Plasma-membrane glycoproteins during spermiogenesis and in the spermatozoa of some Orthoptera

Summary Orthoptera spermatids and spermatozoa from two species of Tettigoniidae and from one of Acrididae were analysed by means of the fluorescent lectins, concanavalin A or wheat-germ agglutinin, with the aim of finding -D mannose· -D glucose· N-acetylglucosamine and sialic acid sugar residues in their plasmamembrane glycoproteins. Labelling with lectins shows remarkable changes occurring in the plasma membrane during spermiogenesis. In early spermatids, the whole cell surface is labelled, but in mature spermatids and spermatozoa, a noticeable fluorescence is restricted to the membrane that covers the acrosome. The end piece of the tail of Acrididae spermatids and spermatozoa fluoresces after wheat-germ agglutinin labelling. The intense labelling of acrosomal area is independent of acrosomal size and shape, as shown by the marked differences observed in the acrosomes of Tettigoniidae compared with Acrididae: in the former, the acrosome is a well-developed structure with an arrow-like shape, but in Acrididae, the acrosome resembles a small vesicle in the anterior tip of the cell. The large amount of some sugar residues in the plasma membrane covering the acrosome is discussed in relation to the features observed in other species, and also in connection with the physiology of the male gamete prior or 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.     

Structure, cytoskeleton, and development of the acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae).

The acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae) is relatively large and complex, consisting of an apical vesicle and two large wing-like extensions that give the spermatozoon the shape of an arrow. The wings have actin microfilaments and microtubules and are covered with a noticeable extracellular material. Actin filaments are present in the acrosome when it first appears in spermatid stages. The acrosome and the acrosomal attachment to the nucleus are more resistant than other structures to the reducing agents DTT and SDS. At the end of spermiogenesis, groups of spermatozoa juxtapose their sperm heads and become joined to form a spermatodesm encircled by an amorphous material. Treatment with the ionophore A23187 rapidly disrupted acrosomes of the free gametes, but acrosomes from spermatozoa contained in the spermatodesm were not disassembled. Packaging of sperm in a spermatodesm appears to protect the acrosome.     

New data on aberrant spermatozoa in the ejaculate of Sus domesticus

This paper describes 16 new types of aberrant spermatozoa observed by scanning electron microscopy in the ejaculate of two healthy, sexually mature Landrace boars. The new anomalies observed were 1) spermatozoa with folded tail and abnormal head; 2) tailless spermatozoa with an abnormal connecting piece; 3) immature spermatozoa with two tails of the same length, fused and coiled; 4) spermatozoa with two tails of the same length, fused and coiled, and a small, rounded head; 5) spermatozoa with two fused tails and a wide head; 6) spermatozoa with three tails of the same length, fused and coiled; 7) immature spermatozoa with two heads and two fused tails; 8) spermatozoa with two heads, one at each tip of the tail; 9) spermatozoa with a short, folded tail and a triangular head; 10) spermatozoa with a short tail lacking the intermediate piece; 11) spermatozoa with a short tail, without the main piece and with a long intermediate piece; 12) spermatozoa with a short tail, without the main piece and with a rough head; 13) spermatozoa with small, rounded head; 14) spermatozoa with small, aberrant heads; 15) spermatozoa with small, bacillary heads; and 16) immature spermatozoa with tapering heads.     

Ultrastructure of the domestic tom cat (Felis domestica ) and tiger (Panthera tigris altaica ) spermatozoa

The ultrastructure of spermatozoa from the domestic tom cat and the Siberian tiger was studied. Semen was collected from anesthetized tom cats and Siberian tigers by electroejaculation. Spermatozoa were fixed and processed for examination by transmission electron microscopy. The principle differences between the spermatozoa from the two species were the head shape, mitochondrial organization in the neck area and structure of the fibrous sheath. Tom cat spermatozoa had an elongated oval-shaped head, while tiger spermatozoa had a more rounded head shape. Circularly oriented mitochondria in the neck area, near the proximal centriole, were frequently observed in tiger cells but rarely observed in tom cat cells. The semicircular ribs of tom cat spermatozoa were larger than the ribs of tiger spermatozoa. Also, the dense fibers (Numbers 3 and 8) of the corresponding microtubule doublets were fused or connected to the longitudinal columns in tiger spermatozoa but showed only occasional attachment in tom cat spermatozoa. These differences could influence results when the tom cat is used as a model for studying tiger semen.     

Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.     

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.     

