Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2.

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.

Subunits of cyclic adenosine 3',5'-monophosphate-dependent protein kinase show differential and distinct expression patterns during germ cell differentiation: alternative polyadenylation in germ cells gives rise to unique smaller-sized mRNA species.

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Identification and characterization of a beta-adrenergic receptor in hamster Sertoli cells.

Sertoli cells cultured from immature hamsters contain a beta-adrenergic receptor which is coupled to the cAMP second messenger system. Thus, isoproterenol, epinephrine, and norepinephrine, which act via beta-adrenergic receptors, all stimulate cAMP accumulation in Sertoli cells cultured for 4-5 days. This cAMP response to isoproterenol is inhibited stereospecifically by the beta-receptor blocker, propranolol. It is also sensitive to inhibition by beta-adrenergic antagonists in this order of potency: nonspecific beta receptor antagonists, propranolol, timolol, hydroxypindolol greater than beta 1 selective antagonists, oxyprenolol, metoprolol much much greater than beta 2 selective antagonist, butoxamine. Butoxamine was at least 1000-fold less sensitive than either the nonspecific or the beta 1 selective antagonists at inhibiting the response of either isoproterenol (nonspecific), dobutamine (beta 1 selective) or zinterol (beta 2 selective). The hamster Sertoli cell beta receptor is, therefore, predominantly of the B1 subtype. This beta receptor mediated increase in cAMP is sensitive to homologous desensitization and is stimulated synergistically by forskolin. In addition, Seroli cells freshly isolated from immature hamsters contain an active beta receptor. However, this beta receptor mediated increase in cAMP is dependent on the type of trypsin used in the cell preparation. In agreement with Kierszenbaum et al (1985), freshly isolated Sertoli cells from immature rats never responded to the catecholamines regardless of the type of trypsin used; indicating an important physiologic difference between rat and hamster Sertoli cells.

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.

Characterization of rat testicular peritubular myoid cells in culture: alpha-smooth muscle isoactin is a specific differentiation marker.

In frozen sections of testes from 20-day-old rats, alpha-smooth muscle (SM) isoactin was prominently immunostained in the peritubular tissue and in vascular walls, but not in areas populated by germinal cells, interstitial cells, or Sertoli cells. Peritubular myoid cell (PMC)-enriched preparations were isolated by two different procedures involving our previously published sequential enzymatic treatment ("conventional peritubular cell [PC]-enriched preparation") and by density-gradient purification of PMC from these preparations. The properties of different populations of PMC in culture were compared with respect to plating efficiency, rates of proliferation, and presence of cytoskeletal proteins. PMC, maintained in culture under defined conditions, contained proteins immunoreactive with monoclonal antibodies against alpha-SM isoactin. This was detected by immunostaining and by Western blots of cell extracts subjected to gel electrophoresis. Neither Sertoli cells, skin fibroblasts, bovine endothelial cells, nor glial cells contained alpha-SM isoactin detectable by the above techniques. We report the ontogeny of alpha-SM isoactin in the peritubular tissue of testes at different stages of gonadal development, and show that it is detectable within 8 days after birth. In addition, we describe immunocytochemical changes that occur during culture in various media of PMC prepared from testes of 20-day-old rats. We compare the use of alpha-SM isoactin as a differentiation marker for PMC with the use of desmin in facilitating the identification of PMC, and in following alterations in phenotype during culture in various culture media. Data presented demonstrate that about 81% of cells in the "conventional PC-enriched preparation," and about 94% of cells in the more purified populations of PMC were positive for alpha-SM isoactin in cells maintained in culture for 18 h after plating. These same PMC also were shown to express vimentin and plasminogen activator inhibitor, type 1. We conclude that alpha-SM isoactin is an excellent specific marker for PMC in seminiferous tubules and in culture.

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)

A novel maturation marker for human Sertoli cells.

The maturation of Sertoli cells at puberty is critical for the initiation and maintenance of spermatogenesis. Little is known about the factors which control Sertoli cell maturation. We have examined Sertoli cells in testicular specimens from 44 subjects, ranging in age from 6 months to 67 years, for reactivity with a mouse monoclonal antibody (M2A) using the immunoperoxidase reaction. We found positive reactivity with prepubertal but not postpubertal Sertoli cells, suggesting that the antigen defined by M2A was a marker of immature Sertoli cells. This marker may be useful for studying the factors which influence Sertoli cell maturation during development of the testis.

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.

Cellular localization of fibronectin gene expression in the seminiferous tubule.

