze the relative surface areas of germ cells adjoining the reconstructed cell to determine the percentage of their surface facing cellular and acellular elements, and these data were compared to data obtained for the rat.

Animals↗
PubMed1986

Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation.

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.

Acrosome↗
PubMed1986

Microfilaments appear in boar spermatozoa during capacitation in vitro.

Boar spermatozoa were incubated in a capacitation medium and examined for the presence of filamentous actin by using the fluorescent probe NBD-phallacidin. F-actin was not observed in uncapacitated sperm, but developed in most regions of the cell during the capacitation period. Fluorescent staining was most intense in the flagellum. When fresh seminal plasma was added to capacitated sperm and the sperm was further incubated, F-actin was no longer observed. In view of previous experiments which indicated that plasma membrane proteins (PMPs), including a major integral PMP, move out of the sperm head into the flagellum during capacitation and that this movement is inhibited by the microfilament poison cytochalasin D (Peterson, Saxena, Saxena, and Russell: Biol. Reprod., in press, '86), we suggest that actin-PMP interactions play a major role in capacitating boar spermatozoa.

Actin Cytoskeleton↗
PubMed1986

Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis.

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.

Acrosome↗
PubMed1986

Increase in the concentration of major boar sperm surface proteins during maturation in the epididymis.

High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.

Animals↗
PubMed1986

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies.

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro.

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.

Animals↗
PubMed1984

Boar sperm cytoplasmic droplets: their ultrastructure, their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes.

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.

Animals↗
PubMed1984

Posttesticular surface modifications and contributions of reproductive tract fluids to the surface polypeptide composition of boar spermatozoa.

Caput and cauda epididymal fluids were found to be exceedingly rich in the numbers and kinds of polypeptides when analyzed by two-dimensional (2-D) gel electrophoresis. Only a few of the major (Coomassie-stained) and minor (silver-stained) epididymal fluid polypeptides were identified on epididymal sperm plasma membranes (PM) and even fewer identified in ejaculated sperm. The 2-D electrophoretic patterns of caput sperm PM differed little from those of cauda sperm PM. Thus, epididymal transit resulted in relatively minor quantitative and qualitative modifications in sperm PM composition. Seminal plasma showed a few major polypeptides from the cauda epididymal fluid, but the major constituents were those polypeptides from the seminal vesicle secretions. Sperm appear to acquire one acidic high molecular weight polypeptide from either the bulbourethral gland or prostate gland, and another major acidic polypeptide of high molecular weight from the seminal vesicle gland. Numerous neutral and basic low molecular weight polypeptides, originating from the seminal vesicles, adhered tightly to sperm. These were major polypeptides and constituted a substantial percentage of the total PM protein. Thus, major contributions to the sperm PM polypeptide profile occurred at ejaculation. This study did not address loosely bound polypeptides but is the first to analyze, in a comprehensive way, the origins of tightly bound sperm polypeptides from a single species.

Animals↗
PubMed1984

Characterization of (Ca2+ + Mg2+) adenosine triphosphatase activity and calcium transport in boar sperm plasma membrane vesicles and their relation to phosphorylation of plasma membrane proteins.

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.

Animals↗
PubMed1984

Determination of the elongate spermatid-Sertoli cell ratio in various mammals.

Criteria were devised for determining the elongate spermatid-Sertoli cell ratio in various mammalian species at the electron microscope level. When data from particular species were pooled, the values were: rabbit, 12.17:1, hamster, 10.75:1; gerbil, 10.64:1; rat, 10.32:1; guinea-pig, 10.10:1; vole, 9.75:1; and monkey, 5.94:1. The elongate spermatid-Sertoli cell ratio is a measure of the workload of the Sertoli cell and is a prime factor determining their efficiency. The higher the ratio, the higher the sperm output is likely to be per given weight of seminiferous tubule parenchyma for a particular species.

Animals↗
PubMed1983

Effect of calmodulin antagonists on CA2+ uptake by boar spermatozoa.

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both 45Ca2+ uptake and net Ca2+ uptake are increased by these drugs. Drug stimulated Ca2+ uptake is blocked by verapamil (1 mM), by ruthenium red (25 microM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca2+ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca2+-ATPase. It is proposed that calmodulin is involved in the control of Ca2+ entry in boar spermatozoa. Most entering Ca2+ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca2+ extrusion does not appear to be regulated by calmodulin.

Animals↗
PubMed1983

Three-dimensional reconstruction of a rat stage V Sertoli cell: I. Methods, basic configuration, and dimensions.

