scispace - formally typeset
Search or ask a question

Showing papers on "Sperm plasma membrane published in 1986"


Journal ArticleDOI
TL;DR: This work has used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusible that result from in vitro hyperactivation and capacitation with bovine serum albumin.
Abstract: We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.

162 citations


Journal ArticleDOI
TL;DR: Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence.

107 citations


Journal ArticleDOI
TL;DR: It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.
Abstract: A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36 degrees C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24 degrees C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24 degrees C to 30 degrees C, but the reasons for this are unclear. It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.

84 citations


Journal ArticleDOI
TL;DR: The data show that increased [Ca2+]i and pHi are necessary for induction of the acrosome reaction and suggest that the 210-kDa protein may play a role in regulating Ca2+ entry into the spermatozoan.
Abstract: Changes in intracellular free Ca2+ ([Ca2+]i) of sea urchin (Strongylocentrotus purpuratus) spermatozoa were measured using the fluorescent Ca2+ indicators fura-2 and indo-1. The intracellular pH (pHi) of sperm was also determined. The fucose sulfate-rich glycoconjugate component of egg jelly induced increases in [Ca2+]i and pHi in sperm and induced the acrosome reaction. Monoclonal antibodies (mAbs) to external domains of a 210-kDa glycoprotein of the sperm plasma membrane induced a 23-fold increase in [Ca2+]i (vs. 9-fold for fucose sulfate-rich glycoconjugate), but the mAbs did not cause the pHi to increase and did not induce the acrosome reaction. When the mAb treatment which induced an increase in [Ca2+]i was combined with an NH4Cl treatment, which increased the pHi, the acrosome reaction was induced. mAb-induced increases in [Ca2+]i were dependent on millimolar concentrations of extracellular Ca2+ and were reversed by placing sperm in Ca2+-free seawater or by chelating Ca2+ with EGTA. The mAb-induced [Ca2+]i increase was sensitive to the pH of the seawater, although mAb binding was not. The data show that increased [Ca2+]i and pHi are necessary for induction of the acrosome reaction and suggest that the 210-kDa protein may play a role in regulating Ca2+ entry into the spermatozoan. These mAbs make it possible to separate the increase in [Ca2+]i from the increase in pHi and may be useful in the elucidation of the regulatory role of Ca2+ in sperm physiology.

75 citations


Journal ArticleDOI
TL;DR: A monoclonal antibody ESA 152 is used in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm, an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells.
Abstract: We have used a monoclonal antibody ESA 152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA 152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA 152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent Mr approximately 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA 152 lateral mobility in the membrane yield diffusion coefficients in the range 10(-9)-10(-8) cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated Fab fragment of the ESA 152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA 152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA 152 diffusibility on both the anterior head and the midpiece.

48 citations


Journal ArticleDOI
TL;DR: In washed but noncapacitated spermatozoa the density of filipin/sterol complexes (FSC) was uniformly high in the PM overlying the acrosome, without any differences between its anterior and equatorial regions.

46 citations


Journal ArticleDOI
TL;DR: Results suggest that capacitation of ram spermatozoa involves loss of specific surface proteins as well as selective adsorption of exogenous fluid components and point to a polypeptide in uterine fluid as an active constituent.
Abstract: Genital tract fluids were collected continuously from conscious ewes through catheters inserted surgically into the uterus and oviducts. Cauda epididymal spermatozoa and fluid were obtained through catheters inserted into the transected vas deferens. The washed spermatozoa were labelled using the surface-specific chloroglycoluril-Na125I procedure. High-resolution electrophoretic analysis of sperm plasma membrane preparations revealed a partial loss of a major surface component (i.e. Mr 97,000) during incubation in uterine and oviduct fluids. This specific loss resulted in a shift in radioactivity distribution toward an Mr 24,000 component which had been previously identified as a sialoglycoprotein. No significant changes in the distribution of radiolabelled surface components were detectable when the spermatozoa were incubated in synthetic medium. Incubation of unlabelled spermatozoa in 125I-labelled uterine fluid showed that adsorption of exogenous fluid components was highly selective; an Mr 16,000 polypeptide was greatly enriched on the sperm surface although it was only a minor component in the incubation fluid. Adsorption of labelled oviduct fluid components was also selective and involved predominantly high molecular weight components (i.e. Mr 140,000, 95,000, 78,000, 53,000). When spermatozoa were incubated in labelled cauda epididymal fluid after exposure to unlabelled uterine and oviduct fluids, several fluid components were incorporated by the plasma membrane, indicating that surface renovation of 'capacitated' spermatozoa may be a more general process rather than a specific event. These results suggest that capacitation of ram spermatozoa involves loss of specific surface proteins as well as selective adsorption of exogenous fluid components and point to a polypeptide in uterine fluid as an active constituent.

