Secreted metalloproteinases in testicular cell culture

It is well known that cultured Sertoli cells secrete plasminogen activators (Lacroix et al., Mol Cell Endocrinol 1977; 9:227-236; Hettle et al., Biol Reprod 1986; 34:895-904). We now show that testicular cells in culture also secrete gelatinolytic metalloproteinases. Gelatin zymographic analysis of concentrated culture medium proteins reveals that Sertoli cells secrete gelatinases of 185 kDa, 110 kDa, 83 kDa, 76 kDa, and 72 kDa in addition to plasminogen activators (PAs). Gelatinase 185 kDa is induced by FSH. Media from Sertoli (epithelial)/peritubular (mesenchymal) cell cocultures contain the Sertoli cell gelatinases and one FSH-stimulated gelatinase of 50 kDa, indicating that gelatinase 50 kDa is regulated by both FSH and cell-cell interactions. A 50-kDa fibronectinolytic activity is also present in the coculture medium from cells grown in the presence of FSH. Casein zymography demonstrates a prominent 30-kDa protease only in media from cocultures. Peritubular cells secrete urokinase-type plasminogen activator (u-PA) and exhibit slight degrading activity at 86 kDa and 74 kDa. The gelatinases are most active in the pH range 7.3-8.5 and are completely or partially inhibited by metal ion chelators indicating that they are metalloproteinases. Our data demonstrate that testicular cells in culture secrete several gelatinases in addition to PAs, and that FSH and coculture conditions regulate some of these secreted proteases. We suggest that the highly regulated secretion of these proteases may well be of physiological importance during testicular development and spermatogenesis.     

Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture.

Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al. Biol Reprod 1990; 43:946-955, 956-964). We have now investigated the regulation of testicular cell type IV collagenase and other metalloproteinases in vitro. Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels. However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mRNA levels. Zymographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-kDa type IV collagenase protein levels. However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloproteinases (83 kDa and 110 kDa and in an activation of the 72-kDa rat type IV collagenase. Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation. Immunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells. Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern. Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 kDa, 83 kDa, and 110 kDa and a triton-insoluble gelatinase of 225 kDa. These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.     

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.     

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.     

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.     

SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.     

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.     

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.     

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.     

Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial-Endothelial Interactions

Abstract: The regulation of microvessel formation and the expression of CNS-specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a coculture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary-like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high-molecular weight urokinase-type plasminogen activator (uPA), low-molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92-, 72-, and 62-kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six- to eightfold decrease in coculture uPA during capillary-like structure formation. Substantially less change in type IV 72-kDa procollagenase activity was seen in cocultures during capillary-like structure formation, but active type IV 62-kDa collagenase activity was significantly increased during capillary-like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS.     

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.     

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.     

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.     

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.     

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.     

Invasive human fibrosarcoma DNA mediated induction of a 92 kDa gelatinase/type IV collagenase leads to an invasive phenotype.

Proteolytic enzymes, such as gelatinase/type IV collagenase, play a pivotal role in cancer invasion and metastasis. Invasive human fibrosarcoma cells (HT1080) secrete two species of gelatinase/type IV collagenase, 68-72 kDa and 92 kDa enzymes. The purpose of this study is to elucidate which species of gelatinase/type IV collagenase plays a more important role in invasion. We have found that HT1080 x human fibroblast hybrids have reduced ability to invade a reconstituted basement membrane (Matrigel) in vitro compared to HT1080 cells, and abundantly secrete only the 68-72 kDa gelatinase/type IV collagenase. These data suggest that the 92 kDa gelatinase/type IV collagenase may be more important in HT1080 cell invasion. We next transfected HT1080 genomic DNA into non-invasive mouse C3H/10T1/2 fibroblast cells, which secrete only 68-72 kDa gelatinase/type IV collagenase. Four invasive transfectants were established. These invasive transfectants secreted the 92 kDa gelatinase/type IV collagenase in addition to the 68-72 kDa gelatinase/type IV collagenase, whereas non-invasive control DNA transfectants did not secrete the 92 kDa gelatinase/type IV collagenase. These results suggest that the induction of the 92 kDa gelatinase/type IV collagenase is important in the invasive phenotype.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)