Showing papers on "Sperm plasma membrane published in 1990"

Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa

Abstract: Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red). The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.

cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals

Abstract: Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.

Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity.

Abstract: Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: Role in the initiation of sperm motility

Abstract: The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions {sup 3}H-tetraphenylphosphonium (TPP) and {sup 14}C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with (K+) greater than 20-40 mMmore » or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.« less

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction.

Abstract: Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.

Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry

Abstract: Mammalian sperm plasma membranes, in contrast to those of mammalian somatic cells, exhibit a significant fraction of lipid that does not diffuse laterally in the plane of the membrane. This nondiffusing fraction results from lipid-lipid interactions. Similar nondiffusing fractions are found in mixed-lipid model systems that contain coexistent gel and fluid domains. These results suggest that the sperm plasma membrane may also exhibit lateral phase segregations of lipids and may contain significant amounts of gel-phase lipid. In this paper we use differential scanning calorimetry to show that, in contrast to the plasma membranes of mammalian somatic cells, the plasma membrane from the anterior region of the head of ram sperm exhibits at least two major endothermic transitions, one centered at approximately 26 degrees C and one centered at approximately 60 degrees C. The heats of these transitions are consistent with gel-to-fluid transitions in model membranes. These transitions are observed both in plasma membrane vesicles and in rehydrated lipid extracts made from these vesicles. These results demonstrate that at physiological temperatures the lipids of the ram sperm plasma membrane are segregated into coexistent fluid and gel domains. Since sperm encounter a wide range of temperatures during their development, these phase transitions may be important in establishing dynamic domains of lipid requisite for epididymal storage and fertilization.

Proteins of seminal fluid and spermatozoa in the trout (Oncorhynchus mykiss): partial characterization and variations.

Abstract: The protein composition of seminal fluid, blood serum, sperm plasma membrane and flagellum of rainbow trout were analysed by SDS-polyacrylamide gel electrophoresis. Immunological identity between proteins of the 2 fluids and sperm components was studied using crossed immunoelectrophoresis, rocket immunoelectrophoresis and immunoblotting. Results indicate that many seminal proteins are antigenically-related to serum proteins, proteins of sperm origin are present in seminal fluid in varying amounts, depending on the animals and sampling time, and several serum-like seminal proteins are bound to spermatozoa.Lipoproteins were isolated from seminal fluid (mean level: 33 μg/ml) and characterized. They were identified as being HDL-like lipoproteins. A possible physiological role is proposed for these seminal lipoproteins.

Ca2+ channels from the sea urchin sperm plasma membrane.

Abstract: Ca2+ influx across the sea urchin sperm plasma membrane is a necessary step during the egg jelly-induced acrosome reaction. There is pharmacological evidence for the involvement of Ca2+ channels in this influx, but their presence has not been directly demonstrated because of the small size of this cell. Sea urchin sperm Ca2+ channels are being studied by fusing isolated plasma membranes into planar lipid bilayers. With this strategy, a Ca2+ channel has been detected with the following characteristics: (a) the channel exhibits a high mainstate conductance (gamma MS) of 172 pS in 50 mM CaCl2 solutions with voltage-dependent decaying to smaller conductance states at negative Em; (b) the channel is blocked by millimolar concentrations of Cd2+, Co2+, and La3+, which also inhibit the egg jelly-induced acrosome reaction; (c) the gamma MS conductance sequence for the tested divalent cations is the following: Ba2+ greater than Sr2+ greater than Ca2+; and (d) the channel discriminates poorly for divalent over monovalent cations (PCa/PNa = 5.9). The sperm Ca2+ channel gamma MS rectifies in symmetrical 10 mM CaCl2, having a maximal slope conductance value of 94 pS at +100 mV applied to the cis side of the bilayer. Under these conditions, a different single-channel activity of lesser conductance became apparent above the gamma MS current at positive membrane potentials. Also in 10 mM Ca2+ solutions, Mg2+ permeates through the main channel when added to the cis side with a PCa/PMg = 2.9, while it blocks when added to the trans side. In 50 mM Ca2+ solutions, the gamma MS open probability has values of 1.0 at voltages more positive than -40 mV and decreases at more negatives potentials, following a Boltzmann function with an E0.5 = -72 mV and an apparent gating charge value of 3.9. These results describe a novel Ca2(+)-selective channel, and suggest that the main channel works as a single multipore assembly.

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

Abstract: The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.

