Biomedical subjects

P S Tung

Publications and source records attributed to P S Tung.

At least 19 recordsLinked to original sources

PubMed1997

A 54-kDa protein related to ras-guanine nucleotide release factor expressed in the rat exocrine pancreas.

The polymerase chain reaction was used to amplify a cDNA encoding the catalytic region of ras-guanine nucleotide release factor (ras-GRF1) from mouse embryonic stem cell mRNA. Antibodies directed against this protein were prepared and affinity purified. Western immunoblotting of rat tissue lysates revealed a 140-kDa protein in brain as expected but, in addition, a strongly immunoreactive 54-kDa protein, p54, was identified in pancreas. Expression of ras-GRF1 in pancreas was confirmed at the RNA level by reverse-transcriptase-coupled polymerase chain reaction analysis; p54 may therefore correspond to a form of ras-GRF1 or a closely related protein. The cellular and subcellular localization of p54 was investigated by enzyme-linked immunocytochemistry and immunogold electron microscopy. In the pancreas, p54 was expressed primarily in acinar cells, where it was localized along the basolateral and apicolateral plasma membranes. Indirect immunofluorescence microscopy of cultured acini further indicated that the plasma membrane localization of p54 was dependent on the maintenance of the acinar histotype and organized acinar structure. When primary acinar cells were permitted to dissipate into monolayer cultures devoid of zymogen granules, ras-GRF1 staining became cytosolic. Our results suggest that ras-GRF1 is involved in the structure and function of the pancreas.

Alternative Splicing↗

PubMed1996

Association of p120 ras GAP with endocytic components and colocalization with epidermal growth factor (EGF) receptor in response to EGF stimulation.

Epidermal growth factor (EGF) stimulation of cells induces down-regulation of EGF receptors through receptor-mediated endocytosis. Simultaneously, activated autophosphorylated EGF receptors become associated with the pTyr-binding domains of various intracellular signaling proteins, including those implicated in the regulation of the plasma membrane-associated small G protein p21 Ras. Included among these EGF receptor-binding proteins is the M(r) 120,000 Ras GTPase-activating protein (GAP). Herein we show by subcellular fractionation, immunoprecipitation and Western blotting, indirect immunofluorescence, and double immunogold labeling and electron microscopy that, in response to EGF stimulation of MDCK cells, GAP becomes first plasma membrane localized and subsequently internalized with EGF receptors. In various subcellular membrane fractions examined, EGF receptor and GAP were physically associated. EGF stimulation also induced association of GAP with p190 Rho-GAP and the GAP-p190 complex coimmunoprecipitated from membrane fractions. Recruitment of SH2 domains to activated receptor tyrosine kinases at the plasma membrane is undoubtedly an important mechanism in controlling the subcellular localization and substrate accessibility of signaling proteins. Our results illustrate that another level of signaling protein regulation may occur by virtue of their association with endosomes.

Animals↗

PubMed1995

CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein.

CaLB was originally observed as a conserved sequence motif in various calcium-responsive signalling proteins and also in p120 Ras GTPase activating protein (p120GAP) (Clark et al. Cell 65: 1043-1051, 1991). Here we show the 43 residue CaLB motif in p120GAP is a functional protein domain that when expressed as a fusion protein in vitro confers Ca(2+)-dependent interactions with cellular membranes and phosphatidylserine and phosphatidylinositol vesicles. p120GAP, but not a mutant lacking the CaLB domain, associates with the particulate fraction of cells in response to elevated intracellular Ca2+ suggesting that p120GAP may be regulated in part by calcium signals. Addition of the p120GAP CaLB domain was able to restore transforming activity and particulate localization to an otherwise transformation-defective and cytosolic mutant v-Sre tyrosine kinase. The CaLB domain appears to be a prevalent protein module that may affect the molecular interactions and subcellular localization of signalling proteins.

3T3 Cells↗

PubMed1994

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 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.

Animals↗

PubMed1993

A murine CDC25/ras-GRF-related protein implicated in Ras regulation.

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development.

Amino Acid Sequence↗

PubMed1993

Proteases are implicated in the changes in the Sertoli cell cytoskeleton elicited by follicle-stimulating hormone or by dibutyryl cyclic AMP.

