Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.     

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa

The rabbit sperm membrane autoantigen RSA-1 is a sialoglycoprotein of 13,000 daltons which first appears on the surface of pachytene spermatocytes. Using specific antiserum to RSA-1 the antigen has been localized by immunofluorescence and immunoperoxidase staining. On testicular cells labeled at 37°C, RSA-1 is seen in patches on the surfaces of pachytene spermatocytes, round spermatids, and over the acrosomal area of later spermatids and spermatozoa. Over the postacrosomal and middle-piece regions of late spermatids and spermatozoa the labeling appears uniform. The uniformity can be seen to stop abruptly at the equatorial segment-postacrosomal border. Labeling cells after fixation gives a uniform distribution of label over the surface where patches were seen at 37°C. The polypeptides recognized by the antiserum used for labeling were identified by immunoadsorbent chromatography and subsequent SDS-PAGE. In testicular cells anti-RSA-1 recognizes the 13,000-dalton form and another component which migrates with the dye front. In ejaculated spermatozoa anti-RSA-1 recognizes a distinct ejaculate complex of higher-molecular-weight proteins containing an 84,000-dalton major band and five minor components.     

Identification of surface autoantigens which appear during spermatogenesis

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS-PAGE. To identify cell-surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo-Gen iodination procedure and run on SDS-PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface-labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface-labeled, staged cell samples were lysed in Triton X-100 and immunoprecipitated with antitestis cell autoantisera. Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.     

Ontogenesis of Spermatozoa Autoantigens in Guinea Pigs

The ontogenetic appearance of three independant spermatozoa autoantigens (S, P and T) has been studied in guinea pig germinal cells by immunofluorescence and comparison with cytology and histological structures during early maturation of seminiferous tubule cells. The maturation of 120 testes from 60 guinea pigs studied from day 1 to day 50 after birth has shown an evolution in 3 periods. During the first, or negative, period (day 1 to day 25), only spermatogonia (from day 1) and spermatocytes I (from day 16) are present. No significant PAS-positive formations are seen and no autoantigen is detected. During the second, or transitional, period (day 26 to 29), spermatocytes II and spermatids appear as well as paranuclear PAS-positive golgian proacrosomal and acrosomal granules. At the same time, the three autoantigens S, P and T are detected on the same PAS-positive formations with a frequency that increases from day to day. During the third, or positive, period (from day 30) all testes present cells with PAS-positive formation, progressive maturation of acrosomes in spermatids and appearance of spermatozoa (present on day 39) leading to the adult structure of seminiferous tubules. The three autoantigens are constantly present during that period. The simultaneous appearance of the 3 antigens in haploid germinal cells (spermatids and possibly spermatocytes II) as an early expression of cytodifferentiation and their total absence from diploid germinal cells (spermatogonia and spermatocytes I) seem to be of biological significance.     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G₁ and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.     

Acrosin and the acrosome in human spermatogenesis

Using the indirect immunofluorescent staining technique, the developmental patterns of (pro) acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.     

Spermiogenesis in the spider,Pisaurina Sp.: A fine structure study

A fine structure study of spermatids and spermatozoa of the spider, Pisaurina sp. demonstrates that early spermiogenesis is similar to other flagellate spermatozoa. An acrosome forms from a Golgi-derived, acrosomal vesicle, a perforatorium indents acromosome and nucleus, a flagellum with a three-plus-nine tubule substructure is formed and nuclear chromatin condenses during spermiogenesis. Divergence from typical spermatozoa includes the presence of a three-tubule substructure of the central flagellar shaft, progressive rounding-up of late spermatids with concomitant incorporation of previously formed flagellum. This evidence is presented in terms of its possible functional significance in fertilization and gamete fusion in spiders.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA).

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.     

ELECTRON MICROSCOPE STUDIES ON SPERM DIFFERENTIATION IN MARINE ANNELID WORMS. I. SPERM FORMATION IN IKEDOSOMA GOGOSHIMENSE

The ultrastructur of spermatozoa and the changes through which they are differentiated during sperm formation in an echiuroid were observed under the electron microscope. Many spermatids are connected to one central cytoplasmic mass and the sperm differentiation proceeds synchronously in one sperm-ball. Dense plate-like structures appear in the cytoplasm of early spermatids and disappear soon. In the process of nuclear condensation, many electron-dense aggregates appear in homogeneously textured chromonema and the aggregates are packed together to form a uniformly dense nucleus. Near the centriole at the opposite side from the central mass, the mitochondria fuse together to form one large middle-piece mitochondrion and the acrosomal vesicle is formed from the Golgi-complex. The differentiating acrosome in the late spermatid moves to the anterior tip of the head. In the completed acrosome, a flocculent substance accumulates in the conspicuously expanded invaginated pocket of the acrosomal vesicle and two kinds of material of different electron density fill the inside of the acrosomal vesicle. The spermatozoa remain connected to the central mass at the lateral side of the head until they become fully mature and are packed into the nephridia before spawning.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats

Summary The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with -elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Characterization of boar sperm cytoskeletal cylicin II as an actin-binding protein

The presence of actin-binding proteins in the perinuclear theca of boar spermatozoa has been investigated, using stepwise extractions of proteins from sperm heads. Proteins extracted with the alkaline buffer 1M Na(2)CO(3), pH 11, were found to contain a 66kDa protein that binds F-actin in actin pelleting assays. Sequence studies and immunological characterization with antibodies specific for human cylicin II identified the 66kDa protein as the homologue of bovine and human cylicin II. Immunocytochemical studies showed the presence of porcine cylicin II in the acrosomal region of round spermatids and in the postacrosomal region of late spermatids and spermatozoa, in agreement with the previously described localization of cylicins. Taken together, the results suggest that cylicin II, a protein of the sperm perinuclear cytoskeleton, is a novel actin-binding protein, which probably plays a role in the actin-related events that occur during spermiogenesis and the early events of fertilization.     