Induction of the Acrosome Reaction in Starfish

Abstract: When starfish spermatozoa reach the jelly coat of homologous eggs, they immediately undergo the acrosome reaction that is a prerequisite for fertilization. Three organic components of the egg jelly are responsible for this phenomenon; namely, an extremely large sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroid saponins named Co-ARIS, and a sperm activating oligopeptide(s) (SAP). ARIS induces the acrosome reaction species-specifically in high Ca2+ or high pH sea water, but it requires Co-ARIS for the induction in normal sea water. The acrosome reaction induced by ARIS and Co-ARIS is not accompanied with a transient increase in the intracellular pH (pHi), which is generally accepted to be inevitable for triggering the acrosome reaction, and proceeds slower than the jelly-induced acrosome reaction. All the three, but nothing else, are required to reconstruct the acrosome reaction-inducing ability of the egg jelly.

Boar sperm membranes antigens. II. Reorganization of an integral membrane antigen during capacitation and acrosome reaction.

Abstract: The dynamics of the cell surface during the process of capacitation is impressively shown by means of a monoclonal antibody directed against the P86/5 antigen. This glycoprotein was located in the sperm plasma membrane using the colloidal gold method in combination with specimen preparation in toto. The antigen is absent at the rostral tip of non-capacitated spermatozoa, but forms clusters over the principal segment and the equatorial segment after induction of capacitation. This formation of microdomains with different properties may be a prerequisite for the onset of the acrosome reaction (AR). During AR the diffusion barrier for the P86/5 antigen breakes down and the antigen occupies now the rostral crescent-like area of the sperm head. These observations are discussed with respect to zona binding and induction of the AR in boar spermatozoa.

Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa.

Abstract: Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.

Changes in the fluidity of the goat sperm plasma membrane in transit from caput to cauda epididymis

Abstract: Highly purified maturing plasma membranes of goat caput-, corpus- and cauda-epididymal spermatozoa, were isolated by an aqueous two-phase polymer method and their fluidity was determined using pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH) as the membrane lipid probes Pyrene following partitioning into the lipid phase of the membrane formed excimers at 480 nm and the amount of excimers formed was markedly higher with the immature caput- than the mature cauda-sperm membrane The fluorescence polarization values obtained with DPH as the lipophilic probe, were lowest in the immature sperm membrane and highest in that of the mature sperm The data with both the probes are consistent with the view that the lipid phase fluidity of the sperm plasma membrane undergoes significant decrease during the epididymal maturation of the male gametes

The Sperm Plasma Membrane A Little More Than Mosaic, a Little Less Than Fluid

Abstract: In this chapter the dynamics of molecular motions in the mammalian sperm plasma membrane will be discussed. Specifically, we will consider the nature of the known or potential forces which control these molecular motions. We will only discuss macromolecules that have been identified as having some physiological role and have been characterized on intact cells using biophysical techniques to gain insight into the organization of the sperm plasma membrane. Information on other cell-surface macromolecules, especially on cilia and flagella, is available in a number of articles (e.g., Brooks, 1985; Vernon et al, 1985, 1987; Olson et al, 1987; Feuchter et al, 1988; Trimmer and Vacquier, 1988).

Boar sperm membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell membrane.

Abstract: A monoclonal antibody, designated mAb P86/5, was generated by immunization of female Balb/c mice with a membrane vesicle fraction composed of the outer acrosomal membrane and plasma membrane (PM-OAM). As determined by fluorescence microscopy and electron microscopy P86/5 recognizes a sperm plasma membrane antigen that is restricted to the sperm head. In intact spermatozoa the P86/5-antigen is distributed over the surface of the sperm head with the exception of the rostral region. By comparing the antibody binding pattern generated at 4 degrees C and 25 degrees C, it could be shown that the P86/5-antigen is capable to diffuse freely within the cell membrane overlying the acrosome whereas its lateral mobility is restricted to the post-acrosomal region. The P86/5-antigen had a molecular weight of about 78 kDa as revealed by SDS-PAGE and western blotting. The glycoprotein nature of the P86/5-antigen was established by lectin affinity chromatography.

