Seminal proteins binding to spermatozoa.
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Animals↗
Biomedical subjects
G Aumüller
Publications and source records attributed to G Aumüller.
Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa.
The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion ("major protein"). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and 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. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10-20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa.
Using commercial monoclonal antibodies against actin and tubulin (alpha and beta), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and - most intensely - in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail. Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.
Actins↗
The major protein of bull seminal plasma: biosynthesis and biological function.
We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle 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 Mr 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 (Esch et 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 physiological process presumably related to the onset of sperm motility.
Amino Acid Sequence↗
Distribution of vimentin-type intermediate filaments in Sertoli cells of the human testis, normal and pathologic.
The presence, distribution and spatial arrangement of vimentin-type intermediate filaments in Sertoli cells from human testis biopsies, were studied in semithin and ultrathin sections using a polyclonal rabbit antiserum. At the ultrastructural level, vimentin immunoreactivity was seen concentrated around the nuclei, along fibrillary material within the cytoplasm and at the ectoplasmic specializations of the Sertoli cell junctions, as well as throughout the periphery of the Sertoli cell processes. It is therefore well suited as a marker for Sertoli cell configuration. In computer-aided 3D reconstructions of 20 serial sections, Sertoli cells displayed particular configurations of intermediate filaments in the different stages of spermatogenesis. Two basic configurations, named AS (before spermiation, stages V, VI, I and II), and PS (after spermiation, stages III and IV) respectively, could be differentiated. In addition to the reconstruction and morphological analysis of vimentin filaments in Sertoli cells from patients with unaltered spermatogenesis (obstructive azoospermia), pathological specimens (spermatogenetic arrest, Sertoli cells only-syndrome) were studied with respect to vimentin immunohistochemistry. The results indicate that vimentin filaments play an important role in the adaptation of Sertoli cells to the varying configurations of neighbouring cells during spermatogenesis as well as under pathological conditions.
Adult↗
Immuno-electron microscopical localisation of alpha-actinin and actin in microvilli of prostatic epithelial cells.
The localisation of alpha-actinin and actin was investigated immunocytochemically in microvilli of rat ventral prostatic epithelial cells fixed by a solution containing 1.25% glutaraldehyde and 1% paraformaldehyde. Both proteins were localised in the microvilli by immunoperoxidase labelling of semithin or ultrathin sections. Almost the whole microvillous core region was labelled by antibodies for actin. Most of the label for alpha-actinin was confined to the region close to the plasma membrane along the length of the microvilli. In addition, positive staining was found at the microvillous tip and weak staining at a small area in the core. These results suggest a role of alpha-actinin in the stabilisation of actin filaments in these regions.
Actinin↗
Ultrastructure and immunohistochemistry of the lateral prostate in aged rats.
Ultrastructural, histological, and immunohistochemical studies were performed on lateral prostates of 1) aged rats from different strains, 2) rats permitted different levels of sexual activity, and 3) castrated rats. Antibodies against the following proteins were used as immunohistochemical markers: SVS II from seminal vesicle, LP 28 from lateral prostate, acid phosphatase isoenzymes from ventral prostate, transglutaminase from coagulating gland, and a commercial monoclonal antibody against cytokeratin. SVS II is a marker of lateral prostatic secretion, while immunoreactions to LP 28 and acid phosphatase (pI 7.1) were cytoplasmic. In aged animals the amount of intracellular secretion is decreased, and focally metaplastic transformation can be visualized by using immunohistochemical markers. Epithelial ultrastructure varied considerably with experimental conditions. Intensive sexual activity resulted in increased polymorphism and increased number of secretory granules within the glandular cells, while castration was followed by a rapid loss of secretory material. Also, in rats older than 10 months, a reduction in the number of secretion granules was common. The epithelium developed a positive immunoreaction to transglutaminase antibodies that were not observed in juvenile glands. Cells, presumably macrophages, which had an intense immunoreactivity for transglutaminase, were increased in number both within and outside the prostatic acini of aged rats. The possible interaction between secretory SVS II, a substrate of transglutaminase, the release of this enzyme from macrophages or its reflux from coagulating glands, the spontaneous cellular exfoliation that is due to decreased androgen levels, and dietary noxae may be of importance in the development of lateral prostatic nonbacterial inflammation in aged rats.
