Jürgen Seitz

Protein Secretion and Secretory Processes in Male Accessory Sex Glands

Publisher Summary This chapter discusses the secretory proteins of the prostate and the seminal vesicles with particular emphasis on the human glands. The male accessory sex glands vary considerably in mammals with respect to their topographical location, size, morphology, and functions, thus reflecting that the diversity of species-specific requirements of these glands for reproduction is due to differences in environment and sexual habits. Secretions from highly developed and specialized accessory sex glands—for example, in rodents, are responsible for the formation of the so called “copulatory plug.” The important groups of proteins characterized are the transglutaminases, the calcium transport modifiers, and the growth factors. Transglutaminase is an enzyme that triggers multifarious biological responses, such as receptor stabilization or polymerization of seminal secretory proteins during the formation of the copulatory plug in rodents, and in immune defense mechanisms. This enzyme is an anchor protein and is released in an apocrine secretion mode from the dorsal prostate and the coagulating gland and may serve as a tool in scrutinizing the special mode of secretion.

Immunohistochemistry of secretory transglutaminase from rodent prostate

SummaryTransglutaminases are Ca2+-dependent intra-and extracellular enzymes catalyzing the cross-linking between proteins and/or polyamines, thereby eliciting divergent physiological effects such as fibrin clot stabilization or hair follicle cross-linking. A secretory transglutaminase (EC 2.3.2.13) was isolated from the coagulating gland of the rat. The protein is highly glycosylated. A fraction purified to homogeneity was used as an antigen to raise polyclonal antibodies in rabbits. These antibodies were used to identify the secretion sites of the protein within the male accessory sex glands as well as to study the immunological relationships of the respective antigen within different organs of different species. In the rat, the coagulating gland and likewise the dorsal prostate gave a positive immunoreaction. In the guinea pig, a closely related protein was detected in the anterior prostate. No cross-reactivity was found with membrane-bound transglutaminase from liver, erythrocytes or blood clotting factor XIIIa. The intraluminal secretion of the aforementioned glands was only weakly stained. No secretory granules were observed in the glandular epithelium but instead bleb-like structures reminiscent of apocrine secretion. A slight background stain of the epithelium remained even in castrated animals where secretion is largely suppressed. The background stain is attributed to a tissue-type, membrane-bound, non-secretory transglutaminase that is not androgen dependent, but instead synthesized only after androgen deprivation.

Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa

SummaryThe 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. Incubation of epididymal spermatozoa in phospholipase-containing solutions removed the acceptor site from the spermatozoa. Separation by polyacrylamide treatment of proteins from epididymal sperm cells extracted by sodium hydroxide or phospholipase treatment, subsequently transblotted on nitrocellulose sheets and directly labeled with gold-tagged major protein, demonstrated a protein duplet with a molecular weight of 65 and 67 kDa, respectively, which appears to represent the specific binder of major protein underneath the sperm surface. Binding of major protein to this ∼66 kDa acceptor site is regarded as a physiological event that may be related to the onset of hyperactivated sperm motility.

Immunohistochemistry of prostasomes from human semen.

To elucidate the origin of so‐called “prostasomes” in human semen, a polyvalent rabbit antiserum was produced against a highly purified preparation of these secretory particles.

Transglutaminases : purification and activity assays

Transglutaminases (TGases) are a widely distributed family of proteins found in many tissues and body fluids of vertebrates. To date the following types have been distinguished: secretory, tissue, epidermal, keratinocyte, and hemocyte TGase as well as factor XIIIa and erythrocyte hand 4.2 TGases are difficult to isolate, as they tend to form irreversible aggregates under native conditions. In this review, the isolation procedures for the different types of TGases are summarized. The most common chromatographic separation methods used for TGase purification are size-exclusion and ion-exchange chromatography. Additionally, other chromatographic methods (hydrophobic-interaction, affinity, adsorption chromatography) and electrophoretic techniques [preparative isoelectric focusing, sodium dodecyl sulphate polyacrylamide gel electrophoresis and zone electrophoresis] are described. Based on the enzymatic function of TGases (cross-linking of a primary amine and peptide bound glutamine), several established activity assays are described.

