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Factors secreted by C6 glioma cells which induce electrical resistances across endothelial monolayers in an in vitro blood-brain barrier model have been partially characterised for the first time. These transendothelial electrical resistances (TEERs) were only evident when cell-free conditioned medium derived from C6 glioma cells was applied to the basolateral surfaces of confluent ECV304 or ECV304-9 cells which are both human umbilical vein endothelial cell lines (HUVEC). Electrical resistance values as high as 600 ohm. sq cm were obtained with this blood-brain barrier model and ultrafiltration techniques suggest that any factor(s) in the conditioned medium responsible for these TEERs have molecular masses of less than 1000 Da. Enzymic proteolysis and heat treatment carried out on the conditioned medium failed to inhibit its effect on the HUVEC monolayers suggesting that these C6 cell-secreted factors are unlikely to be proteins.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-R) is the major rate-limiting enzyme of the mevalonate pathway in many organisms, including yeasts. In the yeast Saccharomyces cerevisiae, there are two isoenzymes of HMG-R (Hmg1p and Hmg2p). Both consist of an anchoring transmembrane domain and a catalytic domain. We have removed the known controlling features of HMG-R by overproducing the catalytic domain of Hmg1p. This overproduction leads to an enhancement of squalene production, implying that HMG-R has been deregulated. The enhancement is apparent under semianaerobic and aerobic conditions. Despite the increase in squalene production, the amount of ergosterol produced by the HMG-R-overproducing yeast was not increased. This result suggests the presence of another regulatory step between squalene and ergosterol formation. Squalene levels generated by cells overproducing the catalytic domain of HMG-R were estimated to be up to 10 times those produced by wild-type cells. The enhancement in squalene production coincided with a reduction in growth rate. This reduction may be a direct consequence of the buildup of high concentrations of squalene and presqualene intermediates of the pathway.

In an effort to obtain a useful in vitro model possessing some of the properties of the blood-brain barrier, we have investigated the properties and interactions of immortalized cell lines. Immortalised human umbilical vein endothelial cells (HUVEC-304), in co-culture with rat C6 glioma cells in a two-chambered assembly, form tight junctional complexes, and develop a permeability barrier having a high transcellular electrical resistance. The endothelial cells generate a barrier with greatest integrity in the presence of glioma cells, or in the presence of glioma cell conditioned medium. Under these conditions, the endothelial cells also display pronounced structural changes which do not occur in the absence of glioma cells. Morphological alterations include a flattening of cell shape from a cuboidal-type to a squamous-type of appearance, and a re-organization of F-actin microfilaments. The integrity of the barrier can be reversibly disrupted by osmotic shock or by tumor necrosis factor-alpha (TNF-alpha). We interpret these observations to indicate that co-cultures of immortalized vascular endothelial and C6 glioma cells provide a model for the investigation of cell-cell interactions required for the generation of a barrier having several properties of the blood-brain barrier.

The actions of an intracellular nitric oxide generator compound on the properties of a co-culture model of the blood-brain barrier are described. Addition of the iron-sulphur cluster nitrosyl Roussin's black salt (RBS, heptanitrosyl-tri-mu3-thioxotetraferrate (1-)) resulted in a rapid and dose-dependent (50-250 microM) decline in the electrical resistance displayed by co-cultures of vascular endothelial cells and C6 glioma cells. The breach in barrier integrity elicited by RBS (250 microM) could be prevented by either haemoglobin (100 microM), methylene blue (200 microM), or by photon-induced inactivation of RBS. In contrast, the nitric oxide synthase inhibitor nitro-L-arginine methyl ester (250 microM) caused no inhibition in the decline in resistance of RBS-exposed cultures. Addition of 8-bromo-guanosine-cyclic monophosphate (500 microM) did not mimic the actions of RBS. Exposure to intense light of co-cultures manifesting a high transcellular electrical resistance resulted in a reduction in tissue resistance which could be prevented by the presence of haemoglobin (100 microM). We conclude that nitric oxide liberated from RBS results in a reversible diminution in the integrity of the endothelial cell barrier in the co-culture system, and we suggest that light-sensitive endogenous nitric oxide generator compounds may be present in intact cells. Possible roles of nitric oxide in blood-brain-barrier function are considered.