Silver-staining patterns of mammalian epididymal spermatozoa

Epididymal spermatozoa from 12 species of mammals were stained using silver nitrate and examined with the light microscope. Silver nitrate differentiates many of the gross morphological features of spermatozoa, including the acrosome, subacrosomal region, perforatorium, postacrosomal sheath, neck, dense outer fibers of the core of the midpiece, annulus, principal piece, and end piece. Silver-staining patterns of spermatozoa reveal both species-specific and strain-specific differences, particularly of the sperm head. The biochemical basis of silver staining may be due in part to the presence of sulfhydryl- and disulfide-rich proteins; however, it cannot be explained entirely by the presence of these moieties. The detail obtained using silver nitrate staining, coupled with the ease and rapidity of the procedure, should be useful to workers in many areas of biological and medical research.     

Fine structure of male genital tracts of some Acrididae and Tettigoniidae (Insecta: Orthoptera)

A morphological, histological and ultrastructural study was carried out on the spermiducts and seminal vesicles of some species of Acrididae and Tettigoniidae. In all the species examined, the spermiducts and seminal vesicles have a monolayered secretory epithelium. Only the species of Acrididae have the sac with a flattened epithelium. Furthermore, in the most distal tubule region of the seminal vesicles of Eyprepocnemis plorans plorans, a rather characteristic secretory mechanism was found: the cytoplasm of the epithelial cells contained a large vesicle delimited by tightly packed microvilli. Numerous small vesicles open into this large vesicle which gradually dilates to merge with the apical plasma membrane releasing its contents into the lumen. Spermiophagic activity was found in all the species investigated. In the Tettigoniidae, this activity was found only in some epithelial cells of the seminal vesicle wall; in the species of the Acrididae the spermiophagic activity was carried out in the spermiduct lumen by an epithelial‐type cellular group. Spermiophagic activity is discussed as well as its role in the reproduction of these insects.     

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.     

Protein constituents of the mouse spermatozoon: I. An electrophoretic characterization

Total protein constituents of the mouse spermatozoon have been fractionated and characterized by polyacrylamide gel electrophoresis. Three spermatozoan fractions were obtained following homogenization with 1% sodium dodecylsulfate (SDS) and sucrose gradient centrifugation: SDS-soluble proteins, SDS-insoluble tail components, and SDS-insoluble head components. Purities of these fractions were assessed at greater than 95% using Nomarksi differential interference microscopy. Subsequently, the SDS-insoluble sperm heads were further fractionated into five protein subclasses by ultracentrifugation and ion-exchange chromatography. SDS-Polyacrylamide gel electrophoresis indicates that each of these spermatozoan fractions contains distinct protein species. Furthermore, the electrophoretic profiles are highly reproducible and show no evidence of cross-contamination or proteolysis. The SDS-soluble fraction, which includes proteins from the plasma membrane, acrosome, axoneme, matrix and cristae of the mitochondria, contains one major 39,000-molecular weight band and numerous minor bands with molecular weights ranging from ～30,000 to greater than 100,000. In contrast, electrophoresis of the SDS-insoluble tail proteins reveals the presence of at least nine prominent bands with apparent molecular weights between 21,000 and 89,000. Ultrastructural analysis suggests that this fraction contains proteins from the outer dense fibers, fibrous sheath, outer mitochondrial membranes, and structural elements of the neck region of the sperm tail. Two subfractions of the SDS-insoluble sperm heads each contain one of the two mouse protamines. In addition, the acidic and moderately basic head fractions each contain a limited number of distinct protein bands with molecular weights ranging from 14,000 to 76,000. These proteins are apparently derived from either the spermatozoan nucleus or the associated perinuclear material, since all other sperm head structures are solubilized during SDS treatment. One- and two-dimensional electrophoresis on acetic acid-urea polyacrylamide gels indicates that the moderately basic fraction may contain minor components that resemble certain histones and/or spermatidal basic nuclear proteins.     

中国蟾蜍属精子形态比较

采用扫描电镜和光学显微镜观察了采自我国的蟾蜍属(Bufo)7种(亚种)的精子形态,对精子各部位量度进行了测量和计算。结果表明,该属7种(亚种)精子的形态基本相同,精子由头部、中片和尾部组成,头部细长微弯且前端渐尖,中片有球状突起,尾部长,由轴纤维、轴丝和波动膜构成。与已有报道的两栖动物的精子形态相比较,蟾蜍属精子与无尾类其他科精子形态差别较大,而与有尾类精子形态相似。本文认为两栖动物精子形态和量度在科间存在明显差异;两栖动物精子形态的差异可能与其繁殖模式有关。     

Head and flagella subcompartmental proteomic analysis of human spermatozoa

Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross‐linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS‐PAGE, 1 mm slices were digested and used for LC‐MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.     

