Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis.

Diazepam binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), which share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. BZD receptors have been found not only in the brain, but also in a variety of peripheral tissues, including the testis. To clarify the role of ODN in the testis, we have investigated the localization of ODN in the rat testis using two different cytochemical approaches: immunocytochemistry and in situ hybridization. Immunocytochemical localization was achieved using rabbit antibodies developed against rat ODN. At the light microscopic level, immunostaining was exclusively located in interstitial cells; the seminiferous tubules were totally unlabeled. In the developing rat, immunostaining in the interstitial cells was first detected in an 18-day-old fetus. The immunolabeling increased as a function of age to reach a plateau at 40 days of age. The ultrastructural localization of ODN was achieved by immunogold staining. The gold particles were exclusively found in the cytoplasm of Leydig cells. HPLC analysis performed in adult rat testicular extracts revealed that immunoreactive material was detected in a peak eluted later than synthetic rat ODN. The cellular distribution of ODN was also studied by in situ hybridization using a 35S-labeled single stranded RNA probe complementary to DBI mRNA. Hybridization signal obtained at the light microscopic level was only detected over interstitial cells. The data obtained clearly indicate that in the rat, Leydig cells synthesize ODN and accumulate ODN-like immunoreactivity. Since Leydig cells have been shown to contain BZD receptors, it might be hypothesized that ODN and/or other DBI-related peptides can play a role in Leydig cell regulation.

Transcription switch of two phosphoglycerate kinase genes during spermatogenesis as determined with mouse testis sections in situ.

In order to elucidate the mechanistic interpretations underlying differential expression of the two phosphoglycerate kinase (PGK) genes during mammalian spermatogenesis, localization of its mRNAs in mouse testis sections was determined by in situ hybridization. MRNA for nonsperm-type PGK-1 was identified in nongerminal Leydig and Sertoli cells, spermatogonia, and spermatocytes, but was not detected in spermatids. In contrast, mRNA for sperm-type PGK-2 was notable in leptotene spermatocytes, becoming most abundant in pachytene spermatocytes. It was amply present in spermatids only up to step 10, completely disappearing after step 12. It is possible to assume that a transcription switch of the two PGK genes ensued following the onset of meiosis. These findings taken together with previous observations indicate that differential expression of the two PGK genes during mammalian spermatogenesis is regulated at the transcriptional and post-transcriptional levels.

Immunohistochemical detection of transforming growth factor-alpha in Leydig cells during the development of the rat testis.

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of Johnson's method for metachromasia (toluidine blue) for testicular interstitial tissue.

A modification of the Johnson's toluidine blue method for identification of testicular interstitial tissue elements is described. This consists in placing the tissue sections in Van Gieson's solution for 2 min after a 3 min immersion in an aqueous toluidine blue solution. Sharp histological distinctions between Leydig cells, mast cells and other connective tissue elements are obtained. The rounded Leydig cells with a large central nucleus and a prominent nucleolus show grayish yellow cytoplasm. The heterochromatin of Leydig cells nuclei stain a deep rose meanwhile its euchromatine is grayish yellow. Mast cells are full of large violet metachromatic granules which obscure nuclear and cytoplasmic features. Finally, fibroblasts and connective tissue collagen fibers stain yellow and pink respectively. An important advantage of this method is that the interstitial tissue components, specially the Leydig cells, retain the stain for a year or more.

Modification of Johnson's method for metachromasia (toluidine blue) for testicular interstitial tissue

A modification of the Johnson's toluidine blue method for identification of testicular interstitial tissue elements is described. This consists in placing the tissue sections in Van Gieson's solution for 2 min after a 3 min immersion in an aqueous toluidine blue solution. Sharp histological distinctions between Leydig cells, mast cells and other connective tissue elements are obtained. The rounded Leydig cells with a large central nucleus and a prominent nucleolus show grayish yellow cytoplasm. The heterochromatin of Leydig cells nuclei stain a deep rose meanwhile it euchromatine is grayish yellow. Mast cells are full of large violet metachromatic granules which obscure nuclear and cytoplasmic features. Finally, fibroblasts and connective tissue collagen fibers stain yellow and pink respectively. An important advantage of this method is that the interstitial tissue components, specially the Leydig cells, retain the stain for a year or more

[Effects of met-enkephalin on the testis. II. Histochemical study]. / Efectos de la met-encefalina sobre el testículo. II. Estudio histoquímico.