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

SummaryUsing commercial monoclonal antibodies against actin and tubulin (α and β), 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.

Ultrastructural localization of uteroglobin immunoreactivity in rabbit lung and endometrium, and rat ventral prostate

SummaryRecent biochemical studies have demonstrated amino acid sequence homologies between uteroglobin from rabbit endometrium and prostatic binding protein from rat ventral prostate. We have studied the ultrastructural distribution of uteroglobin-immunoreactive material in rabbit lung and endometrium and rat ventral prostate using an uteroglobin antibody raised in guinea pigs. Secretory granules of bronchiolar Clara cells, endometrial non-ciliated cells and rat prostate secretory cells gave a positive immunoreaction when this antibody was used. The results indicate a close relationship of immunoreactive epitopes of proteins present in those secretory cells. The functional properties of these proteins (glycoproteins, steroid binding, androgen-dependent secretion) suggest a close functional relationship, for instance a surface action such as coating, capping, masking or lubrication.

Intracellular localization of Prostatic Binding Protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry.

SummaryExtra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by “western blotting” and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl2 in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.

Cytoplasmic Carbonic Anhydrase II of Rat Coagulating Gland Is Secreted via the Apocrine Export Mode

Two different pathways for protein secretion are described for epithelial cells of rat coagulating gland and dorsal prostate: the classical merocrine and the alternative apocrine release mode. Apocrine-secreted proteins are synthesized on cytoplasmic polyribosomes and are subsequently exported in protrusions on the apical cell surface (aposomes). In this article we report the identification and purification to homogeneity of a 29-kD protein from the secretion of rat coagulating gland. N-terminal amino acid sequence analyses revealed 100% identity to rat brain carbonic anhydrase II (CAH II). In addition, the 29-kD protein showed CAH enzyme activity. On Western blot analysis, a polyclonal anti-CAH II antibody raised in rabbit reacted specifically with the rat and human but not bovine CAH II isoforms. Immunohistochemical studies on rat coagulating gland showed strong labeling for CAH II protein in aposomes. Immunoelectron microscopy confined CAH II protein to the cytoplasm and aposomes, whereas no staining was visible in the compartments of the classical merocrine route, the endoplasmic reticulum and Golgi apparatus. The resident cytoplasmic protein lactate dehydrogenase, however, was not found in the secretion. Taken together, the morphological and biochemical data clearly indicate that cytoplasmic CAH II from rat coagulating gland is specifically selected and then secreted via the apocrine pathway.

Distribution of heat-shock protein 60 immunoreactivity in testes of infertile men

Abstract.The immunohistochemical localization of heat-shock protein 60 (hsp60) was investigated in testicular biopsies obtained from 121 adult men with disturbed fertility. In normal unaffected tubules, hsp60 immunoreactivity was localized to spermatogonia, primary spermatocytes and Sertoli cells. In spermatogonia, cytosolic and mitochondrial labelling could be differentiated. In general, the number of stained spermatogonia decreased with the loss of spermatogenic function. A significant (P<0.01) reduction of stained spermatogonia was observed in testes with maturation arrest of spermatogenesis at the level of primary spermatocytes (30.2±21.6%) compared with testes exhibiting normal spermatogenesis. In addition, the decrease in the score correlated significantly with the diminution of cytosolic hsp60 immmunolabelling (coefficient r=0.25, P=0.03). There was a significant difference (P<0.01) in the percentage of cytosolic-stained spermatogonia in testes with a score equal to or greater than 5 (14.7±9.8%) and a score less than 5 (8.9±6.9%). These observations suggest that a low level of hsp60 expression in spermatogonia may lead to a different pattern of protection, which in turn could be involved in low spermatogenic efficiency.