Carnitine and acylcarnitine derivatives have been reported to inhibit cell aggregation (Fritz and Burdzy, 1989, J. Cell. Physiol., 140:18-28). A follow-up of these observations showed that whereas the previously described effects of long-chain acylcarnitines were well replicated, those of carnitine on erythrocytes showed marked variability. The latter phenomenon was traced to the presence of silicates in carnitine solutions derived from the use of sodium hydroxide solutions stored in glass containers for the neutralization of carnitine. The present experiments have led to the discovery that oligomeric forms of silicates are powerful inhibitors of red blood cell aggregation which otherwise occurs in the presence of fibrinogen alone. The active form(s) of silicates in this assay, which appear to be generated by polymerization of silicates in metasilicate solutions on neutralization, are unstable and therefore transient under usual conditions. We estimate that the active oligomeric forms contain between 4 to 18 silicon atoms per molecule. When maintained at -18 degrees C in the presence of carnitine, but not in its absence, the active forms of oligomeric silicates remained stable for months, judging from their ability to inhibit cell aggregation. We conclude that carnitine stabilized the oligomeric form(s) of silicate, or that the species stabilized is an oligomeric silicate-carnitine complex. Comparable concentrations of choline, deoxycarnitine, or gamma-aminobutyrate were less effective in stabilizing the active silicate oligomers. The active forms of the silicate oligomers had Ki values of about 10 microM, calculated as the monomeric form, in inhibiting red blood cell aggregation. The data indicate that free carnitine does not directly inhibit erythrocyte inhibition, as previously interpreted, whereas long-chain acylcarnitine derivatives are active in the absence of silicates. Possible mechanism of actions of silicate oligomers on membranes are discussed.

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 peritubular 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 at this 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.

In the mammalian testis, somatic cells under hormonal regulation greatly influence the different stages of spermatogenesis, both in intermittent breeders and in animals which produce sperm continuously. In turn, specific populations of germinal cells modulate the function of Sertoli cells, the chief somatic cells within mammalian seminiferous tubules. Tubule formation can take place in the absence of germinal cells. Unlike homologous granulosa cells in the ovary, Sertoli cells retain many of their usual functions in germ cell-free animals. Some of the properties of Sertoli cells and their responses to stimulation by androgens or follicle-stimulating hormone are dependent upon information transmitted from neighbouring germinal cells at specific stages of the cycle of the seminiferous epithelium. We review the roles of some of the growth factors and paracrine agents synthesized and secreted by different classes of testicular cells. The potential roles of some of the known factors secreted by Sertoli cells (e.g. activin, inhibin, anti-Müllerian hormones, TGF-beta and somatomedin C) are considered in relation to the control of tubule formation, spermatogonial proliferation and cytodifferentiation, meiosis and the subsequent stages of spermatogenesis. We stress the importance of the unique tubule cytoarchitecture within which cell interactions take place and the changing nature of this cytoarchitecture at different stages of gonadal maturation.

Carnitine plays an essential role in the regulation of long-chain fatty acid metabolism in skeletal and cardiac muscle, a process that is mediated by well-characterized enzymatic mechanisms. Here, Irving Fritz and Edoardo Arrigoni-Martelli review the evidence that carnitine and its O-acyl derivatives also influence membrane fluidity, ion channel functions, smooth muscle contractility, membrane stability and cardiac functions. The authors present the view that direct interactions of carnitine derivatives with cell membranes are independent of reactions catalysed by carnitine acyltransferases. They propose that the novel actions discussed are implicated in the mechanisms by which carnitine and its derivatives protect perfused hearts subjected to ischaemia or to oxidative stress, and help people suffering from certain types of myocardial ischaemia or peripheral arterial disease.