We performed a histochemical study using the Alcian blue-PAS staining method (for mucopolysaccharide), vitamin C, Sudan black (for lipids), and methyl green-pyronine (for nucleic acid). For the study, we utilized 105 male Wistar rats weighing 280-300 gms. Thirty rats comprised the control group, and 75 comprised the study group. Rats in the study group received a single, acute intracardiac dose of met-enkephalin (100 microliters of 50% met-enkephalin solution) and were sacrificed at 15, 30 and 60 minutes following injection, or a chronic intramuscular dose (50 microliters of 40% met-enkephalin solution). We observed that met-enkephalin caused histochemical changes in the rat testis, as evidenced by the accumulation of mucopolysaccharide (early in the study), cytoplasm lipid degeneration, changes in protein synthesis, and a fall in vitamin C stores (in seminal epithelium cell lines, as well as Leydig cells). These changes were more marked in the chronically than in the acutely-treated rats. The foregoing findings demonstrate that enkephalins (endogenous opiates) can cause profound metabolic changes in the rat testis that affect all its metabolic elements [proteins, lipids, polysaccharides and active substances (vitamins...)].

Transplantation of newborn rat testis under the kidney capsule of adult host as a model to study the structure and function of Leydig cells.

Newborn rat testis was transplanted under the kidney capsule of adult castrated and uncastrated male rats to develop and characterize a model system for studies on Leydig cell development. Two weeks after transplantation, the number of Leydig cells and the size of their nuclei in the transplants had increased. Secretion of testosterone was indicated by increased seminal vesicle weights and decreased pituitary LH in the castrated host animals. Pituitary FSH content increased significantly in the uncastrated animals with transplants, which suggested production of an FSH-stimulating factor. Cells with the morphologic features characteristic of fetal- and adult-type Leydig cells were observed in the transplants. The seminiferous tubules with spermatocytes, incipient lumina, and significantly larger average diameters showed more advanced development than those in the normal 2-week-old testis. By the present morphologic and functional criteria, the kidney subcapsular transplantation technique provides a suitable model for studies of fetal and adult Leydig cell development.

Immunolocalization and concentrations of inhibin alpha in the ovine testis and excurrent duct system.

Inhibin was localized in the ovine testis, excurrent ducts, and accessory sex glands by using a rabbit antiserum against a synthetic polypeptide representing the first 30 amino acids of porcine inhibin alpha-subunit. Concentrations of inhibin in fluids entering and leaving the epididymis also were determined in a radioimmunoassay using the same antibody. In the testis, immunostaining of inhibin was conspicuous in the seminiferous epithelium. Leydig cells occasionally were stained and the tunica media of blood vessels always was stained. Intense staining was observed in the epithelia lining the rete testis and ductuli efferentes. Staining also was intense in the epithelium of the initial segment and proximal caput epididymidis, and became less intense along the length of the epididymis. These observations were consistent with concentrations of inhibin in rete testis fluid (8.2 pmol/ml) entering the ductuli efferentes and in cauda epididymal plasma (0.67 pmol/ml) leaving the epididymis. Epithelia of ampullary and vesicular glands and of some prostatic acini were positively stained, but bulbourethral glands were never stained. Adrenal cortex, some proximal convoluted tubules in the kidney, and transitional epithelium of the urethra also were stained. Based on radioimmunoassay data and fluid flow rates for the ram, it was concluded that almost all of the 328 pmol inhibin that enters the ductuli efferentes daily is endocytosed in the proximal parts of the excurrent duct system. The physiological role(s) for inhibin, or inhibin-like peptides, in the excurrent duct system remains speculative.

The actions of calcitonin on the TM3 Leydig cell line and on rat Leydig cell-enriched cultures.