Ultrastructure of testes and spermatogenesis in the trombiculid mite,Hirsutiella zachvatkini (Schluger)

Summary

Spermatogenesis and sperm ultrastructure of the trombiculid mite Hirsutiella zachvatkini (Schluger 1948) have been investigated using transmission electron microscopy and compared with other arachnids studied. Sperm differentiation takes place in groups of synchronously developed germ cells of the two large sac-like paired testes. Each testis is composed of a secretory epithelium, which occupies their medio-ventral regions, and of a germinative epithelium situated in the latero-dorsal parts of testes together with large somatic cells. The germ cells are represented on sections by spermatogonia, spermatocytes, early, middle and late spermatids, and mature spermatozoa. Spermatocytes and spermatids contain two centrioles, which disappear afterwards, and a small Golgi-like structure forming an acrosomal cistema. Mature spermatozoa, which lie both within the meshes of somatic cells and also free in the lumen of testes, are compact oval aflagellate cells provided with peripheral channels. They also contain an acrosome, flattened between the cell membrane and the round electron-dense chromatin body, an oval body of lesser density lying in close proximity to the chromatin body, and a group of 5–7 mitochondria with spherically arranged cristae situated immediately behind the nuclear bodies. An acrosomal filament may be sometimes seen beneath the acrosome in the middle spermatids and disappears in the mature spermatozoa. These findings show that the mode of differentiation and pattern of organization of the male sex cells in trombiculid mites are of rather primitive type compared with other acarine spermatozoa.     

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.     

Intra-acrosomal organization of a 90-kilodalton antigen during spermiogenesis in the rat

The localization of an acrosomal protein was studied using a monoclonal antibody MN7 raised against mouse spermatozoa. MN7 specifically recognized the anterior acrosome of several mammalian (mouse, rat, hamster) spermatozoa fixed with paraformaldehyde. An immunoblot study with periodate treatment showed that MN7 recognized a carbohydrate region of a 90 kDa protein in an extract of mouse and rat cauda epididymal spermatozoa. The change in distribution of the MN7 antigen during acrosome development was investigated in the rat testis using the pre-embedding immunoperoxidase technique. The antigen first appeared in the proacrosomic granules of spermatids in steps 1–2. Small vesicles adjacent to the outer acrosomal membrane and the developing acrosomic system were immunoreactive during steps 4–7. The majority of the antigen was then redistributed to the head-cap portion during steps 8–18, and finally restricted to the anterior acrosome in the step 19-spermatid. These results suggest that the antigen is transported to the acrosome by way of the vesicles that originate from the Golgi apparatus during early spermiogenesis, and are then delivered to the final destination within the acrosome by the intra-acrosomal migration during late spermiogenesis.     

A testis antigen related to the brain D2 adhesion protein

The localization of an antigen immunochemically crossreactive with the rat brain interneuronal adhesion molecule, D2-protein, has in testis been demonstrated by immunoelectrophoresis and immunocytochemistry. This D2-like antigen is localized to spermatids and residual bodies in testis, but it is absent both from spermatogonia and mature spermatozoa and from Sertoli cells. By immunoperoxidase electronmicroscopy D2-like antigen was observed in the head region posterior to the acrosomal membrane of late spermatids. In the nervous system D2-protein is involved in interneuronal adhesion. It is suggested that D2-like antigen in testis may be involved in a similar adhesion between late spermatids and Sertoli cells during spermiation.     

Membrane trafficking machinery components associated with the mammalian acrosome during spermiogenesis.

Active trafficking from the Golgi apparatus is involved in acrosome formation, both by delivering acrosomal contents to the nascent secretory vesicle and by controlling organelle growth and shaping. During murine spermiogenesis, Golgi antigens (giantin, beta-COP, golgin 97, mannosidase II) are detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgi-acrosome flow may be relatively unselective, with Golgi residents retrieved before spermiation is complete. Treatment of spermatogenic cells with brefeldin A, a drug that causes the Golgi apparatus to collapse into the endoplasmic reticulum, disrupted the Golgi in both pachytene spermatocytes and round spermatids. However, this treatment did not affect the acrosomal granule, and some beta-COP labeling on the acrosome of elongating spermatids was maintained. Additionally, N-ethylmaleimide sensitive factor, soluble NSF attachment proteins, and homologues of the t-SNARE syntaxin and of the v-SNARE VAMP/synaptobrevin, as well as members of the rab family of small GTPases, are associated with the acrosome (but not the acrosomal granule) in round and elongated spermatids. This suggests that rab proteins and the SNARE machinery for membrane recognition/docking/fusion may be involved in trafficking during mammalian acrosome biogenesis.