DNA organization in the mature sperm of several orthoptera by the method of polarized fluorescence microscopy.

Mature sperm of Acheta domesticus, Acheta assimilis, Nemobius fasciatus, Nemobius confusus, Melanoplus femur-rubrum, Romalea microptera, Scudderia curvicauda, and Ceuthophilus nigricans were examined for DNA configuration by means of polarized fluorescence microscopy. In all cases, the results suggest that the DNA lies in unsupercoiled array, predominantly parallel to the elongate sperm head axes. Detailed calculations of the factors influencing the degree of polarization of the fluorescent emission from supercoiled DNA are contained in an appendix.

Isolation of a protein subunit from microtubules.

Sea-urchin sperm tails (Strongylocentrotus purpuratus) were obtained by amputation in synthetic sea water and were purified by differential centrifugation. Most of the arms of the outer nine doublets and soluble matrix proteins were removed by this treatment. The central pairs of microtubules were dissolved by dialysis against EDTA at pH 7.5. The extract contained essentially a single component, with a sedimentation constant of 6S, in amounts sufficient to account for the protein content of the central pairs. Incubation of the extract with colchicine-(3)H gave binding levels approaching 0.5-1.0 mole of colchicine per 10(5) g protein. Sucrose-gradient analysis showed that the bound-radioactivity profile coincided with the optical-density profile of the 6S protein. It is concluded that the 6S colchicine-binding protein is a subunit of microtubules.

[Identification and cytochemical study of an extranuclear basic protein in the spermatozoa of decapod crustaceans]. / Mise en evidence et etude cytochimique d'une proteine basique extranucleaire dans les spermatozoides des crustaces decapodes.

Extranuclear basic proteins have been detected in the capsule of the spermatozoa of three species of decapod crustaceans (Nephrops norvegicus L., Macrura; Eupagurus bernhardus L., Anomura; Carcinus maenas Penn., Brachyura). Their properties have been studied by cytochemical methods. Their position inside the capsule of the spermatozoon has been specified with the aid of the electron microscope. Present in a constant fashion in the three species cited, their relative importance is very variable. In contrast to the refringent cone of the spermatozoon of Ascaris, which contains an acid protein, ascaradine, the capsule of the spermatozoon of the three decapod crustaceans studied contains basic proteins which we propose to designate by the general term "decapodine".

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.

Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

Apparent anomalies in nuclear feulgen-DNA contents. Role of systematic microdensitometric errors.

The Feulgen-DNA contents of human leukocytes, sperm, and oral squames were investigated by scanning and integrating microdensitometry, both with and without correction for residual distribution error and glare. Maximally stained sperm had absorbances which at lambdamax exceeded the measuring range of the Vickers M86 microdensitometer; this potential source of error could be avoided either by using shorter hydrolysis times or by measuring at an off-peak wavelength. Small but statistically significant apparent differences between leukocyte types were found in uncorrected but not fully corrected measurements, and some apparent differences disappeared when only one of the residual instrumental errors was eliminated. In uncorrected measurements, the apparent Feulgen-DNA content of maximally stained polymorphs measured at lambdamax was significantly lower than that of squames, while in all experimental series uncorrected measurements showed apparent diploid:haploid ratios significantly greater than two. In fully corrected measurements no significant differences were found between leukocytes and squames, and in four independent estimations the lowest diploid:haploid ratio found was 1.99 +/- 0.05, and the highest 2.03 +/- 0.05. Discrepancies found in uncorrected measurements could be correlated with morphology of the nuclei concerned. Glare particularly affected measurements of relatively compact nuclei such as those of sperm, polymorphs and lymphocytes, while residual distribution error was especially marked with nuclei having a high perimeter:area ratio (e.g. sperm and polymorphs). Uncorrected instrumental errors, especially residual distribution error and glare, probably account for at least some of the previously reported apparent differences between the Feulgen-DNA contents of different cell types. On the basis of our experimental evidence, and a consideration of the published work of others, it appears that within the rather narrow limits of random experimental error there seems little or no reason to postulate either genuine differences in the amounts of DNA present in the cells studied, or nonstoichiometry of a correctly performed Feulgen reaction.

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens.

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm.

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin-phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.

Localization of sulfatoxygalactosylacylalkylglycerol at the surface of rat testicular germinal cells by immunocytochemical techniques: pH dependence of a nonimmunological reaction between immunoglobulin and germinal cells.

The synthesis of sulfatoxygalactosylacylalkylglycerol (SGG) is a marker of germinal cell differentiation during spermatogenesis. Antibodies raised against this lipid have been used to visualize SGG on the surfaces of rat spermatocytes and spermatids. An ionic interaction between SGG and immunoglobulin was shown to occur at physiological pH, resulting in high fluorescence backgrounds for control cells treated with nonimmune sera. Immunofluorescence was therefore performed at alkaline pH such that this interaction was much reduced or eliminated. A method was also developed to detect surface-bound complement fixed in the presence of anti-SGG. SGG was found to be mobile within the plane of the membrane, undergoing ligand-induced "patching" and occasional "capping." However, this phenomenon was independent of temperature.

Immunochemical studies of 22S protein from isolated mitotic apparatus.

Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules.

The localization of glycogen in the spermatozoa of various invertebrate and vertebrate species.

