An electron microscope comparison of plasma membrane vesicles from meristematic and mature soybean root tissue

Plasma membrane vesicles were isolated from homogenates of meristematic and mature soybean root tissue by differential sucrose gradient centrifugation. Vesicles were positively identified by the phosphotungstic acid-chromic acid procedure (PACP). The two preparations were comparable in size class distribution, mitochondrial contamination, and per cent plasma membrane vesicles present. Purity levels were estimated to be greater than 75%. The specificity of PACP was observed for a variety of cell types from both regions. Some variability in PACP staining was offset by careful modulation of the stain protocol and was found to be independent of developmental stage in subcellular fractions. Patchy or discontinuous staining, observed in both intact tissue and in subcellular fractions from both regions, was found to be a function of stain time.     

Dictyosome polarity and membrane differentiation in outer cap cells of the maize root tip

Outer rootcap cells of maize produce large numbers of secretory vesicles that ultimately fuse with the plasma membrane to discharge their product from the cell. As a result of the fusion, these vesicles contribute large quantities of membrane to the cell surface. In the present study, this phenomenon has been investigated using sections stained with phosphotungstic acid at low pH (PACP), a procedure in plant cells that specifically stains the plasma membrane. In the maize root tip, the PACP also stains the membranes of the secretory vesicles derived from Golgi apparatus to about the same density that it stains the plasma membrane. Additionally, the membranes of the secretory vesicles acquire the staining characteristic while still attached to the Golgi apparatus. The staining progresses across the dictyosome from the forming to the maturing pole, thus confirming the marked polarity of these dictyosomes. Interestingly, the PACP staining of Golgi apparatus is confined to the membranes of the secretory vesicles. It is largely absent from the central plates or peripheral tubules and provides an unambiguous example of lateral differentiation of membranes orthogonal to the major polarity axis. In the cytoplasm we could find no vesicles other than secretory vesicles bearing polysaccharide that were PACP positive. Even the occasional coated vesicle seen in the vicinity of the Golgi apparatus did not stain. Thus, if exocytotic vesicles are present in the maize root cap cell, they are formed in a manner where the PACP-staining constituent is not retained by the internalized membrane. The findings confirm dictyosome polarity in the maize root cap, provide evidence for membrane differentiation both across and at right angles to the major polarity axis, and suggest that endocytotic vesicles, if present, exclude the PACP-staining component.     

A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes

Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K⁺. In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K⁺. The level of K⁺-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca²⁺, high ATP concentration in the presence of low Mg²⁺, and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane-enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots.     

Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K⁺-stimulated, vanadate-inhibited Mg²⁺ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

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

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

Effects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membranes and mitochondria to striped bass (Morone saxatilis) sperm

Intact plasma membrane and functional mitochondria are important attributes for the fertilization capacity of fish sperm. In the present study, dimethyl sulfoxide (Me(2)SO) and glycine were investigated in an effort to improve plasma membrane integrity and mitochondrial function in cryopreserved striped bass (Morone saxatilis) sperm. Prior to freezing, no concentration of Me(2)SO (2.5, 5 or 10%) was found to affect (P>0.05) the integrity of plasma membranes after sperm were exposed for 10 min. However, mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. Both fluorescent staining and microscopic examination of the ultrastructure of post-thaw plasma membranes indicated that with increasing Me(2)SO concentration, plasma membranes were better protected, and 10% Me(2)SO had the highest percentage of sperm with plasma membranes intact. However, sperm mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. The inverse relationship between plasma membrane integrity and mitochondrial function, given the Me(2)SO concentration, suggests that care must be taken to select Me(2)SO concentration that will maximize the protection of both plasma membranes and mitochondrial function. The addition of glycine to the cryomedia increased (P<0.05) the percentage of sperm with post-thaw functional mitochondria and ATP content. However glycine did not provide (P<0.05) protection to post-thaw plasma membrane integrity. The highest percentage of sperm with both intact plasma membranes and functional mitochondria was obtained with 7.5% Me(2)SO and 75 mM glycine.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

Critical Staining of Pancreatic Alpha Granules with Phosphotungstic Acid Hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO₄ acidified with 0.3% (w/v) H₂SO₄, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.     

两相法分离地被菊叶片质膜的研究

以地被菊(Dendranthema×grandiflorum Kitamura)叶片为材料,通过水溶性聚合物Dextran T-500和PEG 3350所构成的两相分配体系制备质膜。在一定盐浓度（5 mmol.L-1 NaCl）下选用5种不同的聚合物浓度（5.8%、6.0%、6.2%、6.4%、6.6%,W/W）,研究了地被菊叶片质膜在两相体系中的分配情况,在此基础上进一步研究了不同盐浓度（2、4、5、10、20 mmol.L-1 NaCl）对地被菊叶片质膜的纯度及蛋白产率的影响。标志酶鉴定及磷钨酸染色电镜检测的结果表明,地被菊叶片选用6.2%（W/W）聚合物浓度和7 mmol.L-1 NaCl组成的两相分配体系可获得较高纯度的密实正向型质膜囊泡。     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

Identification of homologous binding proteins in porcine and bovine gametes

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.     

A cytochemical investigation of the host-parasite interface inPisum sativum infected by the downy mildew fungusPeronospora pisi

Summary A cytochemical study involving enzymic digestions, chemical extractions and specific staining methods was made of the host-parasite interface in downy mildew infected pea plants. Enzymic hydrolysis revealed both the penetration and extrahaustorial matrices to have a proteinaceous component, possibly glycoprotein, while the extrahaustorial matrix had cellulose as an additional constituent. Intense silver proteinate staining of both matrices following a prolonged incubation in thiocarbohydrazide indicated the presence of complex carbohydrates. Carbohydrates were confirmed as constituents of both matrices following the complete suppression of silver staining using the aldehyde blocking agents dimedone and sodium borohydride. Both matrices also exhibited a marked affinity for phosphotungstic acid. Following acetylation a complete elimination of phosphotungstate staining resulted, again indicating carbohydrate as a constituent of both matrices. Digestion of the fungal cell wall using an enzyme which hydrolyses -1,3-glucans showed that these carbohydrates are important in its construction. However such enzyme treatment did not affect the structural integrity of either the penetration or extrahaustorial matrix. The extrahaustorial membrane exhibited negligible staining with phosphotungstic-chromic acid treatment, while the host plasmalemma showed a positive but variable staining reaction. A very intense staining reaction characterized the fungal plasmalemma after staining with either phosphotungstic-chromic acid, phosphotungstic acid or silver proteinate.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Membrane histochemistry of Physarum polycephalum myxamoebae

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.