The addition of anti-laminin IgG to the basal surfaces of rat Sertoli cells in culture in a two-chambered assembly results in a perturbation of F-actin arrangements, including disruption of the pericellular circumferal rings, impairments of the Sertoli cell permeability barrier, and subsequently focal defoliation, followed by cell reaggregation. The pentapeptide YIGSR, which competes with the laminin receptor for laminin (Kleinman and Weeks: Curr. Oph. Cell Biol., 1:964-967, 1989; Graf et al.: Biochemistry, 26:6896-6900, 1987) also elicited focal defoliation of Sertoli cells from the extracellular matrix-coated filter in the two-chambered assembly. Addition of YIGSR to Sertoli cell cultures resulted in cell detachment within 2 to 3 h. In contrast, the irrelevant peptide YIGSE had no detectable effects. The anti-laminin IgG was effective only when added to the chamber in which access was readily available to the basal surfaces of Sertoli cells, but YIGSR was effective when added either to the outer chamber or to the inner chamber. These data were interpreted to indicate that the Sertoli cell barrier generated in the two-chambered assembly allowed a relatively rapid diffusion of YIGSR between chambers, but prevented the rapid equilibration of anti-laminin IgG between compartments. Addition of anti-laminin IgG to the basal, but not to the apical surfaces of Sertoli cells, resulted in more rapid rates of equilibration of [3H]-methoxyinulin and [86Rb]Cl across the Sertoli cell monolayer. This evidence of impairment to the integrity of the barrier was detected prior to the disruption of stress fibers and focal defoliation, but after evidence of dissolution of the circumferal F-actin ring, which occurred within 1 h after addition of anti-laminin IgG. We consider the possibility that a transmembrane link exists between extracellular laminin and cytoskeletal elements which modulates the circumferal F-actin ring. We further postulate that this linkage can influence the nature of tight junctional complexes, and thereby the integrity of the Sertoli cell barrier.

Follicle-stimulating hormone (FSH) or dibutyryl cyclic AMP (dbcAMP) elicits striking morphological changes in Sertoli cells in culture in serum-free medium, resulting in a transition from an epithelial type of cell association pattern to that of an astrocytic or fibroblast-like cell, with attenuated cytoplasmic extensions between cells, and with diminished F-actin stained stress fibers. These responses of Sertoli cells do not occur in the presence of normal untreated serum, but they do take place in the presence of acid-treated serum which is depleted of antiproteases. The addition of alpha 2-macroglobulin to serum-free medium or to antiprotease-depleted serum resulted in the blockage of morphological responses of Sertoli cells to FSH or to dbcAMP. Changes in pattern of arrangements of F-actin in Sertoli cells in culture, which occur in response to FSH or to dbcAMP, were also prevented by the presence of alpha 2-macroglobulin. Thus, the diminution in bundles of F-actin containing stress fibers, which otherwise takes place in Sertoli cells stimulated by FSH or by dbcAMP, did not occur in cells in culture in the presence of alpha 2-macroglobulin, in the presence or absence of acid-treated serum. The inhibiting effects of dbcAMP on the migration of Sertoli cells in serum-free medium became nondetectable in medium containing normal untreated serum, but remained evident in Sertoli cells in culture in medium containing acid-treated serum depleted of antiproteases. Addition of alpha 2-macroglobulin blocked the inhibitory effects of dbcAMP on Sertoli cell migration. Similarly, the presence of alpha 2-macroglobulin prevented the inhibitory effects of dbcAMP on the contractility of TM4 cells which had been embedded in collagen type-I and incubated in serum-free medium. We discuss the possibility that cellular proteases may be implicated in the disintegration of microfilament bundles, either by favoring depolymerization of actin filaments; by facilitating breakage of the link of the transmembrane molecular assembly between cytoskeletal extracellular matrix components; or by catalyzing a disruption of the modular organization of one or more of the actin cross-linking proteins. By inference, we postulate that morphological responses of Sertoli cells to FSH require the activation of cellular proteases for one or more of these reactions, and that alpha 2-macroglobulin blocks the Sertoli cell morphological responses to FSH by inhibiting the proteases involved.

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.

Carnitine (gamma-trimethylammonium beta-hydroxy-butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18-28 [1989]). In investigations reported here, we show that carnitine also affects cell-cell adhesion in Dictyostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA-sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA-resistant binding sites. Addition of increasing concentrations of D(+)-carnitine and L(-)-carnitine resulted in a progressive inhibition of both the EDTA-sensitive binding sites and the EDTA-resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl-beta-methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA-resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells.

Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde-fixed human erythrocytes in a non-saturable, temperature-dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.