The major protein of bull seminal plasma: Biosynthesis and biological function

We isolated the major protein of apparent M_r of 15,000–16,000 from seminal plasma as well as from seminal veiscle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively.Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent M_r of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Eschet al., Biochem. Biophys. Res. Comm. 113:861–867, 1983).The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP.Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a phyisological process presumably related to the onset of sperm motility.     

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.     

蓝尾石龙子精子的超微结构

蓝尾石龙子(Eumeces elegans)附睾以2．5％戊二醛和1％锇酸双重固定,按常规制作超薄切片,用H-600透射电镜研究观察精子的超微结构。精子由头部和尾组成,头部由顶体复合体和核组成,尾由颈段、中段、主段和末段组成。头部的顶体囊前部扁平,分为皮质和髓质,顶体下锥由类结晶状的顶体下物质组成,穿孔器顶端尖,、穿孔器基板塞子状,细胞核延长,核内小管缺,核伸展部前端具一电子透明区,核肩圆,核陷窝锥形。颈段具片层结构,近端中心粒和远端中心粒的长轴呈直角,9束外周致密纤维与远端中心粒相应的9束三联微管相联,向后与轴丝相应的9束双联微管相联,中央纤维与2个中央单微管相联。中段短,含有线状嵴的柱状线粒体,由连续的规则小卵状或小梯形致密体组成线粒体间的环状结构,纤维鞘伸入中段,终环紧贴于细胞膜的内表面。线粒体与环状结构的模式为：rs1／mi1,rs2／mi2,rs3／mi3,rs4／mi4,横切面上每圈线粒体数目为10个。主段前面部分具薄的细胞质颗粒区。纤维3和8至主段前端消失。轴丝复合体呈“9 2”型。蓝尾石龙子精子超微结构与已描述的石龙子科种类比较发现,与蜓蜥群和胎生群的石龙子相似;但没有发现石龙子科精子的独征。     

Comparative morphology of primate spermatozoa using scanning electron microscopy. I. Families Hominidae,Pongidae, Cercopithecidae and Cebidae

Spermatozoa from 16 species of primates, representing Families Cercopithecidae Cebidae, Pongidae, and Hominidae, were viewed using scanning electron microscopy (SEM). Various techniques of specimen preparation were employed, especially critical point drying using CO₂, but also critical point drying using N₂O as well as air drying using amyl acetate. Details of ultrastructure and identifying features of the various families are presented, with both relationships and differences being evident. Accompanying light micrographs of several of the species illustrate size relationships of the head, midpiece, and principal piece of the tail. Dimensions of these parts of spermatozoa were calculated from such photographs, and add to the interpretation of overall morphology. SEM is of considerable value in understanding many details of structure in spermatozoa, and should contribute to the study of taxonomic affinities among the various primate groups.     

A comparative study of microspines in the alimentary canal of five families of Orthoptera (Saltatoria)

Microspines in the alimentary tract in five families of grasshoppers and crickets (Orthoptera: Saltatoria) were studied to determine their structures and correlate them with patterns of distribution for potential use in taxonomy and future physiological studies. Short microspines were found in all sections of the foregut of each family, with the caeliferan Acrididae being the most unique, and the ensiferan families forming another group with similarities. Among the Ensifera, the Gryllacrididae and Gryllidae were the most distinctive, especially Stenopelmatus. The hindgut demonstrated another set of relationships, although less distinctive, with the Acrididae, Tettigoniidae, and Gryllacrididae appearing to be one grouping, whereas the Gryllidae and Gryllotalpidae formed another group with similarities.     

Scanning electron microscopic study of spermatozoa from gossypol-treated rats

Summary Spermatozoa from the cauda epididymidis of gossypol-treated rats exhibit distinctive departures from the morphology of spermatozoa from control rats: wrinkled and disorganized cell membrane in the head and tail regions, cell membrane missing from segments of the tail midpiece and principal piece regions, malformed heads, decapitate spermatozoa, retention of a cytoplasmic droplet at variable loci along tail midpieces, and looped tails. The observations suggest that gossypol exerts its contraceptive effect during spermatocytogenesis and spermiogenesis, including the posttesticular development and maturation of spermatozoa in the epididymis.