The cellular location of fibronectin expression within the seminiferous tubule was investigated in order to better understand testicular cell functions and cell-cell interactions. Peritubular cells were shown to actively synthesize and secrete fibronectin in culture by the detection of a radiolabeled 220 kDa secreted protein that is immunologically similar to fibronectin and by the quantitation of fibronectin in peritubular cell conditioned medium with a fibronectin enzyme-linked immunosorbent assay. Sertoli cells did not produce detectable levels of fibronectin when assayed by either of these procedures. A 6.5 kb fibronectin messenger RNA was detected in freshly isolated or cultured peritubular cells, but no fibronectin gene expression was detected in Sertoli cells or developing germinal cells. Combined results imply that the peritubular cells are the only apparent site of fibronectin expression within the seminiferous tubule. During the development of the testis the levels of fibronectin expression increased to a maximum at early puberty (15-day-old rats) and then slowly declined. The results demonstrate that fibronectin can be utilized as a unique functional and biochemical marker for peritubular cells when compared to other cell types in the seminiferous tubule. Production of fibronectin by peritubular cells provides an example of the ability of peritubular cells and Sertoli cells to cooperate in the production of individual components of the basement membrane of the seminiferous tubule. This cellular interaction is an example of a mesenchymal/stromal-epithelial interaction which is postulated to be important for the physiology of many tissues.

Distribution of soltriol [1,25(OH)2-vitamin D3] binding sites in male sex organs of the mouse: an autoradiographic study.

After injection of [3H]-1,25(OH)2-vitamin D3 (soltriol), nuclear labeling is found in Sertoli cells of testes, being highest at the stage of spermiosis, in epithelium of efferent ductules and caput epididymidis and in connective tissue cells of epididymis, in lamina propria and muscular sheath of deferent duct, and in epithelium and muscular sheath of dorsal and ventral prostate of the mouse. This labeling pattern is characteristic for [3H]-soltriol and differs from that for [3H]-dihydrotestosterone and [3H]-estradiol, although with overlap. The nuclear labeling with [3H]-soltriol suggests an action of the hormone on certain processes during spermatogenesis, on sperm maturation, on epididymal fluid resorption, and on secretion and transport of spermatozoa.

The characterisation of non-sperm cells in the ejaculates of fertile men using transmission electron microscopy.

The non-spermatozoal cells (NSC) in the semen samples of 20 fertile human males have been studied by transmission electron microscopy (TEM), in order to accurately distinguish between the different types of cell present and to give a quantitative profile of their relative proportions. The main seminal constituents of the average fertile man were found to be the germinal elements, accounting for 84.0% of the total NSC population. Of this percentage the anucleate bodies, designated "CM" in this study, were the greatest component (43.0%, SEM +/- 4.7). Spermatids were the next commonly occurring cell (22.2%, SEM +/- 2.9), the anucleate cellular masses with organelles, CM(O)'s and the spermatocytes making up the remaining 18.8%. Leucocytes accounted for 13% of the total NSC, respectively: neutrophils - 12% (SEM +/- 4.5); macrophages - 0.9% (SEM +/- 0.3) lymphocytes - 0.1% (SEM +/- 0.1). The remaining 3% consisted of 2.3% (SEM +/- 0.7) epithelial cells and 0.7% (SEM +/- 0.4) Sertoli cells. The principal conclusions reached were: that some normal ejaculates host active phagocytosis and possibly macrophage activation; that anucleate bodies which form a major component of the ejaculate merit further quantitative study and that the shedding of spermatids is an interesting and important aspect of ejaculation.

Sertoli cell ectoplasmic specializations: a type of actin-associated adhesion junction?

In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.

Specific mRNAs in Sertoli and germinal cells of testes from stage synchronized rats.

A treatment which used vitamin A depletion followed by vitamin A repletion was used to synchronize seminiferous tubules to a few related stages of the cycle of the seminiferous epithelium. The success of the synchronization procedure was dependent on the age and size of the rat at the initiation of the experiment (20 days of age and 35-40 g) and the extent to which the vitamin A deficiency had progressed. Administration of retinol was done when the only viable germinal cells in the testis were preleptotene spermatocytes and type A spermatogonia but if the deficiency was prolonged spermatogenesis did not recover. Once established synchrony appeared to be sustained at least through several consecutive cycles. A combination of molecular probes was used to determine if the synchronized testes displayed stage specific variations in Sertoli cell and germinal cell mRNA levels as has been reported for normal asynchronized rats. Sertoli cells in the synchronized testes were shown by quantitative in situ hybridization and by Northern blot analysis to have stage specific variations in the levels of mRNA for transferrin, sulfated glycoprotein-1, and sulfated glycoprotein-2. The mRNA levels in the different stages were qualitatively similar to those in equivalent stages previously reported for testes from asynchronous rats. The germinal cell content of the synchronized testes were examined with Northern blots probed with nick-translated protamine 1 and transition protein 1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical and biochemical characterization of testicular peritubular myoid cells.

