Differential distribution of immunoreactive S-100 protein in mammalian testis

The present study deals with the immunohistochemical localization of S-100 protein in the testes of seven mammalian species including rat, cat, dog, pig, sheep, cattle and horse. Significant differences are demonstrated in the cellular distribution and intensity of immunoreaction for the protein. In bull, ram, boar and cat tests S-100 protein was localized in the cytoplasm and nuclei of Sertoli cells. A particularly intense staining was seen in the modified Sertoli cells of the terminal tubular segment. With the exception of the cat and horse S-100 protein immunoreactivity was additionally found in epithelial cells of the straight testicular tubules and in the epithelial cells of the rete testis. Endothelial cells of capillaries, veins and lymphatic vessels are regularly S-100 immunoreactive in ruminants. Leydig cells were found to be strongly positive for S-100 protein in the cat and rat testes and to a lower degree in pig and horse testes. Finally a distinct immunostaining of peritubular cells was restricted to the testis of dogs and rats. The remarkable species-specific variations of immunoreactivity for S-100 protein in different cell types of the testis support the hypothesis that S-100 protein is a multifunctional protein and may have a different function in testicular physiology.     

S-100 protein in the testis

Summary S-100, a protein originally believed to be unique to the nervous system, has recently been found in extraneural cell types. We report here on the presence of S-100 in the testis, namely in Leydig cells and in lymphatic endothelial cells, using immunohistochemical and immunochemical methods. We show that the protein in the testis is immunologically identical to brain S-100. The S-100-labelled cells in the testis exhibit morphological similarities with other cell types in different tissues known to contain S-100.     

Functional relationships of the mammalian testis and epididymis

The development of knowledge in four areas of research in male reproductive physiology of particular interest is reviewed. The concept of the blood-testis barrier (BTB), which arose following dye exclusion from the seminiferous tubules, has now been established as the differential transfer from interstitial fluid to tubular and rete testis fluids of molecules of physiological importance. The composition of fluid collected mostly from the rete testis of several species, not only reflects the nature of the barrier, but also the secretory capacity of the Sertoli cell. The functional significance of the transfer of molecules into testicular fluid and the composition of the fluid flowing into the epididymis are discussed. Sertoli cells establish the structural basis of the BTB during puberty and divide the seminiferous epitheliuym into basal and adluminal compartments. The Sertoli cell is the prime target for follicle stimulating hormone (FSH). The responses evoked by FSH are discussed, including special mention of androgen binding protein (ABP) and the protein hormone, 'inhibin', with FSH-suppressing properties. The control of FSH in the lamb is mentioned including new evidence to support a tubular source of a feedback agent with significance during the impuberal stage. Finally, some of the biochemical properties of the epididymis and its fluid contents are reviewed and the epididymal sperm are identified as the site of the antifertility action of the 6-chloro-6-deoxy sugars.     

S-100-like immunoreactivity in a planarian

Summary The presence of an S-100-like immunoreactivity was investigated in the planarian Dugesia gonocephala. By microcomplement fixation assay, measurable amounts of S-100-like immunoreactive material (0.11g/mg soluble protein) were detected in planarian high-speed supernatants. The index of immunological dissimilarity between ox S-100 and planarian S-100-like immunoreactive material was higher than that previously calculated between ox S-100 and all the vertebrates tested. By the immunohistochemical PAP method, S-100-like immunoreactivity was only detectable in the cilia of the epidermal cells. Although the biological meaning of S-100-like immunoreactivity in planarian remains to be clarified, the present data introduce new perspectives into the investigation of S-100.     

