Isolation and culture of ram rete testis epithelial cells: structural and biochemical characteristics

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.     

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures.

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.     

Identification of cell types in Sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

Identification of cell types in sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

Summary The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

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.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

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.     

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.     

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.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact

Addition of dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), or cytochalasin D to co-cultures of Sertoli cells and testicular peritubular myoid cells blocks a series of morphogenetic changes which otherwise occur during culture. When Sertoli cells are plated directly onto preexisting layers of peritubular cells maintained under basal conditions, structures form which display many of the characteristics of germ cell-depleted seminiferous tubules. The presence of dbcAMP, MIX, or cytochalasin D, added at varying times after plating Sertoli cells, results in the inhibition of each successive stage of in vitro remodeling: the inhibition of migration of Sertoli cells, the inhibition of initial ridge formation, the blockage of subsequent formation of mounds and nodules of compacted Sertoli cell aggregates, the prevention of the formation of basal lamina and associated layers of extracellular matrix between Sertoli cell aggregates and surrounding peritubular cells, and the inhibition of tubule formation. The presence of dbcAMP also inhibits the migration of peritubular cells, contractions by these cells, and compaction of Sertoli cell aggregates. When intimate cell apposition is prevented by plating the two cell types on either side of a membrane filter, the morphogenetic cascade is blocked, and no formation of a germ cell-depleted seminiferous tubule-like structure occurs. Other effects of dbcAMP on cell shape, cell movement, and cell association patterns during co-culture are described. Possible mechanisms by which dbcAMP, MIX, or cytochalasin D blocks restructuring are discussed. Since each elicits perturbations of the cytoskeleton, we offer the interpretation that cytoskeletal changes may be correlated with the prevention of closely apposing cell compact and the inhibition of basement membrane formation. Interactions observed between Sertoli cells and peritubular cells during co-culture are postulated to be analogous to those occurring in other types of mesenchymal cell-epithelial cell interactions during organogenesis and during tubulogenesis in the fetal testis. Speculatively, the blockage by dbcAMP of the morphogenetic cascade in the co-cultured system may be related to the inhibition by dbcAMP of testis cord formation in organ cultures of fetal gonads reported by others.     

Autoimmune orchitis can be induced by thymic lymphocytes autosensitized against syngeneic sertoli cells in vitro

Thymic lymphocytes from normal inbred Lewis/Wistar rats were cocultured with syngeneic Sertoli cell-peritubular cell preparations in the presence of heterologous or allogeneic serum. Thymic cells cultured in this manner bound to Sertoli cells, became autosensitized , and markedly altered syngeneic Sertoli cell surface properties and remodeling functions in vitro. In contrast, control thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not become sensitized. Coculture of autosensitized thymic cells with syngeneic seminiferous tubule segments, or local transfer of such lymphocytes into syngeneic rat testes, resulted in intratubular infiltration by "light cells." Intratesticular injection of autosensitized thymic cells was followed by derangement of the seminiferous epithelium, and by morphologic changes characteristic of experimental autoimmune orchitis. Thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not elicit these changes. Thymic cells incubated with peritubular cells in heterologous or autologous serum behaved like control thymocytes, and were not sensitized. Data presented indicate that thymic cells are potentially capable of recognizing syngeneic Sertoli cell self-antigens. We speculate that factors normally present in serum may inhibit the recognition by thymic lymphocytes of antigenic determinants present on Sertoli cells. We discuss the possibility that the modulation of interactions between immature thymic lymphocytes and Sertoli cells is implicated in the prevention of autoimmune reactions against the testis.     

Role of laminin in the Morphogenetic cascade during Coculture of sertoli cells with peritubular cells