Structure and Function of Egg-Associated Peptides of Sea Urchins

Abstract: The sea urchin egg is surrounded by a gelatinous extracellular matrix called the jelly coat through which spermatozoa must pass before reaching the plasma membrane of the egg during fertilization (Fig.1). Lillie (1913) observed that the jelly coat, dissolved in sea water, is able to cause a transitory activation of sperm motility and sperm agglutination. Thereafter, many investigators have shown that soluble factors associated with sea urchin eggs stimulate the motility and respiration of sea urchin spermatozoa (Carter, 1931; Cohn, 1918; Gray, 1928; Rothschild, 1956a, 1956b). Hathaway (1963) reported that the factors are diffusible in dialysis, heat-stable, alcohol-soluble and non-volatile. Ohtake (1976a, 1976b) demonstrated that the respiration of sea urchin spermatozoa could be reproducibly stimulated by partially purified egg jelly factors obtained from the sea urchin Pseudocentrotus depressus if extracellular pH was maintained at acidic values. Kopf et al (1979) and Hansbrough and Garbers (1981a) then found that the egg jelly of the sea urchin Strongylocentrotus purpuratus contains a factor which elevates the respiratory rate and cyclic AMP and cyclic GMP concentrations of S. purpuratus spermatozoa. Suzuki and co-workers (1981) have purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus a decapeptide able to stimulate sperm respiration whose sequence is Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly.

Which is the best test to evaluate the integrity of sperm plasma membrane

Abstract: The functional integrity of sperm plasma membrane of 87 semen samples collected from fertile and infertile men was assessed by the hypoosmotic swelling test (HOS). The results were correlated with sperm motility and supravital stains using trypan blue and eosin Y. These data indicate that sperm swelling and staining methods evaluate different properties of spermatozoa. Moreover the present report shows that staining percentages with trypan blue are significantly lower than with eosin Y.

Gamete Interactions and the Initiation of Egg Activation in Sea Urchins

Abstract: The earliest perceivable response of echinoid eggs to the fertilizing sperm is a transient depolarization, the activation or fertilization potential (Steinhardt et al., 1971; Jaffe, 1976; Chambers and de Armendi, 1979). This change in electrical activity involves the appearance of sperm associated ion channels, which depolarize the plasma membrane, as well as the opening of voltage-dependent calcium channels (Chambers and de Armendi, 1979). These changes constitute Phase 1 (Lynn et al., 1988; Chambers, 1989). In voltage clamped eggs, sperm which induce Phase 1 either enter or fail to enter the egg. If sperm penetration occurs, the inward current of Phase 1, initiating Phase 2, continues to increase; if sperm penetration fails to occur the inward current is abruptly severed. During Phase 2 a large, rapid and transient increase in intracellular free calcium (Steinhardt and Epel, 1974; Steinhardt et al., 1977; Whitaker and Steinhardt, 1982; Jaffe, 1983) propagates in the form of a wave from the point of gamete interaction to the opposite pole of the egg (Eisen et al., 1984; Swan and Whitaker, 1986; Yoshimoto et al., 1987). This wave of increased intracellular calcium is initiated following a latent period (Phase 1) of approximately 12 sec and stimulates the egg from its quiescent state to proliferation and embryogenesis (Chambers, 1989).

Maturation-specific type II cyclic AMP-dependent protein kinase in goat sperm plasma membrane.

Abstract: Highly purified plasma membranes (PMs) isolated by aqueous two-phase polymer methods from goat sperm undergoing epididymal maturation, have been analyzed for the isoenzymes of cyclic AMP-dependent protein kinase (RC). The mature and the immature spermatozoa showed profound differences in the profile of the isoenzymes of RC solubilized from the isolated PMs with 0.1% Triton X-100. The immature sperm PM consists of only type I RC in contrast to the mature sperm membrane which possesses both the type I and II isoenzymes. The type II kinase represents nearly 30% of the total membrane-bound RC of the mature cells. The analysis of the surface topography of these isoenzymes of the maturing spermatozoa by using diazonium salt of sulfanilic acid as the surface probe shows that the PM-bound RC(s) are oriented primarily on the external surface of these intact cells. The data demonstrate that type II RC is a maturation-specific ecto-kinase as it appears on the sperm surface specifically during the maturation of spermatozoa in the epididymis.

Isolation and partial characterization of sperm plasma membrane of masu salmon, Oncorhynchus masou

Abstract: 1. 1. Spermatozoa of masu salmon Oncorhynchus masou were disrupted by ultrasonication and their plasma membranes were isolated by discontinuous sucrose gradient centrifugation 2. 2. One band recovered on the top of 27% sucrose was identified as the sperm plasma membrane fraction by electron microscopy and enzyme analyses. 3. 3. Lipid analyses by TLC revealed the absence of neutral glycolipids in the sperm plasma membrane.