Acid Phosphatase↗
Cytochemistry and biochemistry of acid phosphatases VII: Immunohistochemistry of canine prostatic acid phosphatase.
Acid phosphatase (E.C. 3.1.3.2.) has been isolated from canine prostatic gland homogenates by gel permeation chromatography (AcA34 or G150), by affinity chromatography (con A-Sepharose), or by using fluid phase liquid chromatography (FPLC) using Superose 12 and Mono P columns. Acid phosphatase-enriched fractions were submitted to analytical SDS-PAGE or to analytical isoelectric focusing. A protein with a molecular weight of 30 kD (on SDS gels) was used for immunization of rabbits. The antiserum produced was cross-reactive with prostatic acid phosphatase (canine and human) as shown by immunoblotting. When applied to paraffin or plastic sections of normal canine prostate, a positive immunoreaction was found exclusively in the secretory cells. In experimentally altered glands (castration and/or hormone treatment), a varying pattern of immunoreactive cells was found. In canine prostatic carcinomas, intensively reacting cell clusters were found along with nonreactive cells. The antiserum was also slightly cross-reactive with the respective human antigen, but the cross-reactivity of an antiserum prepared against human prostatic secretory acid phosphatase with canine prostatic acid phosphatase was far more pronounced.
Acid Phosphatase↗
Pharmacologically induced ultrastructural and immunohistochemical changes in the prostate of the castrated dog.
The effects of an aromatase inhibitor and of an antiandrogen on the ultrastructure and the expression of a secretory protein (acid phosphatase) and marker proteins for basal cells (keratin) and fibroblasts (vimentin) were studied in the prostate of castrated, androstenedione-treated dogs. Androstenedione treatment partially restored the normal appearance of the gland and also some secretory activity. In the central portion of the gland, basal cell hyperplasia developed instead of secretory activity after androstenedione treatment. Administration of the aromatase inhibitor reduced the number of secretory cells but did not completely suppress the latter. There was some proliferation of the connective tissue surrounding the atrophic acini. Combined treatment with aromatase inhibitor and antiandrogen resulted in a general atrophy of prostatic acini that was less intense relative to the changes observed after castration. Residual secretory activity, detected in specimens treated exclusively with aromatase inhibitor, were lacking after combined treatment. The influence of all regimens on the ultrastructure of smooth muscle cells was comparably discrete, whereas regional differences in the arrangement pattern of the epithelium and the fibromuscular stroma were impressive. The ultrastructural findings support previous results of a synergic inhibitory effect of aromatase inhibitor and antiandrogens on the canine prostate.
Acid Phosphatase↗
Establishment, growth properties, and morphological characteristics of permanent human small cell lung cancer cell lines.
Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in establishing and permanently growing SCLC. Serum-free SIT medium and SIT2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R 10 medium was superior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.
Bone Marrow↗
Immunohistochemistry of secretory proteins in the bull seminal vesicle.
Antibodies against several proteins isolated from bovine seminal fluid (ribonucleases, bull seminal proteinase inhibitor BUSI II, seminal antimicrobial protein SAP) were used to identify the secretion sites of the respective proteins within the genital tract of the bull. Consistent positive immunoreactions were achieved with most of the antisera in the seminal vesicle epithelium, while only weak or dispersed immunoreactions were found in the epididymis, ductus deferens or the prostate. Comparison of serial sections of bull seminal vesicle stained with different antisera gave incongruous distribution patterns of positively reacting cells. This was interpreted as a sign of either a differential secretion cycle for different proteins in these cells or, more likely, a fixation or processing artifact. There was no clear cut evidence for secretion of seminal antimicrobial protein by seminal vesicle epithelium, but rather a resorption of intraluminally concentrated SAP. The secretion site of this important protein remains obscure, unless cRNA probes for in situ hybridisation studies are available.
