F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.     

Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.     

Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis.

The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.     

Mouse sperm protein sp56 is a component of the acrosomal matrix

Previously, we identified the guinea pig sperm acrosomal matrix glycoprotein AM67 and demonstrated that it is most closely related to mouse sperm sp56, initially reported to be a cell-surface protein. On the contrary, our studies demonstrated that sp56 is an intra-acrosomal component. Based upon the homology between guinea pig AM67 and mouse sp56, we hypothesized that sp56 was part of the acrosomal matrix, a structure that had yet to be demonstrated to exist in mouse sperm. In this paper, we show that sp56 first appeared in late meiotic cells and accumulated during spermiogenesis, the haploid stage of spermatogenic cell development. Using affinity-purified anti-peptide antisera, we determined that the molecular weight of sp56 in cauda epididymal sperm approximated that of guinea pig AM67 ( approximately 67 000 M:(r)) and that sp56 was present in a high molecular weight, disulfide-linked complex. The forms of sp56 in pachytene spermatocytes and spermatids had higher molecular weights than was found for the sperm form; the size differences were apparently due to alterations in carbohydrate side chains. The sp56 complex could not be solubilized by the nonionic detergent Triton X-100 but remained associated with the dorsal surface of the mouse sperm head, demonstrating that sp56 is a component of the mouse sperm acrosomal matrix.     

Developmental expression of G protein alpha subunits in mouse spermatogenic cells: evidence that G alpha i is associated with the developing acrosome.

Guanine nucleotide-binding proteins (G proteins) are important signal transducing molecules found in all cells. G proteins are associated with the plasma membrane/outer acrosomal membrane region of acrosome-intact sperm and at least one G protein is involved in the zona pellucida-induced acrosome reaction. With the goal of elucidating the functions of these proteins during spermatogenesis, we investigated the types of G proteins present in spermatogenic cells and when they first become associated with the developing acrosome. Using bacterial toxin-catalyzed [32P]ADP-ribosylation in conjunction with immunoprecipitation and immunofluorescence utilizing antibodies directed against specific regions of various G protein isotypes, the alpha subunits of Gi1, Gi2, Gi3, and G(o) were detected in mouse spermatocytes and spermatids. An antiserum recognizing a conserved sequence of G alpha i subtypes localized to the proacrosomal granules of spermatocytes and the developing acrosome of spermatids. Levels of G alpha o diminished as spermatocytes developed into spermatids such that G alpha o was not detected in cauda epididymal sperm. Immunoreactivity using G alpha o-specific antisera did not display a distinct regionalization within any of the spermatogenic cell types. G alpha s was not detected in the developing spermatogenic cells or sperm. The association of G alpha i with the developing acrosome suggests a role for G proteins may have a role in acrosome biogenesis as well as being part of a complex required later for signal transduction leading to acrosomal exocytosis.     

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

Characterization of fucosyltransferase activity during mouse spermatogenesis: evidence for a cell surface fucosyltransferase

Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltransferase activity of mixed germ cells displayed an apparent Vmax of 17 pmol (mg of protein)-1 min-1 and an apparent Km of approximately 13 microM for GDP-L-[14C]fucose in the presence of saturating amounts of asialofetuin at 33 degrees C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization.     

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.     

Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents.

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.     

Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine-conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin-stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis-derived material were lectin-positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea-pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin-stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.     

Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.     

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.     

Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.     

Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha-ras-activated oncogene.

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.     

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.