45 citations


Journal ArticleDOI
TL;DR: This paper shows that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse.
Abstract: The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.

41 citations


Journal ArticleDOI
TL;DR: Data are interpreted to mean that the seminal inhibitor acceptor sites on the sperm surface of incubated sperm function in the in vitro binding to the zona pellucida.
Abstract: Murine cauda epididymal sperm preincubated in either a modified Krebs-Ringer or M 199 solution bind to cumulus-free, zona pellucida-intact eggs. Pretreatment of such eggs with an affinity purified preparation of the seminal inhibitor binding component (acceptor), isolated from epididymal sperm, reduces in a concentration dependent manner, the number of sperm that bind. Treatment of cauda sperm, preincubated in either of the above two media, with the seminal inhibitor, also reduces the number of sperm able to bind. Incubation of cauda sperm in the Krebs-Ringer solution for up to 4 h does not affect their ability to bind the seminal inhibitor. Omission of bovine serum albumin from the preincubation medium results in a significant reduction in sperm binding. These data are interpreted to mean that the seminal inhibitor acceptor sites on the sperm surface of incubated sperm function in the in vitro binding to the zona pellucida.

36 citations


Journal ArticleDOI
01 Sep 1986-Lipids
TL;DR: The membrane fraction that was released from caudal boar sperm undergoing an in vitro acrosome-like reaction was isolated and characterized with respect to density, marker enzymes and lipid composition, which will allow a better understanding of the mechanism of the sperm acrosomes reaction.
Abstract: Prior to fertilization, mammalian sperm must undergo the acrosome reaction, which involves modifications of the plasma and outer acrosomal membranes followed by vesiculation and release of the membranes. The membrane fraction that was released from caudal boar sperm undergoing an in vitro acrosome-like reaction was isolated and characterized with respect to density, marker enzymes and lipid composition. This membrane had a lower phospholipid/protein ratio (mg/mg) than the sperm plasma membrane, whereas both membranes had similar molar sterol/phospholipid ratios. The major phospholipid was sphingomyelin, followed by phosphatidylethanolamine and phosphatidylcholine, whereas in the plasma membrane the order was reversed; the two major phosphoglycerides contained alkylacyl and alkenylacyl species in addition to the diacyl species. The released membrane also contained lower amounts of cholesterol sulfate and unsaturated fatty acids than the plasma membranes. These results, in combination with our studies on the changes of the sperm membranes during maturation and acrosome reaction, will allow a better understanding of the mechanism of the sperm acrosome reaction.

28 citations


Journal ArticleDOI
TL;DR: In this paper, the integration and fate of the sperm plasma membrane following its incorporation into the oocyte plasma membrane were examined, and it was shown that sperm plasmalemma provided less than 10% of the total surface membrane.