Testicular peritubular myoid cells, which have properties similar to those of vascular smooth-muscle cells, secrete a variety of metalloproteinases when maintained in culture in a chemically defined medium. The predominant metalloproteinases secreted were identified as latent type IV procollagenases having molecular masses of 72 kDa and 75 kDa, as detected in Western immunoblots with specific antibodies against type IV procollagenase. When peritubular cells were stimulated by dibutyryl cyclic AMP, forskolin or cholera toxin, they secreted increased amounts of type IV procollagenase. However, little if any of the active type IV collagenase, having a lower molecular mass of 66 kDa, could be detected under these conditions. Addition of low concentrations of cytochalasin D to peritubular cells in monoculture resulted in conversion of the latent type IV collagenase into its active form, assessed with antibody-specificity studies and by the appearance of the 66 kDa protein. In contrast, Sertoli cells in culture did not manifest an increased conversion of type IV procollagenase into type IV collagenase in the presence of cytochalasin D, even though cytochalasin D addition invariably resulted in a disruption of the microfilament assembly in each of these gonadal somatic cell populations. When peritubular cells were co-cultured with Sertoli cells, addition of cytochalasin D no longer resulted in formation of increased amounts of the active form of type IV collagenase. Sertoli cells and peritubular cells each secreted a tissue inhibitor of metalloproteinase type 2, detected with a specific antibody in a Western immunoblot to have a molecular mass of 21 kDa. We conclude that cytochalasin D acts on mesenchymal-type peritubular cells, but not on epithelial-type Sertoli cells, to enhance the conversion of latent type IV procollagenase into active type IV collagenase. This conversion of type IV procollagenase into type IV collagenase by peritubular cells was inhibited by factor(s) secreted by Sertoli cells. Interactions between Sertoli cells and peritubular cells are postulated to modulate net proteinase activities in discrete regions of the testis.

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.

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.

The membrane attack complex of complement (MAC) plays an important role in the mediation of proteinuria in experimental membranous nephropathy induced by Heymann antiserum. SP-40,40 is a recently described serum protein which appears to inhibit the formation of cytolytic MAC in a manner analogous to S protein/vitronectin. SP-40,40 is homologous to proteins originally isolated from rat and ram seminal fluid (sulfated glycoprotein 2 and clusterin, respectively). By current convention, these proteins are considered clusterin homologues. The objective of this study was to examine the participation of rat clusterin in passive Heymann nephritis. Using an antibody to rat clusterin as an immunofluorescent probe, clusterin deposits were demonstrated along the glomerular capillary wall in an identical pattern to rat C3 and C5b-9. Decomplementation using cobra venom factor prevented proteinuria and intraglomerular MAC formation. The epimembranous clusterin were not detected in the complement-depleted animals. The role of clusterin in the mediation of glomerular injury remains unknown, but it is probably related to in situ formation of the terminal complement cascade where it may play a regulatory role.

Sulphated glycoprotein 2 (SGP-2) is the major secreted protein product of rat Sertoli cells; likewise, clusterin is a major constituent of ram rete testis fluid. Isolation and sequencing of the intact subunits and peptides derived from clusterin show that it is the ram homologue of rat SGP-2. Human serum protein 40,40 (SP-40,40), a component of the SC5b-9 complex of complement, has recently been reported to be the human homologue of rat SGP-2. Analysis of the amino acid sequences of rat SGP-2 and human SP-40,40 show that both of these proteins have a significant relationship to the heavy chain of myosin. The regions of highest sequence similarity correspond to the major amphipathic domains in SGP-2/SP-40,40 and the long alpha-helical-tail domain of myosin, which forms a rod-like structure. SGP-2 has anomalous sedimentation behaviour which indicates that it probably exists in an extended conformation. A putative dinucleotide-binding structure has been identified in the longest stretch of identity between SGP-2 and SP-40,40. Elucidation of these features of SGP-2 and SP-40,40 may help to direct future studies into the role of these proteins in the reproductive and complement systems.

Messenger RNA for tissue-type plasminogen activator has been detected in RNA extracts from rat Sertoli cells in culture. Relative levels are increased in Sertoli cells stimulated by follicle-stimulating hormone or by dibutyryl cyclic AMP (dbcAMP) and decreased in cells maintained in the presence of transforming growth factor beta, type 1 (TGF-beta 1). Messenger RNA for plasminogen activator inhibitor, type 1 (PAI-1) has been detected in RNA extracts from rat peritubular myoid cells. Relative levels are increased in peritubular cells stimulated by TGF-beta 1, and decreased by the presence of dbcAMP in the medium. Data are interpreted to indicate that net protease activities in the seminiferous tubule are regulated at transcriptional levels by endocrine and paracrine agents.

