Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Adrenomedullin inhibits the contraction of cultured rat testicular peritubular myoid cells induced by endothelin-1

The present study documents that adrenomedullin (AM), a vasoactive peptide originally identified in pheochromocytoma tissue and present in the testis, in vitro affects the function of testicular peritubular myoid cells (TPMC), a contractile cell type located in the seminiferous tubule wall. AM stimulated cAMP production by cultured TPMC taken from 16-day-old rats, and this effect was completely inhibited by the AM antagonist AM-(22-52) and partially by the CGRP (calcitonin gene-related peptide) antagonist CGRP-(8-37). Studies on TPMC contractile activity documented that AM inhibits TPMC contraction induced by endothelin-1 (ET-1) and that its effect is antagonized by AM-(22-52). Neutralizing AM produced by TPMC with the addition of anti-AM antibody induced a significant increase of ET-1-induced contraction. When exposed to the protein kinase A inhibitor H-89, AM inhibitory activity on ET-1-induced TPMC contraction was suppressed, whereas the nitric oxide synthase inhibitor N:(G)-nitro-L-arginine methyl esther did not modify AM activity. In conclusion, our study indicates that AM stimulates cAMP production and inhibits the contraction induced by ET-1 in TPMC in vitro, and that AM produced by TPMC has an autocrine effect. We propose that AM may have a role in the control of seminiferous tubule contraction.     

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.     

Pleiotropic actions of melatonin in testicular peritubular myoid cells of immature Syrian hamsters

BackgroundPeritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs.MethodsWe established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors.ResultsExogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin.ConclusionMelatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis.General significanceIf transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.     

Rat Sertoli cell extracellular matrix regulates glycosaminoglycan synthesis by peritubular myoid cells in vitro

We have previously reported metabolic cooperation between Sertoli and peritubular myoid cells in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). This study concerns Sertoli cell ECM-peritubular myoid cell interactions in terms of GAG synthesis. We have examined the responses of hormones and other regulatory agents such as a combination of follicle-stimulating hormone (FSH), insulin, retinol, and testosterone (FIRT) on peritubular myoid cells, and tested if Sertoli cell ECM or serum factor substitute for the stimulation by FIRT. Testicular peritubular myoid cells cultured on Sertoli cell ECM showed significant increases in the levels of cell- and ECM-associated GAG over that when cultured on uncoated plastic. This indicates a specific cell-substratum interaction between Sertoli cell ECM and peritubular myoid cells in the testis in terms of GAG synthesis. Moreover, in terms of cell-associated GAG synthesis, peritubular myoid cells cultured on Sertoli cell ECM or on plastic in the presence of serum substituted for the stimulatory response of FIRT on peritubular myoid cells cultured on uncoated plastic. The data are discussed in relation to the possible role of cell-substratum interaction in maintaining peritubular myoid cell functions. © 1993 Wiley-Liss, Inc.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

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.     

Membrane associated proteoglycans in rat testicular peritubular cells

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG) Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (K_av=0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.Abbreviations PG Proteoglycans - CSPG Chondroitin Sulfate Proteoglycans - HSPG Heparan Sulfate Proteoglycans - HSCSPG Heparan and Chondroitin Sulfate Proteoglycans - DNAse I Deoxyribonuclease I - DMEM Dulbeccos modified Eagle's medium - H/D HAM F12/DMEM - ECM Extracellular Matrix - PBS Phosphate Buffered Saline - PI Phosphatidylinositol - GPI Glycosyl Phosphatidylinositol - PI-PLC Phosphatidylinositol Specific Phospholipase C - TBS Tris Buffered Saline - STI Soybean Trypsin Inhibitor - GAG Glycosaminoglycans - HA Hyaluronic Acid     

Cell-substratum and cell-cell interactions promote testicular peritubular myoid cell histotypic expression in vitro

Peritubular cells, prepared from seminiferous tubules from testes of 20-day-old-rats, were seeded onto different substrata and cultured under varying conditions. When plated onto polystyrene or glass surfaces, peritubular cells assumed a typical fibroblast-like cell shape and cell association pattern, together with a fibroblast-like migration behavior. They maintained high rates of proliferation even after achieving confluency. In contrast, when peritubular cells were plated onto a seminiferous tubule biomatrix (ST-biomatrix) surface, they spread to form a continuous cell layer having a myoepithelioid histotype similar to that of peritubular myoid cells in the intact seminiferous tubule. The characteristics of the myoepithelioid histotype described include a squamous, polyhedral cell shape; a cobblestone-like cell association pattern, with closely apposing or slightly overlapping cell borders, and a very low mitotic index. When peritubular cells were plated onto laminin, collagen, fibronectin, heparin, or a liver biomatrix, a fibroblast-like pattern resulted, indicating that ECM components listed and liver biomatrix are unable to substitute for ST-biomatrix in maintaining normal myoepithelioid characteristics in vitro. In cocultures of Sertoli cells plated on top of peritubular cells, the peritubular cells directly in contact with Sertoli cell aggregates developed a myoepithelioid histotype, whereas peritubular cells in regions not in direct contact had a fibroblast-like histotype. The data are discussed in relation to the possible role of cell-cell interactions, and cell-substratum interactions, in the acquisition and stabilization of the histotype of peritubular cells in the seminiferous tubule during development.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue

We have studied the distribution of histochemically detectable alkaline phosphatase in cultures of seminiferous tubule fragments and of peritubular cells from prepubertal rats. The same material also was immunohistochemically evaluated for the presence of desmin-containing intermediate filaments. The comparative analysis of alkaline phosphatase and desmin positivity shows that alkaline phosphatase histochemistry selectively detects desmin-containing contractile cells in tubular and peritubular cell cultures. We propose alkaline phosphatase as a novel marker for myoid cells that can be of help in screening, defining, and eventually standardizing the exact composition of peritubular cell cultures, a model that is of increasing interest in the study of cellular interactions in the testis.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Purification and characterization of testicular transferrin secreted by rat Sertoli cells.