Studies demonstrating calcitonin receptors on Leydig cells have suggested that these cells may be one of the many sites affected by this peptide. To investigate this possibility, the effect of synthetic salmon calcitonin on the TM3 Leydig cell line (derived from immature mouse Leydig cells) and on primary Leydig cell-enriched preparations was examined. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in TM3 cells. In addition, the hormone stimulated the basal secretion of testosterone in both TM3 cell- and Leydig cell-enriched cultures and potentiated the action of hCG on Leydig cell-enriched cultures. Synthetic salmon calcitonin also increased the concentration of androgen and estrogen receptors in cultured TM3 Leydig cells by 2- and 4-fold, respectively, when added to the culture medium (1 micrograms/ml). The fact that 8-bromo-cyclic AMP decreased both androgen and estrogen receptor concentrations suggested that the effect of calcitonin on sex steroid receptors is not mediated by its effect on cyclic AMP in these cells. The possibility that the action of calcitonin on steroid receptors might be mediated by another messenger such as calcium (Ca2+) was therefore considered. Progressively lowering the concentration of Ca2+ in the culture medium of the cells from 1.5 mM to less than 0.01 mM decreased the concentration of both androgen and estrogen receptors. Returning the Ca2+ concentration to normal levels (1.5 mM) restored steroid receptor levels. Receptor levels were also decreased when the extracellular Ca2+ concentration was lowered to 0.5 mM, and treatment with the Ca2+ ionophore, A23187 (1 microM), restored receptor levels to normal. The calcium channel blocker, verapamil, decreased the androgen receptor concentration but unexpectedly increased the concentration of estrogen receptors. It was concluded that calcitonin stimulates cAMP formation and testosterone secretion, and increases the concentration of sex steroid receptors. These observations provide evidence that the previously demonstrated calcitonin receptors on Leydig cells may be coupled to several biologic responses in this cell type.

Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis.

The basement membranes of developing Leydig cells in fetal and newborn testis of rat were studied by ultrastructural and immunocytochemical methods. Fetal-type Leydig cells in prenatal rats were organized in irregularly outlined groups in the interstitium and were extensively surrounded by ultrastructurally identifiable basement membranes and immunocytochemically localized laminin and collagen type IV. Prenatal Leydig cell precursors had small patches of laminin and collagen type IV on their surfaces, which indicated that changes in extracellular matrix took place during their differentiation to mature fetal-type Leydig cells. Additionally, ultrastructural evidence was obtained for a basement membrane surrounding the fetal human Leydig cells similar to that in fetal rats. Soon after birth the rat fetal-type cells gathered into distinct clusters surrounded by delicate envelope cells and a discontinuous basement membrane. Basement-membrane structures, laminin, and collagen type IV were observed between the clustered cells as well. The basement membranes covering large cell surface areas of the fetal-type Leydig cells in fetal and newborn rats differed from those of the adult-type cells, which, according to our earlier study, are covered only by small patches of basement membrane. The difference between the basement membranes of the fetal- and adult-type rat Leydig cells further supports the concept of two different Leydig cell populations. The earlier findings of the epithelial nature of the Leydig cells agree with the observation of basement membranes in the Leydig cells.

Immunohistological determination of oestrogen receptor, progesterone receptor, and intermediate filaments in Leydig cell tumours, Leydig cell hyperplasia, and normal Leydig cells of the human testis.

Testicular Leydig cell tumours are able to produce oestrogens and can be induced by exogeneous oestrogen administration. Oestrogen and progesterone receptors, cytokeratin, vimentin, and proliferative activity were determined immunohistologically in human testes in six Leydig cell tumours, 14 cases of Leydig cell hyperplasia, and 13 cases with normal Leydig cells. While both steroid receptors were detected in about 70 per cent of the tumour cells in cryostat sections, no reaction was observed in normal Leydig cells. This supports the hypothesis of an enhanced receptor state in a Leydig cell subpopulation as a basic pathophysiological factor in the development of Leydig cell tumours. On cryostat sections, all tumours co-express cytokeratin and vimentin. Neither the receptors nor the intermediate filaments could be detected reliably in paraffin sections. The low proliferative activity of Leydig cell tumours corresponds to their benign clinical course.

Leydig cells do not have Fc receptors.

It has been reported that Leydig cells have Fc receptors, which traditionally have been considered markers specific for macrophages and polymorphonuclear leukocytes. The purpose of this investigation was to study further this phenomenon and also to determine if Leydig cells and macrophages could be separated from each other either by density gradient centrifugation using Percoll or by differential detachment with trypsin treatment of cultures of crude interstitial preparations. Interstitial cells were obtained by collagenase digestion of rat testis and established in culture. These cultures were reacted for 3 beta-steroid dehydrogenase and Fc receptor and viewed with phase contrast microscopy. No individual cells were positive for both steroid dehydrogenase activity and Fc receptors. The order in which the cells were stained for these two markers did not influence the results. Trypsin treatment of these crude interstitial cultures removed over 90% of the Leydig cells and approximately 20% of the macrophages. Macrophages were located in the same portion of Percoll gradients as the less dense (Population I) Leydig cells, while Leydig cells found in the dense area of the gradient (Population II) were not contaminated with macrophages. These studies indicate that Leydig cells do not have Fc receptors and that a subpopulation of Leydig cells can be isolated free of macrophages using density gradient centrifugation.

