Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization

We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT-1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the acrosome reaction. These rearrangements of cell surface molecules may act to regulate cell surface function during fertilization.     

Distinct cytoskeletal domains revealed in sperm cells

Antibodies against different cytoskeletal proteins were used to study the cytoskeletal organization of human spermatozoa. A positive staining with actin antibodies was seen in both the acrosomal cap region and the principal piece region of the tail. However, no staining was obtained with nitrobenzoxadiazol-phallacidin, suggesting that most of the actin was in the nonpolymerized form. Most of the myosin immunoreactivity was confirmed to a narrow band in the neck region of spermatozoa. Tubulin was located to the entire tail, whereas vimentin was only seen in a discrete band-like structure encircling the sperm head, apparently coinciding with the equatorial segment region. Surface staining of the spermatozoa with fluorochrome-coupled Helix pomatia agglutinin revealed a similar band-like structure that co-distributed with the vimentin- specific staining. Instead, other lectin conjugates used labeled either the acrosomal cap region (peanut and soybean agglutinins), both the acrosomal cap and the postacrosomal region of the head (concanavalin A), or the whole sperm cell surface membrane (wheat germ and lens culinaris agglutinins and ricinus communis agglutinin l). In lectin blotting experiments, the Helix pomatia agglutinin-binding was assigned to a 80,000-mol-wt polypeptide which, together with vimentin, also resisted treatment with Triton X-100. Only the acrosomal cap and the principal piece of the tail were decorated with rabbit and hydridoma antibodies against an immunoanalogue of erythrocyte alpha-spectrin (p230). p230 appeared to be the major calmodulin-binding polypeptide in spermatozoa, as shown by a direct overlay assay of electrophoretic blots of spermatozoa with 125I-calmodulin. The results indicate that spermatozoa have a highly specialized cytoskeletal organization and that the distribution of actin, spectrin, and vimentin can be correlated with distinct surface specializations of the sperm cells. This suggest that cytoskeleton may regulate the maintenance of these surface assemblies and, hence, affect the spermatozoan function.     

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the 1A4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase. The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment. It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.     

Regional heterogeneity of human spermatozoa detected with monoclonal antibodies

The regional antigenic heterogeneity of human spermatozoa is confirmed with 6 monoclonal antibodies raised against ejaculated human spermatozoa. The topographical localization of the antigenic determinants suggests the existence of at least 6 domains on the human spermatozoon. Different fixatives had severe detrimental effects on the antigen-antibody binding. On live human spermatozoa, each antibody bound to a distinct region: acrosome, equatorial segment, entire tail, neck, midpiece and terminal piece. The antigens detected on the acrosome, equatorial segment and entire tail were surface components, whereas the other three were intracellular structures. The determinant present along the entire tail was a sperm-coating antigen. The molecular weights of the recognized antigens were estimated with the Western blot technique. Immunostaining of individual ejaculates established that the percentages of positive cells were 12-56% for the acrosome, 8-35% for the equatorial segment, 90-100% for the entire tail, 20-52% for the neck, 9-35% for the midpiece and 36-90% for the terminal piece. In addition, labelling of motile and immotile spermatozoa showed differences in the percentages of positive cells, with 5 out of 6 monoclonal antibodies, or in the fluorescence intensity, with the last one labelling the entire tail.     

Hamster sperm cross react with antiactin.

Hamster sperm extracts contain a polypeptide which comigrates with muscle action on polyacrylamide gel electrophoresis. On double diffusion precipitation plates, sperm extracts form a single precipitin band with an antibody to muscle actin (antiactin) and show a reaction of identity with muscle actin. Indirect immunofluorescent microscopy revealed that antiactin binds along the concave margin and equatorial segment in the acrosomal region, in the connecting piece of the neck, and in the principal piece of the tail. These results are evidence that hamster sperm contain actin. The possible significance of these observations in fertilization is considered.     

Immunogold localization of the regulatory subunit of a type II cAMP- dependent protein kinase tightly associated with mammalian sperm flagella

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.     