Androgen binding protein (ABP) in rabbit testis and epididymis

ABP has been measured in 105,000 g supernatants of testis and epididymls from rabbits of different ages and compared with a similar androgen binding protein (TeBG) in rabbit serum. Whereas the concentration of ABP in the caput epididymidis increased markedly from immaturity to adulthood, serum TeBG decreased, indicating that ABP and TeBG are regulated by different hormonal mechanisms.The concentration of ABP (pmoles/mg protein) in sexually mature rabbits was much higher in the epididymis than in the testis. Within the epididymis most of the ABP was concentrated within the caput, and very low amounts were found in the cauda, indicating that binding activity of ABP is destroyed as it passes through the epididymis.In addition to ABP (R_f ~0.7), rabbit epididymal supernatant contains a larger binding protein for 5α-dihydrotestosterone (DHT; 17β-hydroxy-5α-androstan-3-one) with slower electrophoretic mobility (R_f ~0.4) and a more rapid sedimentation rate on sucrose gradients (7S). This component is most probably the intracellular androgen receptor in the rabbit e pididymis.     

Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.     

S-100 protein stimulates cellular proliferation

Summary S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [³H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95–31.25 g/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 g/ml). Maximum stimulation of PBL was at 31.25 g/ml. PBL were not inhibited even at high concentrations of S-100p (125 g/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an autocrine tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

Perspectives in S-100 protein biology: Review article

The S-100 protein family constitutes a subgroup of Ca²⁺-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a₀, S-100a and S-100b, plus a number of structurally related proteins displaying 28–55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca²⁺-induced Ca²⁺ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca²⁺. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities.     

Cerebrospinal fluid S-100 protein levels in neurological pathologies

The aim of this paper was to evaluate S-100 concentration in cerebrospinal fluid (CSF) from patients with different neurological disorders, and in subjects with no proven neurological pathology, in order to study possible differences in their protein concentrations. The total number of patient-samples examined was 119 (58 males and 61 females; mean age 35 yrs, 1-79 yrs). Based on the final diagnoses, nine patient groups were studied: a control group, meningitis, acute lymphatic leukemia (ALL), dementia, hydrocephalia, polyneuropathy-motor neuron disease, acute cerebral infarction (ACI), and patients diagnosed with multiple sclerosis. S-100 protein concentrations were measured by the Sangtec 100 two-site immunoradiometric assay. The highest S-100 levels in CSF were found in the dementia group, ACI group, bacterial-fungal and lymphocytic meningitis groups (Kruskal-Wallis test). The S-100 concentrations in these groups were significantly higher compared with the control group (Mann-Whitney U test, p<0.05, p<0.01) and the multiple sclerosis group (p<0.05, p<0.01). No other significant differences were found between groups. Our results suggest that the high protein levels in CSF found in these pathologies may reflect the presence of brain damage. However, the levels need to be considered individually, as they depend on several factors, such as age, severity of brain damage or interval between the onset of brain damage and the taking of the sample.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Characterization of an androgen binding protein (ABP) in rat testis and epididymis

An androgen binding protein (ABP) was demonstrated in the 105,000 g supernatant of rat testis homogenate after charcoal extraction of endogenous steroids. Testis ABP proved to be identical to an ABP previously described in rat epididymis. It contained saturable high-affinity sites which exhibited binding specificity for dihydrotestosterone (6) and testosterone when measured by polyacrylamide gel electrophoresis or by competitive binding using charcoal adsorption. Binding to ABP was not affected by ribonuclease or neuraminidase but was decreased by the disulfide reducing agent, dithiothreitol and the sulfhydryl reagent, N-ethylmaleimide. Binding was abolished by treatment with proteolytic enzyme. The mean molecular radius of ABP was 2.92 nm as determined by the retardation of electrophoretic mobility in polyacrylamide gels of decreasing pore size. Assuming a partial specific volume of 0.66–0.74 the molecular weight was 86,000–91,000 for a spherical molecule. ABP binding was stable after treating at 45° C for 20 min. but was destroyed at 60° C. Binding was maximal between pH 7.5 and 9.0 and decreased at pH below 7.0.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Immunohistochemical demonstration of vimentin and S-100 protein in the kidneys

Vimentin and S-100 protein expression was studied in the kidneys of adult sheep and goat using immunohistochemistry. Vimentin was detected in the podocytes, mesangial cells of the glomerulus, in the endothelium of renal capillaries and renal stromal cells. In collecting tubules, ducts and nerves of the renal papilla, S-100 protein was expressed.