Identification and characterization of arginine vasopressin receptors in the clonal murine Leydig-derived TM3 cell line.

Specific arginine vasopressin (AVP) binding sites were identified and characterized using Leydig cell membranes prepared from a clonal murine Leydig-derived cell line, TM3. 3H-AVP binding data analyses demonstrated that the radioligand binds to a high affinity, low capacity, homogeneous class of sites with a dissociation constant of 0.5 nM. Characterization of these AVP binding sites included competition studies. Displacement of 3H-AVP binding with high affinity by unlabelled AVP, LVP and the V1 antagonist, d(CH2)5Tyr(Me)AVP, indicated that the Leydig cell AVP receptor is of the V1 type. Furthermore, AVP did not increase adenylate cyclase activity in TM3 membranes, a finding consistent with the V1 type of AVP receptor. No competition with 3H-AVP was found with the V2 agonist, dVDAVP, or the selective oxytocin agonist, [Thr4,Gly7]oxytocin. No specific binding for oxytocin was found in Leydig cell membranes. No specific binding for either 3H-AVP or 3H-oxytocin was observed in membranes prepared from the Sertoli cell line or peritubular cell line. These findings indicate that murine Leydig cells have specific AVP binding sites of the V1 type. These AVP sites are not coupled to the adenylate cyclase system.

Immunocytochemical demonstration of substance P in hamster Leydig cells during ontogenesis.

The cellular localization of substance P immunoreactivity was demonstrated at the light microscopical level in the hamster testis during fetal and postnatal development. A selective immunostaining was observed both of fetal and adult generation of Leydig cells. The comparison of the immunocytochemical findings with the ultrastructural characteristics of Leydig cells provided evidence that Leydig cells besides their androgen-producing capacity also had an neuropeptide producing function. The possible role of substance P in the local paracrine control of gametogenesis was discussed.

Identification of immunoreactive gastrin-releasing peptide related substances in adult rat Leydig cells.

Purified adult rat Leydig cells were found to produce gastrin-releasing peptide (GRP) by radioimmunoassay (RIA). Gel chromatography of the extracted material showed a single peak of GRP immunoreactivity. Further high pressure liquid chromatography (HPLC) analysis resolved the extract into two peaks that closely resembled the C-terminal fragment of GRP, GRP18-27 and GRP14-27. Immunohistochemical studies revealed specific staining for GRP in the Leydig cells of adult rat testis. These results demonstrate, by a number of independent criteria, that rat Leydig cells contain substances which behave like authentic GRP-like peptides. Since the peptides appear to be of local origin, a paracrine function within the rat testis is suggested.

Sex steroid levels and Leydig cell ultrastructure of the male common sheath-tail bat, Taphozous georgianus.

Male sheath-tail bats were collected from central Queensland over a 12-month period. Plasma testosterone levels peaked in August, coincident with an increase in the volume of the accessory glands and ampulla/seminal vesicle secretion. Peak spermatogenesis occurred in summer and autumn and declined in the face of maximal testosterone levels in winter. Levels of androstenedione and 5 alpha-dihydrotestosterone were high compared with testosterone levels and showed no significant seasonal changes. Ultrastructural examination of Leydig cell cytoplasm revealed numerous lipid droplets and mitochondria, and an abundant smooth endoplasmic reticulum. There were no seasonal changes in Leydig cell ultrastructure. The anomalous reproductive pattern in this species is consistent with the imposition of a cold-induced winter spermatogenic shutdown, on a framework of continuous spermatogenesis, with spring peaks in testosterone and accessory gland activity.

Quantification of P450scc, P450(17) alpha, and iron sulfur protein reductase in Leydig cells and adrenals of inbred strains of mice.