Journal ArticleDOI
TL;DR: The findings suggest that the ability of boar sperm to attach to porcine oocytes develops as the result of the addition of one or more of these PMPs to sperm during epididymal transit.
Abstract: Cauda boar sperm but not caput sperm bind to isolated porcine oocytes, and several cauda sperm plasma membrane proteins (PMPs) with high affinity for isolated zonae have been identified. These PMPs migrate with apparent molecular weights near 70, 45 and 30 K [Peterson et al, Gamete Res 12: 91–100, 1985]. When caput plasma membranes (PM) were fractionated on dextran sulfate (DS) agarose and compared to cauda PM similarly fractionated, less protein with high affinity for DS bound to the column, and significant differences in the PMPs eluted with increasing concentration of NaCl were observed. The PMPs at ∼70, ∼45, and ∼30 K, noted above, and two bands near ∼20K were present in fractions from cauda PM but were absent or present in only small quantities in caput PM. Cauda PM fraction eluted at high salt were effective in blocking the binding of sperm to isolated oocytes; caput PM fractions were not. Antibodies to the cauda PMPs eluted at high salt, which blocks the binding of capacitated sperm to eggs and fertilization in vitro, were absorbed by cauda PMV but not by caput PMV. These findings suggest that the ability of boar sperm to attach to porcine oocytes develops as the result of the addition of one or more of these PMPs to sperm during epididymal transit.

Journal ArticleDOI
TL;DR: The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididcyal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.
Abstract: Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA-I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA-I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.

Journal ArticleDOI
TL;DR: Observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.
Abstract: Electron microscopic and cytochemical investigations were carried out on inseminated Arbacia oocytes comparing structural and chemical properties of their microvillous surface and fertilization cones. Early fertilization cones (up to 4 min postinsemination) were relatively small, smooth surface projections of cytoplasm that engulfed the incorporated sperm nucleus. However, in contrast to surrounding microvillous areas of the oocyte surface, enlarged fertilization cones (8 to 15 min postinsemination) had a distinctive crenated appearance that persisted until their regression. When examined by various cytochemical techniques, membrane delimiting fertilization cones had a much lower affinity for agents that stain negatively charged and carbohydrate moieties (cationized ferritin, concanavalin A, ruthenium red, and alcian blue) than did other regions of the oocyte surface. This difference in surface properties of membrane delimiting the site of sperm-egg fusion was not due solely to incorporated sperm plasma membrane and did not occur when inseminated oocytes were incubated with cytochalasin B. Little or no difference in the membrane of the fertilization cone versus microvillous areas was observed when inseminated eggs were freeze-fractured or prepared with agents (filipin and polymixin B) to demonstrate β-hydroxysterols and anionic phospholipids. These observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.

Journal ArticleDOI
TL;DR: A simple rapid quantitative method has been developed for the estimation of sperm ecto-SH groups on the basis of their high affinity binding to the mercurial: [203Hg]p-chloromercuriphenylsulfonic acid (PCMPS) used as a surface probe.

Book ChapterDOI
01 Jan 1986
TL;DR: The exchange of information between a cell and its environment is frequently mediated by ionic movements through the plasma membrane, and the physiology of sea urchin sperm is a representative case in which this phenomenon occurs.
Abstract: The exchange of information between a cell and its environment is frequently mediated by ionic movements through the plasma membrane. The physiology of sea urchin sperm is a representative case in which this phenomenon occurs. For instance, when these cells are spawned into sea water, their motility and respiration activate (Ohtake, 1976; Nishioka and Cross, 1978; Christen et al., 1982). This activation is mostly dependent on the transport of Na+ and H+ through the plasma membrane (Nishioka and Cross, 1978). The sperm acrosome reaction, a prerequisite for egg fertilization, is also modulated by ionic fluxes (Schakmann et al., 1978). This reaction in sea urchin sperm consists of the exocytosis of the acrosomal vesicle located at the anterior region of the sperm head (Dan, 1952; Summers et al., 1975) and leads to the exposure of a protein required for sperm-egg binding (Vacquier and Moy, 1977; Glabe and Lennarz, 1979) and of lytic enzymes that digest the coat of the egg (Levine and Walsh, 1979; Green and Summers, 1980). Also during the process, the intracellular cyclic nucleotides increase (Garbers and Kopf, 1980), and an actin-containing acrosomal tubule is formed (Tilney et al., 1973).