A model of a rat stage V Sertoli cell was reconstructed from semiserial sections. Seventy-five montages were made from 96 grids that supported the 223 sections needed to traverse the reconstructed cell. Section thickness (184.6 nm) and spacing of sections were calculated based on the diameter of spherical germ cell nuclei included within the sections. The outline of the cell border was traced on acetate sheets and from there traced on Plexiglas; then the Plexiglas was cut with a hot wire. The model, enlarged to a magnification of 7,800 times, was made from five parts that may be disassembled. The cell dimensions, as determined from the model, were 89.8 micron in the centripetal axis, 41.2 micron in the longitudinal axis, and 29.5 micron in the circumferential axis. The cell was irregularly columnar in shape with a base resting on the basal lamina and lateral surfaces in contact with adjacent Sertoli cells and round germ cells. Lateral processes were of three types: 1) conical processes extending from the lateral surface near its base; 2) cup-shaped, sheet-like processes partially encompassing round germ cells; and 3) flattened, sheet-like processes extending between round germ cells. Elongate spermatids occupied deep, irregularly shaped cylindrical recesses oriented in the centripetal axis of the Sertoli cell. The walls of the Sertoli cell forming these cylinders were thick basally and sheet-like and very thin near the lumen where they anastomosed and also composed the lateral walls of the cell. The tapered luminal extensions of the sheet-like, cylindrical processes were termed apical processes. The volume of the reconstructed cell was calculated to be 6,012 micron3. This study has provided the first unencumbered view of the external profile of a Sertoli cell. The model represents one of two major configurations that the Sertoli cell assumes during the spermatogenic cycle.

Animals↗
PubMed1983

Three-dimensional reconstruction of a rat stage V Sertoli cell: II. Morphometry of Sertoli--Sertoli and Sertoli--germ-cell relationships.

Sertoli--Sertoli and Sertoli--germ-cell configurational relationships were studied using morphometric techniques and direct measurements as obtained from micrographs used to reconstruct a model of a rat stage V Sertoli cell. Regional areas of the Sertoli cell surface, which faced germ cells, other Sertoli cells, or noncellular structures, were expressed as relative surface area percentages; and the absolute surface areas for these regional areas were calculated. The surface areas of the reconstructed cell, in its unmagnified state, was found to be 12,163 micron2. Cell processes were enumerated and studied using morphometric techniques. The surface area of the reconstructed Sertoli cell facing germ cells and Sertoli cells was also determined. Five Sertoli cells showed extensive contact with the reconstructed cell at the level of the Sertoli--Sertoli junctional contact region. This contact region averaged 3.51 micron in width. The relative and absolute surface area of subsurface ectoplasmic specialization of the Sertoli cell that faced germ cells and other Sertoli cells was calculated, and the extent of penetration of step 17 spermatids into the Sertoli crypts was determined. Surface relationships of the reconstructed cell to cellular and noncellular elements were depicted on outline drawings of the Sertoli cell.

Animals↗
PubMed1983

Three-dimensional reconstruction of a rat stage V Sertoli cell: III. A study of specific cellular relationships.

Specific Sertoli--Sertoli and Sertoli--germ-cell contacts and/or junctions were investigated employing micrographs used to reconstruct serially a model of a rat stage V Sertoli cell. The Sertoli--Sertoli junctional contact areas occurred in a belt-like arrangement near the base of the Sertoli cell. This configuration is consistent with their proposed function as a sealing element limiting the passage of materials toward the tubular lumen. Sertoli ectoplasmic specializations also formed a continuous belt, or band, around the reconstructed cell at the junctional contact area. Eighteen Sertoli--Sertoli tubulobulbar complexes were found; some (12 in number) invaginated the reconstructed cell, while others (6) emanated from it. Of 37 round germ cells that were sectioned in their entirety and adjoined the reconstructed cell, 23 displayed desmosome-gap junctions with either the reconstructed cell or an adjoining cell. Since there were multiple junctions connecting some germ cells to Sertoli cells, the total number of junctions was much greater (35). Desmosome-gap junctions of the Sertoli cell were numerous connecting pachytene spermatocytes, less numerous connecting type B spermatogonia, and even less numerous connecting step 5 spermatids; and none was seen joining Sertoli cells with elongate spermatids. Most desmosome-gap junctions join germ cells to the body of the Sertoli cell at its basal aspect. Their numbers and position indicate that they play a role in the maintenance of the integrity of the seminiferous epithelium and may provide a route for cell-to-cell communication. Ectoplasmic specializations of the reconstructed cell were seen facing only 3 of 37 round germ cells, and 7 ectoplasmic specializations from adjoining Sertoli cells faced these germ cells, all of which were step 5 spermatids. That there were no ectoplasmic specializations facing pachytene cells indicates that ectoplasmic specializations are not acquired as these cells pass through Sertoli--Sertoli junctions, but are acquired later in spermatogenesis.

Animals↗