Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.     

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.     

Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis

Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86–97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one-dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197–224, 1965]. Furthermore, two-dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (? M_r 57,000, pI 6.45) and to round spermatids (? M_r 39,500, pI 4.85), respectively.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Radio-iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells

Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1–8) have been iodinated using 1,2,3,6,tetracholoro-3α, 6α-diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two-dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25–30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN-catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.     

Human spermatogenic cell marker proteins detected by two-dimensional electrophoresis

Highly homogeneous populations of human pachytene spetmatocytes and round spermatids have been obtained from normal adult testis using unit gravity (STA-PUT) sedimentation. Contaminating Leydig cells have been removed by density centrifugation in discontinuous Percoll gradients to yield resultant germ cell purities of 90–95% for pachytene spermatocytes and 89–96% for round spermatids. The total cellular polypeptide composition of separated human germ cells has been analyzed by two-dimensional polyacrylamide gel electrophoresis to compare 1) human and mouse pachytene spermatocytes (species specificity), 2) samples of human spermatocytes obtained from different individuals (allo specificity), and 3) pachytene spermatocytes and round spermatids from the same patients (stage specificity). Mouse and human germ cells have been found to exhibit extensive homology, but identified marker proteins limited to human spermatocytes include a group of polypeptides at p45/5.9 as well as a protein at p67/5.2. Proteins unique to mouse germ cells include component p65/5.5. Comparisons between different preparations of human pachytene spermatocytes have revealed about 90% electrophoretic homology, but some striking allotypic variations have been noted including the proteins at p45/5.9. Finally, presumptive stage-specific spermatogenic cell markers have been identified including the p45/5.9 polypeptides that are present only in human spermatocytes. Although the physiological roles of particular marker proteins have not yet been determined, the present findings indicate that purified spermatogenic cell populations may be analyzed biochemically to identify constituents important in the regulation of sperm development in man.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Enrichment of primary pachytene spermatocytes from the human testis

Normal adult human testis has been separated using a combination of mechanical and enzymatic procedures to yield a suspension of viable single cells. The predominant cell types comprising this suspension are as follows: primary pachytene spermatocytes (7% of total cells), round spermatids (17%), residual bodies and condensing spermatids (31%), and Leydig cells (15%). Separated human germ cells viewed by Nomarski differential interference microscopy closely resemble mouse spermatogenic cells in relative size and appearance. Isolation of an enriched population of human pachytene spermatocytes has been achieved using unit gravity sedimentation (STA-PUT) according to protocols originally developed for murine tissue. Pachytene cells are enriched to 75% and are contaminated only with Leydig cells and binucleated spermatid symplasts. Ultrastructural examination of isolated human pachytene spermatocytes indicates that these cells, as well as isolated round spermatids, exhibit a normal in situ morphology. Spermatocytes, for example, show numerous synaptonemal complexes, nuclear pores, annulate lamellae, and dictyosome-like saccules. Round spermatids after isolation exhibit peripheral mitochondria, annulate lamellae, developing acrosomes, and other morphological features characteristic of early spermiogenesis. Therefore, enriched populations of human spermatogenic cells seem suitable for analysis using immunofluorescent, autoradiographic, or serological methods. In particular, isolated human spermatocytes should be useful for the analysis of molecular events involved in meiosis and should facilitate investigations concerning the pathophysiology of certain human infertility conditions.     

Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse

Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine [( 35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-10(6) cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constituents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.     

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

Human autoantibodies to spermatogenic antigens and Sertoli cells

Immunofluorescence staining using human autoantibodies is a simple and reliable method for investigation of meiotic and post-meiotic cells. Patients suffering from autoimmune diseases often produce circulating autoantibodies to antigens of germ cells and Sertoli cells. Four hundred human autoimmune sera were screened by indirect immunofluorescence on mouse seminiferous tubule cells. Autoantibodies of several specificities were found: one group reacted with organelles of meiotic prophase spermatocytes or spermatozoa. Included in this group were autoantibodies to synaptonemal complexes, sex vesicle, acrosome, and sperm tail. A second group of autoantibodies was found to stain different spermatogenic cell types uniformly, such as round spermatids or Sertoli cells.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Expression of heat shock proteins by isolated mouse spermatogenic cells.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.