Animals↗
[Immotile cilia syndrome].
The case of a patient with immotile cilia is reported. This syndrome is characterized by infertility caused by immotile spermatozoa and by bronchiectases owing to inadequate function of ciliated cells. Electron microscope studies showed gross abnormalities of the axial threads in the flagella of the spermatozoa examined.
Adult↗
Immunoelectron microscopic evidence for different compartments in the secretory vacuoles of the rat seminal vesicles.
Immunoelectron microscopy of the rat seminal vesicle was performed using specific antibodies to secretory proteins. Proteins were precipitated from rat seminal vesicle secretion and were separated by SDS-polyacrylamide gel electrophoresis. Among the great number of bands the two most prominent bands were selected and designated SVS II and IV. Their apparent molecular weights were 48 kDa and 16.5 kDa respectively. The bands were excised from the gels and used for antibody production in rabbits. The respective antisera were used for immunohistochemical studies both at the light and electron microscopic levels in the rat seminal vesicle and the different prostatic lobes in infantile, adult and castrated animals. A positive immunoreaction was observed in seminal vesicle and lateral prostatic epithelium of the intact adult rat, while it was lacking in prepubertal and castrated animals. The subcellular distribution of both proteins was clearly different: SVS II was exclusively confined to the electron dense core of the secretory vacuoles, while SVS IV was detected only in the clear halo surrounding the central granule. It is suggested that the spatial arrangement of both proteins in the seminal vesicle secretion vacuole reflects a particular functional significance of each of these proteins. These proteins may serve as a tool in the study of regulation of androgen-dependent protein synthesis.
Androgens↗
Functional characteristics of nuclear 5 alpha-reductase from rat ventral prostate.
The subcellular distribution and functional characteristics of 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3.1.22) from rat ventral prostate were studied and compared to the 5 alpha-reductase from female rat liver. Tissue fractionation retained main enzymic activity in the microsomal fraction of rat liver, while 5 alpha-reductase from rat prostate was localized in the nuclear membrane with a specific activity 160 times that of the initial homogenate. The purity of nuclear envelopes was checked by electron microscopy. Solubilization experiments indicated that the hepatic 5 alpha-reductase is attached to the endoplasmic reticulum as a peripheral protein, while the nuclear prostatic enzyme is an integral membrane protein. Incubation experiments with phospholipases suggested a decisive role of the surrounding phospholipids for the prostatic enzyme activity. To elucidate the characteristics of hydrogen transfer of the enzyme, the effect of flavins and different other cofactors on 5 alpha-reductase activity in isolated prostatic nuclei were studied. Our findings indicate that in rat ventral prostate the conversion of testosterone to 5 alpha-dihydrotestosterone proceeds by a direct hydrogen transfer from NADPH to testosterone. Concerning these parameters the behaviour of hepatic 5 alpha-reductase is absolutely different from the prostatic enzyme. The localization of 5 alpha-reductase within the nuclear envelope of rat ventral prostate as an integral membrane protein seems to be of physiological significance with regard to the action of androgens.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Immunohistochemical and ultrastructural study of Sertoli cells in androgen insensitivity.
The ultrastructure of a perfusion-fixed gonad in a case of androgen insensitivity was studied using thin sections and freeze-fracture replicas. The distribution and arrangement of intermediate filaments in Sertoli cells was visualized immunohistochemically using an antibody against vimentin. Leydig cells lacked Reinke crystals, but contained all of the cytoplasmic organelles involved in steroid synthesis and additionally several lysosomes. The basement membrane and the basal lamina of the testicular tubules were considerably thickened. The testicular tubules consisted of gonocytes and Sertoli cells which had an immature nuclear structure, incomplete development of intercellular junctions and a primitive distribution pattern of intermediate cytoplasmic filaments. The previously reported differences in electron density of Sertoli cell cytoplasm are a non-specific feature without significance to Sertoli cell maturation.