Testicular peritubular cells (PC) in culture in serum-rich Eagle's minimal essential medium (MEM) on a polystyrene substratum proliferate and form fibroblast-like monolayers. The cells assume a flattened shape, and F-actin microfilaments are assembled to form prominent stress fibers. When PC grown under these conditions are dispersed and replated at a low density, a subsequent shift from serum-rich MEM to serum-free MEM results in dramatic changes. Within an hour, the cells round up, the F-actin microfilament assemblies, together with the cytoskeleton, become disrupted, and the degree of contractility is diminished. Under these conditions, addition of transforming growth factor-beta (TGF-beta) results in a more rapid recovery than that observed in cells maintained in basal MEM alone. The presence of TGF-beta results in an increased percentage of cells with flattened shapes during periods between 1 and 6 h after the shift to serum-free MEM. Concomitantly, PC treated with TGF-beta form and maintain well-organized, prominent stress fibers composed of F-actin microfilament bundles. In addition, the degree of contractility of PC embedded in collagen gels and cultured in serum-free MEM is markedly enhanced in cells stimulated by TGF-beta. Treatment of cells with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) or catecholeamines results in a rounding up of PC in culture, associated with a disruption of microfilament assemblies. Addition of TGF-beta prevents these effects of dbcAMP and beta-agonists, and permits PC to contract. We discuss the physiological significance of observations presented, consider possible mechanisms of action of TGF-beta on PC, and put forward the hypothesis that TGF-beta is one of the paracrine factors in the seminiferous tubule that influence interactions between Sertoli cells and peritubular myoid cells.

The addition of androgens (testosterone or dihydrotestosterone) resulted in decreased levels of detectable plasminogen activator activity in the medium when Sertoli cells were maintained in culture in a serum-free chemically defined medium in a two-chambered assembly. This occurred in the presence or absence of extracellular matrix or peritubular cells in the system. In the complete two-chambered assembly, addition of androgens simultaneously resulted in a small but significant increase in the integrity of the Sertoli cell barrier separating the two chambers, as indicated by slower rates of equilibration of [3H]methoxyinulin between inner and outer chambers. These responses to steroids appeared to be androgen specific, since other steroids examined (17 beta-estradiol, progesterone, and dexamethasone) had no detectable effects on levels of plasminogen activator activities or on barrier function. We confirmed that when FSH or (Bu)2cAMP is added to stimulate plasminogen activator secretion by Sertoli cells, the integrity of the barrier is decreased, provided no antiproteases are present in the serum-free medium. Simultaneous addition of androgens inhibited these effects of (Bu)2cAMP on Sertoli cells, but did not influence the responses of Sertoli cells to FSH. We compare actions of androgens on Sertoli cells in culture under various conditions and discuss the possible physiological significance of the inhibition of plasminogen activator activity.

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.

The competence of the seminiferous tubule barrier was evaluated by determining the sucrose space in isolated testis preparations obtained from adult wild-type mice and from mutants having defects in spermatogenesis. The distribution of [3H]sucrose in testes from wild-type mice was in a space which constituted 18.5% of the [14C]urea space. The corresponding sucrose space in testes from Tfm/Y and Sxr/+ sterile mutants was elevated to 57.7% and 52.0%, respectively. Independent approaches to detect an impairment in the seminiferous tubule barrier of testes from Tfm/Y mice consisted of estimations of the osmotic barrier to hypertonic LiCl, using histological techniques, and determination of the testis sucrose space in vivo. With both approaches, results obtained supported the presence of an impaired barrier in testes of Tfm/Y mice. The sucrose space was within normal limits in testes of the other mutants examined, which were defective in spermatogenesis. The results do not provide any evidence for an impaired barrier in testes of mice having Movbr/Y and Gy/Y mutations, or the t6twl genotype.

Clusterin has been purified from ram rete testis fluid by conventional techniques and by immunoaffinity chromatography. The molecule is characterized as a glycoprotein having a molecular mass of approximately 80,000 Da and an isoelectric point of 3.6. The purified protein retains the capacity to elicit clustering of cells in an in vitro assay. Under reducing conditions in the presence of sodium dodecyl sulfate, clusterin dissociates into subunits of about 40,000 Da. Heterogeneities in apparent molecular mass were eliminated after treatment of clusterin with neuraminidase. Gel filtration chromatography revealed that clusterin exists in dimeric and tetrameric forms under conditions of neutral pH and low salt concentrations. In the presence of 6 M urea, only the monomeric form is evident, with an apparent molecular mass of approximately 85,000 Da. Clusterin, which was found to contain 4.5% glucosamine, binds to concanavalin A-Sepharose and also to wheat germ agglutinin Sepharose. The amino acid composition of clusterin is reported. The possible cellular source of clusterin in rete testis fluid is discussed. It is shown that Sertoli cells in the seminiferous tubule are one potential source, since primary cultures of rat Sertoli cells secrete a protein having the same immunochemical and physical properties as clusterin isolated from ram rete testis fluid. Possible functions of clusterin are discussed.