Book ChapterDOI
01 Jan 1986
TL;DR: Development of a vaccine based on sperm antigens represents a promising approach to contraception and the feasibility of this approach is based upon two evidences: the studies of involuntary immunoinfertility in humans and the experimental immunization studies.
Abstract: Development of a vaccine based on sperm antigens represents a promising approach to contraception. The feasibility of this approach is based upon two evidences. The first type of evidence comes from the studies of involuntary immunoinfertility in humans. Presence of antisperm antibodies have been reported to be one of the major causes of immunoinfertility in both men and women.1 Second type of evidence is provided by the experimental immunization studies. Immunization of male and female animals of various species with extracts of sperm or mature testis results in a significant reduction in fertility by causing either fertilization failure or preimplantation embryo mortality or both. 2–5 The whole sperm cannot be used as an immunogen for the development of an antisperm vaccine due to the presence of numerous antigens on germ cells which could be shared with other somatic tissues. The utility of an antigen as an immunocontraceptive vaccine is contingent upon its tissue-specificity, its homogeneity, strong immunogenicity and its involvement in fertilization and fertility.

Book ChapterDOI
TL;DR: The sperm plasma membrane has been the object of intense research activity in recent years and a large amount of data has accumulated, particularly on its structure and biochemistry, that is now beginning to yield information that may be generally useful in cell biology.
Abstract: The sperm plasma membrane has been the object of intense research activity in recent years (Moore, 1985). As a consequence, a large amount of data has accumulated, particularly on its structure and biochemistry, that is now beginning to yield information that may be generally useful in cell biology. Of particular interest are the restricted structural domains that overlie specific regions of the cell (such as the acrosome, the mid-piece mitochondria, etc.).

Book ChapterDOI
TL;DR: P pH i is discussed as it is an important regulatory component of sperm motility and the acrosome reaction and methods available for measurement of pH i include uptake of radiolabeled weak acids and bases, 31 P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein that undergo spectral or fluorescent shifts with changes in pH i.
Abstract: Publisher Summary This chapter discusses methods used to measure ion changes important to activation of sea urchin sperm motility and the acrosome reaction. The methods used to measure pH i , ion fluxes, and membrane potentials in sea urchin sperm are described. Methods available for measurement of pH i include uptake of radiolabeled weak acids and bases, 31 P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein that undergo spectral or fluorescent shifts with changes in pH i . Simple ionic manipulation, regulation of pH i , and [Ca 2+ ] are of fundamental importance to sperm physiology. pH i is discussed as it is an important regulatory component of sperm motility and the acrosome reaction. pH i changes in sea urchin sperm correlate with or are linked with other cation movements also. In particular, a Na + /H + exchange is a component of the sperm plasma membrane that can either raise or lower the pH i . Induction of the acrosome reaction results in multiple changes in the sperm cation composition. Na + and Ca 2+ enter the sperm and K + (and H + ) exit. Of these Ca 2+ entry is a crucial requirement for the acrosome reaction regardless of the method used to initiate it.

Book ChapterDOI
01 Jan 1986
TL;DR: The “Acrosome Reaction” refers to a series of changes in the acrosome leading to its eventual loss, with the concomitant release of its content of hydrolytic enzymes and the exposure of a new limiting membrane over the anterior portion of the sperm head, both of which are essential for fertilisation.
Abstract: The “Acrosome Reaction” refers to a series of changes in the acrosome leading to its eventual loss, with the concomitant release of its content of hydrolytic enzymes and the exposure of a new limiting membrane over the anterior portion of the sperm head, both of which are essential for fertilisation (see Austin and Bavister, 1975; Austin, 1977; Bedford and Cooper, 1978). It proceeds by fusion of the sperm plasma membrane with the outer acrosomal membrane directly beneath it, giving rise to membrane vesicles which have been clearly shown in the boar to arise from two membranes with different structures (Russell et al., 1979a). The pores so created allow the escape of soluble enzymes (see Section II.D.9).