The relationship of maximal testosterone production to the amounts of cholesterol side-chain cleavage (P450scc), 17 alpha-hydroxylase/C17-20 lyase (P450(17) alpha), and iron sulfur protein (ISP) reductase was determined in Leydig cells from four inbred strains of mice (RF/J, SWR/J, C3H/He, and DBA/2). The amounts of P450scc, P450(17) alpha, and ISP reductase were also determined in adrenal glands of the same mice. cAMP-stimulated testosterone production and P450scc protein were high in RF/J and SWR/J compared to C3H/He and DBA/2 Leydig cells. A significant correlation between the amount of this enzyme and the capacity for testosterone production was found (r = 0.89; P less than 0.0005). ISP reductase was highest in RF/J, SWR/J, and C3H/He Leydig cells, which are significantly different from DBA/2. No significant differences in the amount of P450(17) alpha in Leydig cells from the four strains could be detected, and neither ISP reductase nor P450(17) alpha correlated with testosterone production. To ascertain if tissue-specific factors affect the expression of these enzymes, P450scc, ISP reductase, and P450(17) alpha were quantitated in adrenals from the same mice. P450scc and ISP reductase were expressed differently in adrenals compared to Leydig cells; levels of both proteins were high in C3H/He and RF/J adrenals compared to SWR/J and DBA/2. P450scc and ISP reductase were coordinately expressed in the adrenal, unlike in Leydig cells. P450(17) alpha was not detected in mouse adrenal glands. The results of this study suggest that strain-related differences in the capacity of Leydig cells for testosterone production may be determined by the amount of P450scc per Leydig cell. The expression of P450scc and ISP reductase in Leydig cells and adrenal glands appears to be influenced by both genetic and tissue-specific factors.

Isolation and partial characterization of an interleukin-1-like factor from rat testis interstitial fluid.

Testicular interstitial fluid (ISF) was collected by in vivo perfusion of rat testes and analyzed for the presence of interleukin-1 (IL-1) activity by utilizing a murine thymocyte proliferation assay. IS obtained from nine rats were all positive with dose-response curves of IL-1 activity similar to those produced by rat testicular aqueous extracts, rat macrophage IL-1 and human recombinant IL-1 alpha. Chromato-focusing of pooled ISF revealed a single peak of IL-1 activity with an estimated isoelectric point of 6.1-6.3. HPLC size exclusion chromatography demonstrated two active peaks with apparent molecular ratios Mr of 15,000-18,000 and 5000-7000, respectively. The molecular properties of the 15,000-18,000 Mr component are very similar to those of an IL-1-like factor previously isolated from seminiferous tubules. Our results indicate that the testicular IL-1-like factor is secreted by the seminiferous tubules into the interstitial tissue. Its function in the testicular interstitium is unknown but it might be relevant for the tendency to testicular relapse of childhood lymphocytic leukemia.

Testicular immunosuppressive protein.

Auto-, allo- and xenografts of various endocrine tissues survive for prolonged periods in the testicular interstitium. The reason for transplant survival outside the blood-testis barrier has been obscure. In the present paper we describe a high molecular weight (Mr = 130,000), heat- and pH-labile immunosuppressive factor with an isoelectric point of 6.3-7.3 in extracellular fluid collected from the rat testicular interstitium. The results show that the testicular immunosuppressive agent is not a steroid, but a protein. This testicular immunosuppressive protein may contribute to the immune privilege in the testicular interstitium.

Oxytocin in Leydig cells: an immunocytochemical study of Percoll-purified cells from rat testes.

Testicular interstitial cells, from rats aged 35 days, were dispersed with collagenase and separated through Percoll into 5 fractions (I-V); fraction I being the least dense. Measurement of basal testosterone production, histo-enzymological staining for 3 beta-hydroxysteroid dehydrogenase activity and electron microscopy indicated that the majority of Leydig cells were found in fraction IV (corresponding to a density of 1.076-1.097 g/ml). In addition, cells from this fraction responded to hCG treatment in a dose-dependent manner on day 0 and remained responsive after being cultured for 1 day. Immunostaining for oxytocin indicated that this fraction also contained the majority of the oxytocin-immunoreactive cells. On day 1 of culture, 56% of the cell population from fraction IV were positively stained for the steroidogenic enzyme and 75% immunoreactive for oxytocin. This overlap indicates that the Leydig cells were also the oxytocin immunoreactive cells.