With the periodic acid-thiosemicarbazide-silver proteinate procedure for the detection of polysaccharides in thin sections, glycogen is localized in the cavities of centrioles and basal bodies, within the axoneme (and surrounding it), in mitochondria, and in the "packing" cytoplasm of the middle piece of spermatozoa of several invertebrate and vertebrate species. The cytochemical localization of glycogen is verified by extraction with alpha-amylase solution. These findings establish the existence of stored glycogen in sperm. The polysaccharide presumably serves as an endogenous source of energy in the absence of extracellular metabolites, under either aerobic or anaerobic conditions. Other hypotheses on the physiological significance of intracellular glycogen stores in sperm are discussed. Sperm that store glycogen contain some enzymes of glycogen metabolism. In the presence of glucose-1-phosphate, ATP, and Mg(++) ions, an amylophosphorylase catalyzes the in vivo synthesis of glycogen. The newly formed product resembles gamma-particles, and is digestible with alpha-amylase.

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins.

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.

Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor: purification and localization on surfaces of spermatogenic cells and sperm.

We have found that the rat testis contains a cell surface galactosyl receptor that is antigenically related to the minor species of rat liver asialoglycoprotein receptor (ASGP-r) and has binding affinity for galactose coupled to agarose. In immunoblotting experiments, rat testis galactosyl receptor (RTG-r) is recognized by antiserum raised against the minor ASGP-r species of rat liver (designated rat hepatic lectin-2/3, RHL-2/3). Antiserum raised against the major species RHL-1 does not recognize an antigenic protein equivalent to RTG-r. Triton X-100-extracted rat liver and testes preparations fractionated by affinity chromatography on galactose-agarose and resolved by SDS-PAGE under reducing conditions, show that rat liver contains both the major (RHL-1) and minor (RHL-2/3) ASGP-r species whereas rat testis displays only a receptor species comigrating with RHL-2/3. RTG-r was present throughout testicular development. The receptor was found in seminiferous tubules, cultured Sertoli and spermatogenic cells, and epididymal sperm. Indirect immunofluorescent studies show RHL-2/3-like immunoreactivity on the surface of Sertoli cell, meiotic prophase spermatocytes, spermatids, and epididymal sperm. In spermatids and sperm, the immunoreactivity is restricted to the plasma membrane overlying the dorsal portion of the head. Because of RTG-r has galactose binding affinity, is present on surfaces of Sertoli and developing meiotic and postmeiotic spermatogenic cells, and overlies a region of the intact acrosome on epididymal sperm, RTG-r may have a role in spermatogenesis and in events leading to sperm-egg recognition.

Three-dimensional reconstruction of an actin bundle.

We present the three-dimensional structure of an actin filament bundle from the sperm of Limulus. The bundle is a motile structure which by changing its twist, converts from a coiled to an extended form. The bundle is composed of actin plus two auxiliary proteins of molecular masses 50 and 60 kD. Fraying the bundle with potassium thiocyanate created three classes of filaments: actin, actin plus the 60-kD protein, and actin plus both the auxiliary proteins. We examined these filaments by transmission electron microscopy and scanning transmission electron microscopy (STEM). Three-dimensional reconstructions from electron micrographs allowed us to visualize the actin subunit and the 60- and 50-kD subunits bound to it. The actin subunit appears to be bilobed with dimensions 70 X 40 X 35 A. The inner lobe of the actin subunit, located at 20 A radius, is a prolate ellipsoid, 50 X 25 A; the outer actin lobe, at 30 A radius, is a 35-A-diam spheroid. Attached to the inner lobe of actin is the 60-kD protein, an oblate spheroid, 55 X 40 A, at 50 A radius. The armlike 50-kD protein, at 55 A radius, links the 60-kD protein on one of actin's twin strands to the outer lobe of the actin subunit on the opposite strand. We speculate that the 60-kD protein may be a bundling protein and that the 50-kD protein may be responsible for the change in twist of the filaments which causes extension of the bundle.

Detection of complex carbohydrates in the Golgi apparatus of rat cells.

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.

Lack of dynein arms in immotile human spermatozoa.

Sermatozoa from two brothers who are not twins were found to be straight and immotile. Examinations of the sperm showed that oxygen consumption and lactic acid production were normal; viability tests showed that the percentage of dead sperm was not increased. The ultrastructural appearance of the sperm tail was normal except for a complete lack of dynein arms and some irregularities in the arrangement of the accessory fibers and the longitudinal columns of the fibrous sheath. The mitochondrial apparatus and the sperm head conform to the conventional model. According to the sliding-filament hypothesis first proposed by Afzelius (1959. J. Biophys. Biochem. Cytol. 5:269.), the arms are responsible for the bending movements of the tail. The simplest explanation for the simultaneous lack of arms and sperm motility appears to be that the two brothers have a genetic disorder involving production, assembly, or attachment of the dynein arms.

Mouse sperm basic nuclear protein. Electrophoretic characterization and fate after fertilization.

Mouse sperm were labeled in vivo with [3H]arginine. The sperm were then followed autoradiographically from the time of label incorporation until after fertilization. The label was completely lost from the sperm head after fertilization, during the oocyte's second meiotic division. That the [3H]arginine was incorporated into a sperm-specific basic protein was demonstrated by fractionating acid extracts of epididymal and ejaculated sperm with polyacrylamide gel electrophoresis. All the histone fractions were resolved in the epididymal extracts, but in addition a band was present that migrated faster than histone F2al and slower than the salmon protamine used as a marker. This new fraction (proposed name: musculine) was also present in ejaculated sperm; it was shown to be the only fraction that was labeled. Musculine therefore represents the end product of a histone transition in mice. It is, however, according to our electrophoretic characterization, not identical to the classical fish protamines. Rather, musculine resembles bovine sperm nuclear protein. Since the loss of this fraction from the sperm head was coincident with the rearrangement of the male genome, before its resumption of transcription, it is suggested that musculine is involved in the control of chromatin that accompanies spermiogenesis and fertilization.

Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis.

Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [3H]4 and sodium metaperiodate/NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa greater than 90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt X proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.