Sertoli cells synthesize and secrete a transferrin-like protein (testicular transferrin) [Skinner & Griswold (1980) J. Biol. Chem. 255, 1923-1925]. The purpose of the present study was to purify and characterize testicular transferrin and to compare it with serum transferrin. Testicular transferrin was obtained from the medium of cultured rat Sertoli cells, whereas serum transferrin was obtained from rat serum. Both proteins were purified with the use of phenyl-Sepharose hydrophobic chromatography and transferrin immunoaffinity chromatography. The purified proteins were shown to have similar molecular masses (75 000 Da) and amino acid compositions. The pattern of tryptic peptides from testicular and serum transferrin were found to be essentially the same when analysed by reverse-phase high-pressure liquid chromatography. The carbohydrate composition of both transferrins was determined by several colorimetric assays and g.l.c. Testicular transferrin, isolated from cell culture medium, had increased amounts of glucose, galactose and glucosamine. Serum transferrin that was incubated with cell culture medium also had a large amount of associated glucose. The results show that testicular transferrin and serum transferrin are structurally very similar and are possibly products of the same gene expressed in two different tissues, the testis and liver. However, the amount of carbohydrate associated with these two proteins is different.     

Extracellular matrix components and testicular peritubular cells influence the rate and pattern of Sertoli cell migration in vitro

We report the patterns of migration of Sertoli cells plated on specific substrata, and the influences of testicular peritubular cells on these processes. Data presented indicate that while peritubular cells readily spread when explanted onto Type I collagen, Sertoli cells do not. A delay of 4 to 6 days occurs after Sertoli cells are plated before they begin to migrate randomly to form plaque-like monolayers on Type I collagen. These processes are dependent upon the synthesis and subsequent deposition of laminin and/or Type IV collagen by Sertoli cells, and are independent of fibronectin. A different behavior occurs when reconstituted mixtures of purified Sertoli cells and pertiubular cells are sparsely plated onto Type I collagen. Peritubular cells rapidly spread to form chains of cells between Sertoli cell aggregates. Sertoli cells then migrate on the surfaces of the peritubular cells, culminating in the formation of cable-like structures between aggregates. Evidence is presented that the Sertoli cell migration to form "cables" under these conditions is dependent upon fibronectin synthesized by peritubular cells, and is independent of the presence of laminin or Type IV collagen. We discuss the possible relevance of these data to the role which precursors of peritubular cells may play in determining the behavior of Sertoli cell precursors in vivo during tubulogenesis, or in the remodelling of the seminiferous tubule which occurs during different stages of the cycle of the seminiferous epithelium in spermatogenesis.     

On the character of the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis

Postnatal differentiation of the peritubular myoid cells in mouse testis is hormone dependent. In order to analyse the differentiation of the peritubular tissue, an attempt was made to develop an experimental model system utilizing an in vitro method. Fragments obtained from adult, 7- or 10-day-old mice, were cultured in McCoy's modified 5a medium for 9–19 days. The fragments and monolayers that grew from them were examined with the electron microscope at the end of the culture period. Monolayers originating from either mature or immature testicular expiants were comparable in appearance. They were composed of spindle-shaped cells that contained abundant profiles of granular endoplasmic reticulum and free ribosomes, as well as arrays of 40–60 Å thick filaments and associated dense bodies. In these respects they resembled smooth muscle cells in culture, in developmental, and in pathological conditions. Examination of the peritubular tissue in the testicular explants indicated that the monolayer of myoid cells originated from the fibroblasts rather than the peritubular myoid cells. Peritubular cells in explants from mature rats retained their myoid features at the end of the culture period but myoid cell differentiation failed to progress in expiants obtained from immature animals. Additional work is necessary in order to establish the suitability of these preliminary culture attempts to support normal development before conclusions may be drawn concerning the role of hormones in myoid cell differentiation. The role of microfilaments as a contractile organelle of cells is discussed.     

Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates.

Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of [3H]-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

Comparison of actin-filament bundles in myoid cells and Sertoli cells of the rat,golden hamster and mouse

Testes of adult rats, golden hamsters and mice were fixed with paraformaldehyde. Seminiferous tubules were then isolated by collagenase dissociation, stained with fluorescent phallotoxin, and viewed in a confocal laser microscope to observe actin filaments. Bundles of actin filaments in the myoid cells, especially in the rat, were arranged at right angles to each other in relation to the longitudinal axis of the tubule. In the hamster, circumferentially directed bundles were more frequent than longitudinally directed bundles. The actin bundles in the mouse were thinner than those in the rat and hamster, and their lattice network was less prominent. Nuclei of the myoid cells were elliptical and their short diameters were parallel to the long axis of the seminiferous tubules in the animals examined. Areas of myoid cells and of basal junctional portions of Sertoli cells were measured and compared in all animals studied. There were significant differences in the areas among the three species. The golden hamster showed the largest value for myoid-cell area, and the mean value for Sertoli-cell area was highest in the mouse.