With indirect immunofluorescent microscopic techniques, we have shown that fibronectin is distributed primarily in or along the basal lamina of the seminiferous tubule boundary tissue in sections of testes from 20-day-old rats. Purified rat Sertoli cell-enriched aggregates, maintained in culture in the presence or absence of serum, exhibit no detectable immunofluorescence with fibronectin antibody, whereas purified peritubular cells in culture do have a positive reaction to fibronectin antibody. Peritubular cells in culture incorporate [35S] methionine into fibronectin which can be immunoprecipitated with a fibronectin antiserum, but Sertoli cells do not. We have used various criteria to estimate the degree of purity of Sertoli cell-enriched preparations. The presence of peritubular myoid cells in conventional Sertoli cell-enriched aggregates, cultured in the presence or absence of serum, can be detected with transmission electron microscopic examination, by the Feulgen staining procedure, and by the immunocytochemical identification of fibronectin. We describe a technique to purify Sertoli cells in conventional Sertoli cell-enriched preparations by treatment with hyaluronidase, resulting in a lesser number of peritubular cells by the above criteria, even in preparations cultured in the presence of serum. Data presented suggest that some of the products previously attributed exclusively to Sertoli cells in Sertoli cell-enriched preparations, particularly those cultured in the presence of serum, may have been contributed by peritubular cells.

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.

Factors have been identified that influence the integrity of the barrier generated by Sertoli cells (SC) in culture in a two-chambered assembly. The permeability of the barrier was assessed by determining rates of equilibration of [3H]methoxyinulin or [86Rb]Cl across the Sertoli cell monolayer. The complete system consisted of a confluent monolayer of SC maintained on an extracellular matrix (Matrigel)-coated filter together with peritubular cells on the opposite side of the filter. In confirmation of previous results, levels of plasminogen activator (PA) activity secreted were increased by treatment of SC with FSH or with cAMP derivatives [(Bu)2cAMP (dbcAMP)]. PA levels in the culture medium were inversely related to times required for 50% equilibration of [3H]methoxyinulin across the SC monolayer. Thus, elevated PA levels, elicited by stimulation with FSH or dbcAMP, were associated with a decreased integrity of the barrier generated by SC preparations maintained in serum-free medium in the complete system. The increase in permeability of the barrier in SC elicited by FSH dbcAMP could be prevented, however, by the addition of various antiproteases. FSH actions on barrier function were complex. Effects of FSH that favored barrier integrity were most readily detected when proteolytic activity was inhibited. The addition of intact serum increased the integrity of the barrier, but acid-treated serum depleted of antiproteases had no such effect. We advance the hypothesis that proteases are implicated in modulation of the formation and maintenance of the seminiferous tubule barrier by SC.

Radiolabeled proteins secreted into the medium by rat Sertoli cells in primary culture have been examined for specific interactions with polyclonal and monoclonal antibodies directed against serologically detectable H-Y antigen(s). None of the proteins secreted by Sertoli cells reacted specifically with H-Y antibodies, as determined with immunoprecipitation procedures and immunoabsorbent affinity chromatography, followed by SDS gel electrophoresis. Radioactivity profiles of proteins obtained after reaction with H-Y antibodies were similar to those observed after treatment with nonimmune sera or with irrelevant antibodies. We obtained comparable findings with proteins secreted by the mouse cell line TM4, which is of presumptive Sertoli cell origin, and with proteins present in ram rete testis fluid. These and other findings presented do not support the contention that Sertoli cells secrete a protein having the properties of serologically detectable H-Y antigen as previously described.