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.

Actins↗

PubMed1993

Interactions of Sertoli cells with laminin are essential to maintain integrity of the cytoskeleton and barrier functions of cells in culture in the two-chambered assembly.

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.

Actins↗

PubMed1992

Competition between cell-substratum interactions and cell-cell interactions.

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.

Animals↗

PubMed1991

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium.

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.

Animals↗

PubMed1991

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

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

Animals↗

PubMed1991

High-resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development.

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.

Animals↗

PubMed1990

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

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

Actins↗

PubMed1990

Transforming growth factor-beta elicits shape changes and increases contractility of testicular peritubular cells.

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.

Animals↗

PubMed1988

Topography and behavior of Sertoli cells in sparse culture during the transitional remodeling phase.

We report observations on the behavior of Sertoli cells in sparse culture during the period from the time of plating to the time of initial confluence (the transitional remodeling phase). Changes in shape, structure, and polarity of cells, as well as changes in migration patterns and cell-cell association patterns, have been followed during the transitional remodeling phase with the aid of topographical markers. These markers are based upon differences between ultrastructural features of the basolateral and apicolateral surfaces. The basolateral surface is characterized by plasmalemmal blebs, whereas the apicolateral surface is characterized by filopodial extensions. Structural differences observed in situ remain evident in Sertoli cells isolated by sequential enzymatic treatments that are described. Another marker is provided by laminin-binding sites, which are detected exclusively on the blebbed, basolateral surfaces of freshly prepared Sertoli cell aggregates. The orientation described is sustained during the initial radial migration of Sertoli cells explanted on uncoated glass coverslips. Under these conditions, blebs are detected only on the dorsal surfaces, and filopodial extensions are evident only on the ventral surfaces. In contrast, Sertoli cells sparsely plated on a reconstituted basement membrane (air-dried Matrigel) migrate rapidly, display an extraordinary capacity to form elaborate cytoplasmic extensions for cell-cell and cell-substratum contacts, and readily retract blebs and filopodial extensions. These cells do not form mosaic borders, whereas cells plated on uncoated glass do form a monolayer with mosaic-like borders. Cells sparsely seeded on gelated Matrigel migrate preferentially at gaps between adjacent cell explants, and develop a compact cell-cell association pattern. These cells display few, if any, cytoplasmic extensions. We compare the behavior of Sertoli cells sparsely plated on Matrigel with the behavior of Sertoli cells in situ during different stages of development.

Actins↗

PubMed1988

Rat testicular peritubular cells in culture secrete an inhibitor of plasminogen activator activity.

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.

Animals↗

PubMed1988

Modulation of Sertoli cell functions in the two-chamber assembly by peritubular cells and extracellular matrix.

A two-chamber assembly has been employed to investigate influences of peritubular cells (PC) and extracellular matrix (Matrigel) on barrier formation by Sertoli cells (SC) in culture and on transferrin production. The kinetics of transport of [3H]inulin across a Millipore filter were essentially the same in the presence or absence of Matrigel or PC. In contrast, a SC monolayer retarded the diffusion of [3H]inulin, increasing the estimated time for 50% equilibration from about 4 h to approximately 12 h. Matrigel and PC each independently further increased the equilibration time, with the largest effects elicited by the presence of Matrigel (approximately 21 h). Data have been interpreted to indicate that these two components, especially extracellular matrix, facilitate the formation of a functional barrier by SC in the two-chamber system. PC assume a more important role than Matrigel in the modulation of transferrin secretion by SC. Transferrin concentrations were higher in the inner chamber, corresponding to those in the adluminal compartment, but transferrin masses were higher in the outer chamber under the conditions described. We report the effects of the presence and absence of Matrigel, PC, and FSH on levels of transferrin secreted by SC. Addition of FSH resulted in increased transferrin secretion by SC maintained under all conditions examined. We compare our data with those previously reported by others and attempt to provide a basis for the differences observed. We discuss the properties of the system and outline major advantages and limits of the two-chamber assembly in investigations on the polarity and properties of SC in culture.

Animals↗

PubMed1987

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

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

1-Methyl-3-isobutylxanthine↗

PubMed1987

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

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

Animals↗