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At least 37 records · Page 2Linked to original sources
PubMed1984

Changes in testicular function induced by short-term exposure of the rat testis to heat: further evidence for interaction of germ cells, Sertoli cells and Leydig cells.

This study was designed to determine the effects of a short episode of testicular heating (43 degrees C for 15 min) on spermatogenesis and Sertoli and Leydig cell function. Rats killed at intervals up to 156 days after heating were assessed by histological examination, and by measurement of serum FSH and LH, and by tests of Sertoli cell function consisting of fluid production, androgen binding protein (ABP) content of the ligated and unligated tests, together with the binding of [125I]FSH. Leydig cell function was assessed by in vitro testosterone production, serum testosterone levels and [125I]hCG binding to testes homogenates. Testis weight declined 7 days after heating to 70% of control and remained lower until 82 days, whereas epididymal weight did not decrease significantly until 26 days and also recovered by 82 days. Fluid production was significantly lower in heated testes at 26 days and returned to normal at 56 days. ABP production measured as the difference between the ABP content of ligated and unligated testes was significantly reduced at 14 and 26 days, but subsequently recovered. Serum FSH levels were significantly elevated from 14-26 days in the heat treated group and the binding of [125I]FSH was reduced at 26 days post-heating. Basal and stimulated in vitro T production was significantly increased in the heat-treated testes at 14 days and subsequently returned to normal whilst [125I]hCG binding was significantly lower in the heat-treated testes from 7-26 days. Serum T and LH did not alter significantly during the study. Primary spermatocytes and young spermatids were the most heat sensitive germ cell type and a reduction in spermatogenesis was noted from 7 to 26 days, although recovery appeared complete by 56 days and thereafter. These results demonstrate that the transient spermatogenic disruption induced by heating is accompanied by significant alterations in Sertoli and Leydig cell function which are identical to those produced in other models of spermatogenic dysfunction. The results suggest that the duration of these changes appears to correlate closely with alterations occurring in the germ cell compartment.

Androgen-Binding Protein↗
PubMed1983

Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells.

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Animals↗
PubMedDate not supplied

Secretion of androgen binding protein by Sertoli cells is influenced by contact with germ cells.

Sertoli cells were cultured alone or with germ cells to evaluate the effect of the association with germ cells on the secretory activity of Sertoli cells. Secretion of androgen-binding protein, which is specifically secreted by Sertoli cells, was measured under several experimental conditions. The following experimental models were utilized: 1) cultures of explants of seminiferous epithelium from prepubertal animals in which germ cells adherent to Sertoli cells are present (Sertoli cell enriched cultures); 2) monolayers formed only by Sertoli cells, obtained by removing germ cells from Sertoli cell enriched cultures, and 3) cocultures of Sertoli cell only cultures and germ cell populations at defined stages of differentiation. The results obtained indicated that FSH-induced ABP secretion was greatly reduced in Sertoli cell only cultures as compared to enriched Sertoli cell cultures, and that this difference was stable throughout the first eight days of culture. In addition, cocultures of Sertoli cell only cultures with germ cells induced an increase of ABP when cocultured germ cells were at differentiation stages, such as pachytene spermatocytes, which are able to recognize and firmly adhere to the Sertoli cell monolayers. Cocultures with round spermatids, which do not adhere to Sertoli cells, did not increase the amount of FSH-induced ABP production. The addition of nongerminal cells such as lymphocytes and fibroblasts were also not effective in stimulating ABP secretion. Surface interaction between Sertoli cells and cocultured germ cells seemed to be necessary for this FSH-induced ABP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen-Binding Protein↗
PubMed1989

Influence of germ cells on Sertoli cell secretory activity in direct and indirect co-culture with Sertoli cells from rats of different ages.