Seminiferous peritubular cells have previously been shown to secrete a protein termed P-Mod-S which modulates the functions of Sertoli cells. The present study provides an initial characterization of P-Mod-S and examines the actions of P-Mod-S on Sertoli cells. Gel filtration chromatography demonstrates that P-Mod-S has an apparent molecular weight of 70 000 that could not be dissociated to a lower molecular weight form. A 40- to 90-fold purification of P-Mod-S was obtained with a predicted half maximal effective concentration for Sertoli cells of less than 10(-9) M. Through an analysis of the actions of P-Mod-S on Sertoli cells it is demonstrated that P-Mod-S stimulates the Sertoli cell to a greater extent than any single hormone or vitamin known to influence the cell. P-Mod-S maximally stimulates testicular transferrin and androgen-binding protein production by Sertoli cells, but does not stimulate levels of plasminogen activator activity. P-Mod-S also appears to induce the synthesis of several proteins that are not detected in control non-treated Sertoli cell cultures. One such protein whose synthesis was stimulated by P-Mod-S treatment of Sertoli cells was a component having a molecular mass of 20 kDa. This 20 kDa Sertoli cell-secreted protein was specifically immunoprecipitated with an antibody against an epididymal lactalbumin-like protein. This implies that P-Mod-S can induce Sertoli cells to synthesize and secrete a lactalbumin-like protein. P-Mod-S was found not to contain mitogenic activity. Data presented indicate that testicular peritubular cells synthesize and secrete a 70 kDa non mitogenic paracrine factor termed P-Mod-S which has a dramatic influence on Sertoli cell functions. Results are discussed with respect to modulation of epithelial (Sertoli) cell functions by components produced by mesenchymal (peritubular) cells.

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.

We confirm that basal rates of formation of plasminogen activator by cells in cultured tubule segments at stages of the cycle associated with spermiation (stages VII and VIII) are higher than those by cells in tubule segments at any other stages of the cycle of the seminiferous epithelium. We demonstrate that addition of cAMP derivatives or follicle-stimulating hormone in the presence of a phosphodiesterase inhibitor results in a large stimulation of plasminogen activator formation by tubule segments at all stages of the cycle. The greatest percentage increase (approximately 100-fold) is observed in cells in tubule segments having lowest basal rates of plasminogen activator formation (stages IX-VI). Even under stimulated conditions, however, the amounts of plasminogen activator produced by cultured tubule segments at stages VII and VIII remain greater than those produced by cultured tubule segments at other stages of the cycle, and these differences persist during organ culture for 48 h. Insulin and testosterone do not alter rates of formation of plasminogen activator. We conclude that Sertoli cells, the primary source of formation of plasminogen activator in the testis, are metabolically heterogeneous in the seminiferous tubule and that the germ cell association patterns in various stages of the cycle modulate Sertoli cell functions. We discuss the data in relation to the tissue restructuring within the seminiferous tubule which occurs during spermatogenesis and the possible role of plasminogen activator in these processes.

Highest concentrations of follicle-stimulating hormone (FSH) are required to elicit maximal increases in cAMP formation by isolated Sertoli cells in culture, and progressively lower concentrations are required to increase three late responses maximally (estradiol synthesis from testosterone, DNA synthesis, and synthesis of androgen-binding protein, respectively). Identical dose-response curve relations are observed when cholera toxin is used as the stimulatory agent. Results do not rule out the possibility that cAMP could mediate the various responses of Sertoli cells to FSH.

We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-in-activated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influence of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1 : 8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.

Sertoli cells isolated from the testes of 18 to 20-day old rats synthesize estradiol-17 beta when grown in primary culture in the presence of testosterone (5 X 10(-7) M). After an initial lag phase of about 2 hours, follicle-stimulating hormone (FSH) stimulates synthesis of estradiol up to 50-fold, synthesis being approximately linear for 24 hours when medium is changed every 2-6 hours. Luteinizing hormone (LH) causes only a marginal stimulation, at concentrations consistent with contamination with FSH. Dibutyryl cyclic 3',5'-adenosine monophosphate (dbcAMP) (10(-4)M) mimicks the effect of FSH. No significant synthesis occurs in the absence of steroid substrate or in the presence of pregnenolone (5 X 10(-7) M). When cells are pre-incubated for 24 hours with FSH in the absence of testosterone, addition of testosterone results in an immediated increase in estradiol synthesis without a lag phase, suggesting that FSH or dbcAMP are capable of inducing the conditions necessary for stimulation in the absence of testosterone. These observations provide the first direct evidence of estradiol-17 beta synthesis by Sertoli cells from normal animals, and offer evidence that synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and cyclic AMP.