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.

Polyamine profiles in rat testis, germ cells and Sertoli cells during testicular maturation.

Polyamine cellular concentrations (putrescine, spermidine and spermine) in the rat testis and testicular cell types were determined by fluorescence spectroscopy of their dansyl derivatives. A method is described to separate dansylated polyamines by high performance liquid chromatography in less than 12 minutes. In rat Sertoli cells, polyamine concentrations (per mg DNA) were greater than those in germ cells and the testis. The concentrations of all three polyamines increased with age. Concentrations of spermidine and spermine in germ cells also increased with age and leveled off after 27 to 35 days. On the other hand, higher putrescine levels were found in the testis of young rats (13 to 22 days) while the greatest spermidine and spermine contents were observed in the testis from rats of 31 to 35 days old. Of great interest, Sertoli cells from all age groups studied released a relatively large quantity of putrescine and a smaller amount of spermidine, but no spermine, into culture media. The amount of polyamine released by Sertoli cells varied with the age of the animal. Sertoli cells from 27-day-old rats released the greatest quantity of putrescine on a per mg DNA basis. The release of putrescine increased after hypotonic treatment that removed contaminating germ cells from the remaining Sertoli cells. It is concluded that cellular polyamine levels in the rat testis, germ cells and cultured Sertoli cells and the amount of polyamines released by Sertoli cells were age-dependent during the first wave of spermatogenesis.

Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor: purification and localization on surfaces of spermatogenic cells and sperm.

We have found that the rat testis contains a cell surface galactosyl receptor that is antigenically related to the minor species of rat liver asialoglycoprotein receptor (ASGP-r) and has binding affinity for galactose coupled to agarose. In immunoblotting experiments, rat testis galactosyl receptor (RTG-r) is recognized by antiserum raised against the minor ASGP-r species of rat liver (designated rat hepatic lectin-2/3, RHL-2/3). Antiserum raised against the major species RHL-1 does not recognize an antigenic protein equivalent to RTG-r. Triton X-100-extracted rat liver and testes preparations fractionated by affinity chromatography on galactose-agarose and resolved by SDS-PAGE under reducing conditions, show that rat liver contains both the major (RHL-1) and minor (RHL-2/3) ASGP-r species whereas rat testis displays only a receptor species comigrating with RHL-2/3. RTG-r was present throughout testicular development. The receptor was found in seminiferous tubules, cultured Sertoli and spermatogenic cells, and epididymal sperm. Indirect immunofluorescent studies show RHL-2/3-like immunoreactivity on the surface of Sertoli cell, meiotic prophase spermatocytes, spermatids, and epididymal sperm. In spermatids and sperm, the immunoreactivity is restricted to the plasma membrane overlying the dorsal portion of the head. Because of RTG-r has galactose binding affinity, is present on surfaces of Sertoli and developing meiotic and postmeiotic spermatogenic cells, and overlies a region of the intact acrosome on epididymal sperm, RTG-r may have a role in spermatogenesis and in events leading to sperm-egg recognition.

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells.

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Localization of immunoreactive testibumin in the testis and epididymis of adult rats.

Testibumin is a glycoprotein previously isolated from the spent media of primary Sertoli cell-enriched cultures prepared from 20-day-old rats. Immunoassayable testibumin is found in the highest concentrations in testis, epididymis and fluids of the male reproductive tract in adult rats. In the present study, light microscopy was used to show that immunostainable testibumin in paraffin sections of rat testis was localized along the base of the seminiferous epithelium and in finger-like projections from the base of the epithelium, corresponding to the position of the Sertoli cells. The immunostaining of Sertoli cells was shown to be specific since either purified testibumin or crude Sertoli cell-enriched culture medium could compete with antibody for binding sites in tissue sections. The observations using light microscopy were confirmed when Sertoli cells were examined by electron microscopy using a pre-embedding immunostaining technique. The epithelium of the epididymis also contained immunoreactive testibumin which was localized in the caput, corpus and cauda. Immunostainable testibumin was also localized in the corpora lutea of the rat ovary and in the epithelium of the uterine endometrium. These observations are consistent with previous reports that immunoreactive testibumin is present in these organs as demonstrated by radio-immunoassays. We conclude that (i) the immunolocalization of testibumin in Sertoli cells adds to a growing list of observations suggesting that it is made in this cell type; (ii) the other sites of testibumin synthesis in the male are uncertain but the ovary and uterus are possible sites in the female; (iii) electron microscopy following pre-embedding immunostaining and epitope selection can be used as an adjunct to conventional immunocytochemistry to localize proteins in Sertoli cells.