Monoclonal antibodies to bull sperm surface antigens

Four monoclonal antibodies were generated during the present investigation. Mice were immunized with washed ejaculated bull spermatozoa. Spleen cells from the immunized mice were fused with myeloma cells (SP2/0) and four different hybridoma clones were obtained, producing specific monoclonal antibodies. These antibodies were designated as SP₂A₅, SP₁A₂, SP₁C₄ and SP₁C₆ respectively. All belonged to the IgG sub-class 1. The specificity of these monoclonal antibodies was tested using both ELISA (enzyme-linked immunosorbent assay) and indirect immunofluorescence staining techniques. Quantitative estimation of antibody-antigen reaction was done by optical density measurements. Ejaculated bull spermatozoa were always reactive for each ELISA procedure. Other test cells including spleen, testicular, ovarian, uterine and pancreatic cells from both bull and rabbit were non-reactive. Bull testicular spermatozoa were also non-reactive. However, seminal fluid (without sperm) was reactive. All four monoclonal antibodies were reactive to the midpiece of the ejaculated bull spermatozoa. In addition, SP₂A₅ was also reactive to the acrosomal area and SP₁A₂ was reactive to the acrosomal and the post-acrosomal area. Cytoplasmic droplets of ejaculated spermatozoa from bull and human were also possibly reactive to one antibody (SP₁C₆). The results clearly suggest the heterogeneous nature of the sperm cell plasma membrane and precise molecular alteration in the plasma membrane components (antigens) as the sperm cells differentiate and mature during their transit through the epididymis.     

Mitochondrial sheath movement and detachment in mammalian,but not nonmammalian,sperm induced by disulfide bond reduction

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe Mito Tracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 mM Mito-Tracker. Exposure of bull sperm to 0.1–10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization. Mol. Reprod. Dev. 47:79–86, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of an 80-kilodalton bull sperm protein identified as PH-20

This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60(src) from the Rous Sarcoma Virus When subjected to two-dimensional electrophoresis, this 80-kDa protein migrated as several isoforms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequence analysis of a peptide obtained following trypsin digestion of the bull sperm protein showed homology to the PH-20/hyaluronidase precursor sperm protein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased immunolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react lost their labeling almost completely. As for the PH-20 protein, the 80-kDa bull sperm protein possesses a hyaluronidase activity that is higher at pH 7.0 than at pH 4.0 in an in-gel assay. Unlike what has been observed in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C or with trypsin. However, this protein was not sedimented by a 100,000 x g centrifugation after nitrogen cavitation, which suggests that the 80-kDa protein is loosely attached to the sperm membrane by a yet-unknown mechanism. These results suggest that the 80-kDa bull sperm protein shares many homologies with the sperm PH-20 protein reported in the literature and, most likely, is the bull sperm homologue of the PH-20.     

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.     

Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.     

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.     

The tip of the coiled-coil rod determines the filament formation of smooth muscle and nonmuscle myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.     

Differential distribution of tubulin epitopes in human spermatozoa

Two monoclonal antibodies (16 D3 and 24 E3) were used to map tubulin domains in human spermatozoa by indirect immunofluorescence. Their specificity to tubulin in these cells was established by Western blotting. Whereas 16 D3 uniformly stained the principal piece of the flagellum, the staining provided by 24 E3 decreased along the tail to become very weak 30 micron further away from the midpiece. This latter antibody also reacted with the proximal centriole as well as the midpiece, but not all spermatozoa stained identically at this level indicating heterogeneity within the population of sperm cells from a given donor. 16 D3 reacted weakly with the head, and the staining was interrupted after a bright spot in the neck. The study of a pathological case (the short tail spermatozoon) with an abnormal arrangement of dense fibers was consistent with a correlation between the distribution of the epitope defined by 24 E3 and that of peri-axenomal structures. The existence of tubulin domains interacting with these structures is postulated.     

Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.     

Cytoskeletal proteins F-actin and β-dystrobrevin are altered by the cryopreservation process in bull sperm

The cryopreservation process has an important impact on sperm structure and physiology. The negative effects have been mainly observed on the plasma membrane, which is directly stabilized by the cytoskeleton. Since cytoskeleton proteins are osmosensitive and thermosensitive, the aim of this study was to evaluate the damage caused to the bull sperm cytoskeleton by cryopreservation (freezing-thawing). Fresh and frozen-thawed bull semen samples were exposed to a treatment with the neutral detergent Brij 36-T. Electron microscopy evidenced important damages at the sperm perinuclear theca after the protein extraction protocol; the perinuclear theca was partially solubilized, the perinuclear theca substructure disappeared in the cryopreserved samples. Furthermore, the sperm head's shape was significantly altered on the cryopreserved samples. Fluorescence analysis showed a decrease of the intensity of actin and dystrobrevin on the frozen-thawed samples. Western blot assays revealed a stronger signal for actin and β-dystrobrevin in the frozen-thawed sperm samples than in the fresh ones. Our results suggest that the cryopreservation process highly alters the sperm cytoskeleton stability, causing its proteins to become more fragile and therefore more susceptible to be extracted.     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.