Rabbit polyclonal antibodies, directed specifically against clusterin purified from ram rete testis fluid, were employed in an investigation of the biosynthesis of clusterin by cultures of rete testis epithelial cells and by Sertoli cells prepared from testes of adult rams. Cells in serum-free medium were incubated in the presence of either [35S]methionine, [3H]leucine, or [3H]glucosamine, and radiolabeled proteins secreted were immunoprecipitated. The pellet was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the gels were then fluorographed. In other experiments, protein bands were transferred to nitrocellulose and visualized immunochemically. Under non-reducing conditions, a single band was detected, having a molecular weight of 80,000. Under reducing conditions, doublet bands were detected, having approximate molecular weights of 40,000 (major band) and 37,000 (minor band). These properties were indistinguishable from those obtained with authentic samples of pure clusterin subjected to gel electrophoresis and Western immunoblot procedures. Amounts of clusterin synthesized by rete testis cells in culture, quantitatively determined with a sandwich enzyme-linked immunosorbent assay procedure, were approximately 4 micrograms/micrograms cell DNA/48 h. Immunocytochemical localization investigations, using monoclonal antibodies against clusterin, revealed the presence of clusterin in the perinuclear of juxtanuclear regions, in both rete testis epithelial cells and Sertoli cells in culture. The possible functions of clusterin produced by rete testis epithelial cells and by Sertoli cells are discussed.

We have developed the concept that mechanisms evolved very early for the modulation of spermatogenesis in response to changes in the external environment, and that these ancient control mechanisms were retained during subsequent evolution. In nearly all animals, the regulation of germinal cell development is postulated to be mediated through the control of gonadal somatic cell functions, associated with the creation and maintenance of an optimal milieu within the spermary of seminiferous tubule in which gametogenesis takes place. In primitive organisms, a small number of stages intervenes between environmental stimuli and subsequent alteration of gonadal somatic cell functions. In contrast, in more complex organisms, the number of intervening stages is greatly amplified and modulated via neuroendocrine mechanisms involving receptors, transducers, and various sorts of relays and messengers. The evolution of these neural and endocrine controls appears to have occurred in lock-step with the evolution of increasing layers of complexity of regulators of spermatogenesis. This is not unduly surprising, since the requirement to have functionally fertile male and female partners of the same species together at the same time and place would require considerable integration of behavioral and recognition mechanisms during courtship and mating. The nature of these neural mechanisms is likely to prove no less complex than that of mechanisms in the gonad required for successful gamatogenesis. The neuroendocrine regulation of spermatogenesis in starfish and in chordates is postulated to act in a manner completely homologous to the ways in which external environmental stimuli influence spermatogenesis in more primitive organisms. In both sets of cases, the gonadal somatic cells (nurse cells) are the ultimate targets which mediate the effective turning on or turning off of spermatogenesis. The hormone-responsive nurse cells are postulated to achieve this simply by creating a microenvironment in the vicinity of germinal cells which permits the expression of program required for development, or by failing to do so. In mammals, Sertoli cells and peritubular cells, only when optimally stimulated by hormones and paracine factors, are thought to form a functional unit which provides this necessary microenvironment. In less complex organisms, other nurse cell arrangements exist to nourish the syncytia of developing germ cells with the mixture of nutrients, salts, etc. required for a gametogenesis to take place in a protected milieu.

Rat testicular peritubular cells in culture secrete an inhibitor of plasminogen activator (PA) activity. Conditioned serum-free medium from secondary cultures of peritubular cells (PcMEM) was concentrated and then fractionated by gel exclusion chromatography. Under native or denaturing conditions, PA inhibitor (PA-I) activity appeared in fractions having a molecular weight of approximately 55,000. The PA-I inhibited the tissue-type plasminogen activator, and also that of the two-chain form of urokinase, but not the one-chain form. Addition of guanidine HCl (4 M) to PcMEM resulted in a large increase of inhibitory activity. The 55,000 molecular weight PA-I band in PcMEM reacted with antibodies against plasminogen activator inhibitors produced by bovine vascular endothelial cells, or by human fibrosarcoma cells, as detected by immunoadsorption experiments, by immunoblotting, and by reverse fibrin autography. We describe other characteristics of the protease inhibitor produced by testicular peritubular cells, and we discuss its possible functions in the control of PA activity in the seminiferous tubule at different stages of spermatogenesis.