Adult↗
Biochemical and immunological relationships of prostatic and leukocytic acid phosphatases and their subcellular localization.
Human acid phosphatases from various sources were purified by a new rapid procedure including chromatofocusing and gel permeation chromatography on an FPLC system. Isoenzymes 2a and 4 (Mr 105,000 and 76,000, respectively) are the prevalent acid phosphatases in prostate, leukocytes and bone marrow. Additional forms are proteolytic products formed during isolation procedure. The source of elevated acid phosphatase in blood serum in metastasizing prostatic carcinoma can be either prostate itself, the tumour or, more likely, bone marrow cells destroyed during the invasion by the tumour cells. Distribution of isoenzymes is not organ specific. It was found using an antiserum against isoenzyme 4 that immunoreactivity is confined to the specific secretory granules both of prostatic epithelium and leukocytes.
Acid Phosphatase↗
Effects of sexual rest or sexual activity on the structure and function of the ventral prostate of the rat.
Previous studies have shown that sexual activity increases the weight of the accessory sex glands significantly, while the organ weights correlate inversely with the assayable androgen receptor concentrations in the prostate of sexually active versus sexually resting rats. In an effort to determine the structural basis of this phenomenon, the ventral prostates of adult rats kept with female rats for 4-6 months (HE-rats) were compared to those kept in groups of 5 males in one cage (HO-rats) for the same period. As an estimate of the secretory function of the gland the concentration of prostatic binding protein (PBP) was determined in prostatic cytosols using a highly specific ELISA. Catecholamines were measured by means of HPLC and subsequent electrochemical detection. Morphological studies included immunocytochemistry of PBP, visualization of adrenergic nerves, stereological analysis of stromal and glandular compartments of the prostate, and electron microscopy of the epithelium. The main findings were as follows: 1) The prostates of HE-rats were 35% heavier than those of the HO-rats. 2) The content in secretion was in the same range in both HE-rats and HO-rats (1.5 and 1.44 mg PBP per 1 mg DNA). 3) Immunocytochemistry and electron microscopy demonstrated a very homogeneous secretion within the glandular lumen of HO-rats with a diminished amount of secretory material within the glandular cells. In HE-rats the glandular lumina were clearly larger in diameter and intraluminal secretion was less homogeneously stained. The height of the epithelium was increased and the individual secretory cells contained several secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid Phosphatase↗
Cytochemistry and biochemistry of acid phosphatases V: Electrophoretic studies on the heterogeneity of acid phosphatases from human prostate, seminal fluid, and leukocytes.
Comparison of zymograms of acid phosphatases (orthophosphoric monoester phosphohydrolase, acid optimum, E.C.3.1.3.2) from human prostate, leukocytes, seminal vesicles, and seminal fluid, separated by analytical isoelectric focusing (IEF), resulted in the identification of three individual groups, particularly of the prostate. These groups contain three molecular forms at different molecular weights and activities in varying quantities and combinations. The molecular weights, estimated by SDS gel electrophoresis, were 1) 86,000, 2) 76,000, and 3) 46,000/50,000 (in a doublet) daltons. None of these isoenzymes were restricted to the prostate, but they were present in very high concentrations in the prostate. Compared to the prostate, the seminal vesicles and isolated leukocytes had very closely related zymograms of acid phosphatases. No specific inhibitor has been found that would selectively inhibit one particular isoenzyme without affecting the others. Protein titration and staining activity of IEF gels at different pH values showed that the isoenzymes from all three groups have high hydrolytic activity of orthophosphoric monoesters beyond the acidic pH range of pH 4-5. Incubation of acidic isoforms of acid phosphatases with neuraminidase did not result in the formation of a homogeneous stem molecule. Further analysis of the secretory moiety using the western blotting method showed a binding of peroxidase-conjugated concanavalin A (Con A), indicating that these isoenzymes are glycoproteins. Moreover, isoenzymes extracted from prostate, seminal fluid, and human leukocytes are immunologically identical.
Acid Phosphatase↗