Observations summarized in this article demonstrate an essential role of laminin during the restructuring processes that occur during coculture of Sertoli cells with testicular peritubular cells. The data presented indicate that laminin becomes detectable on the free surfaces of Sertoli cells only after reaggregation of Sertoli cells begins, coincident with the initiation of repolarization at a specific stage of the morphogenetic cascade. We infer that laminin deposited at this time serves as a cohesion molecule that permits peritubular cells to come into close contact with Sertoli cells and subsequently to spread along the free surfaces of Sertoli cells. These conclusions and inferences are based on the following experiments. Cycloheximide-treated peritubular cells in culture in MEM containing cycloheximide readily attach to laminin-coated polystyrene surfaces. By contrast, added peritubular cells do not attach onto monolayers of Sertoli cells in monoculture or onto Sertoli cells plated on top of peritubular cells and maintained in coculture for periods of up to 48 h in cocultures maintained for 6 days, however, labeled peritbular cells readily adhere to the free surfaces of reaggregated Sertoli cells. Laminin, but not fibronectin, appears on the free surfaces of the reaggregated Sertoli cells atthis time, coinciding with the period of initial mound formation. The addition of antilaminin IgG, but not antifibronectin IgG, blocks the attachment of cycloheximide-treated peritubular cells to laminin-coated plates and also blocks the subsequent migration of peritubular cells required to form a monolayer. Similarly, anti-laminin IgG inhibits the attachment and spreading of labeled peritubular cells seeded on the free surfaces of reaggregated Sertoli cells in mounds generated during the morphogenetic cascade. We interpret the combined data to indicate that the appearance of laminin on the free surfaces of Sertoli cells is required to permit peritubular cells to adhere and subsequently to migrate on Sertoli cell surfaces, resulting in the formation of a tubule-like structure. © 1994 Wiley-Liss, Inc.     

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.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.     

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.     

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.     

Transforming growth factor beta gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions

The potential role of transforming growth factor beta (TGF beta) as a mediator of cell-cell interactions within the seminiferous tubule was investigated through an examination of the local production and action of TGF beta. Sertoli cells and peritubular (myoid) cells were isolated and cultured under serum-free conditions. Secreted proteins from Sertoli cells and peritubular cells were found to contain a component that bound to TGF beta receptors in RRA. Reverse-phase chromatography of Sertoli cell and peritubular cell secreted proteins fractionated a protein with similar biochemical properties as TGF beta 1. This fractionated protein also contained TGF beta bioactivity in its ability to inhibit growth of an epidermal growth factor-dependent cell line. Both peritubular cells and Sertoli cells contained a 2.4 kilobase mRNA species that hybridized in a Northern blot analysis with a TGF beta 1 cDNA probe. TGF beta 1 gene expression was not detected in freshly isolated germ cells. TGF beta 1 alone was not found to influence Sertoli cell nor peritubular cell proliferation with cells isolated from a midpubertal stage of development. The effects of hormones and TGF beta on Sertoli cell differentiation and function were assessed through an examination of transferrin production by Sertoli cells. TGF beta 1 had no effect on transferrin production nor the ability of hormones to influence transferrin production. The presence of peritubular cells in a coculture with Sertoli cells also did not affect the inability of TGF beta 1 to act on Sertoli cells. Although Sertoli cell function did not appear to be influenced by TGF beta 1, peritubular cells responded to TGF beta 1 through an increase in the production of a number of radiolabeled secreted proteins. TGF beta 1 also had relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of Sertoli cells and peritubular cells responded to TGF beta 1 by the formation of large cell clusters with ball-like structures. Data indicate that TGF beta may have an important role in influencing the differentiation and migration of peritubular cells. Observations demonstrate the local production of TGF beta within the seminiferous tubule by Sertoli cells and peritubular cells and suggest that TGF beta may have a role as a paracrine-autocrine factor involved in the maintenance of testicular function.     

Identification of type IV collagenase in rat testicular cell culture: influence of peritubular-Sertoli cell interactions

In the testis, interactions between peritubular cells (mesenchyme) and Sertoli cells (epithelium), together with proteolytic remodeling of the extracellular matrix, may play a central role in testicular development, morphogenesis, and spermatogenesis. In this study we demonstrate that a metalloproteinase of 72 kDa present in rat Sertoli cell and Sertoli-peritubular cell coculture medium is activated by p-aminophenylmercuric acetate (p-APMA) to a lower molecular mass form, indicating that it is likely to be a latent collagenase. Immunoblots using antibodies to three different domains of type IV collagenase show that the 72-kDa protease and a 76-kDa protease are type IV pro-collagenases. Sertoli cells cultured alone produce basal levels of type IV collagenase that can be immunolocalized in the cytoplasm of cultured cells. Peritubular cells cultured alone produce much less type IV collagenase. However, Sertoli and peritubular cells in coculture do produce type IV pro-collagenase, and in cultures consisting predominantly of peritubular cells, the activated form of type IV collagenase was detected by both zymography and immunoblotting. Cells growing during the transitional phase (from cell attachment to confluence) secrete more metalloproteinases than during the confluent phase. In contrast, plasminogen activator levels are unaffected by time in culture. These results show that rat testicular cells in culture produce and secrete type IV collagenase, and that the secretion and activation of this enzyme and other metalloproteases is regulated by the ratio of mesenchymal cells to epithelial cells and time in culture.