We examined the effect of direct and indirect Sertoli-germ cell co-culture on androgen binding protein (ABP) and transferrin (TRF) secretion by Sertoli cells (Sc) from 10-, 18-, and 26-day-old rats. Addition of germ cells (Gc), mainly (greater than 80%) pachytene spermatocytes, directly to Sc monolayers enhanced basal and follicle-stimulating hormone (FSH) + testosterone-stimulated ABP and TRF secretion at all three ages. When the Gc were co-cultured indirectly with Sc (separated by a Nucleopore filter), only 50% of the direct stimulatory effect was found at 18- and 26-day-old groups, whereas no difference between direct and indirect co-culture was noted with Sc from 10-day-old rats. With 18- and 26-day-old rat Sc, the Gc effect on ABP and TRF secretion declined after 6 days of Sc culture, reaching the level of Sc-only cultures after 10 days, whereas the direct effect was maintained throughout the entire culture period. With Sc from 10-day-old animals, both direct and indirect effect of Gc decreased after 6 days but the levels of ABP and TRF secretion remained above those of Sc-only cultures. The viability and number of Gc in indirect co-cultures were maintained significantly higher than in Gc-only control cultures. The direct and indirect Gc effect was completely reversed 48 h after the Gc were removed from Sc cultures of 18- and 26-day-old rats, whereas in Sc cultures from 10-day-old rats 40% of the stimulatory effect remained after 48 h of Gc removal. We conclude that Gc can influence Sc secretory activity through both direct contact and some released factor(s). These two pathways may have different relevance at different ages during sexual maturation.

Aging↗
PubMed1986

Inhibition of RNA and DNA synthesis in Sertoli cells by co-culture with spermatogenic cells.

Sertoli cell monolayers were prepared from 30-day-old rat testes and cultured for 7 days to eliminate contaminant germ cells. Some of these monolayers were co-cultured with a spermatogenic cell preparation enriched in pachytene spermatocytes and round spermatids (fraction 3 from a Percoll gradient), isolated from 30-day-old rat testes. After co-culture for 4 to 48 h, germ cells were removed. RNA synthetic activity in rat Sertoli cell cultures alone was 216,800 +/- 66,480 dpm [3H]uridine.2h-1 X 10(6) cells-1 (mean +/- SD) compared to 98,390 +/- 23,595 in rat Sertoli cells which had been co-cultured with germ cells of fraction 3 for 24 h (P less than 0.01). By contrast, RNA synthesis in Sertoli cell monolayers prepared from immature pigs were unaffected by co-culture with rat germ cells. A similar inhibitory effect of germ cells was observed in rat Sertoli cells stimulated with FSH or testosterone. Culture medium, conditioned by 20 h culture of a fresh preparation of rat spermatogenic cells of fraction 3, was active in inducing the inhibitory effect on RNA synthesis in rat Sertoli cells. Co-culture of rat Sertoli cells with germ cells of this fraction also decreased the incorporative of [3H]thymidine into DNA in rat Sertoli cells, from 9061 +/- 3339 to 4766 +/- 526 dpm.2h-1 X 10(6) cells-1 (P less than 0.01), but no such change was found in pig Sertoli cells. A different spermatogenic cell preparation, partially deprived of pachytene spermatocytes (fraction 5), stimulated rat Sertoli cell DNA synthesis (Sertoli alone 7833 +/- 2550, Sertoli cells which had been in co-culture with germ cells of fraction 5, 13,300 +/- 2279 dpm.2h-1 X 10(6) cells-1, P less than 0.05). These inhibitory actions of some germ cells on Sertoli cells were observed together with the previously reported simultaneous stimulatory effect of Sertoli cells on germ cells. These Sertoli cell-germ cell interactions of detected in culture may represent regulatory influences operating in vivo.

Animals↗
PubMed1995

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells.

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5 x 10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 micrograms/cm2) in 0.5 ml McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 ml serum-free medium with or without various hormones or other compounds. PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed. Monkey Sertoli cells were also capable of secreting PAI-1. Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1999

Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells.

Sertoli cell differentiation is initiated in the embryo to promote testicular development and male sex determination. In the adult, Sertoli cells are critical for maintenance of the spermatogenic process. Previously, Sertoli cell differentiation has been shown to be regulated in part by basic helix-loop-helix (bHLH) transcription factors. This was based on the observation that promoters of a number of Sertoli cell genes contained bHLH-responsive E-box response elements and that overexpression of Id, a negatively acting HLH protein, down-regulates Sertoli cell differentiated functions. Analysis of Sertoli cell bHLH proteins demonstrated the expression of REBalpha in Sertoli cells. REBalpha and REBbeta are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REBalpha and REBbeta has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REBalpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REBalpha or REBbeta demonstrated that Sertoli cells express only REBalpha, not REBbeta. REBbeta was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REBalpha is not hormone responsive. REBalpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REBalpha is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REBalpha in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REBalpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REBalpha isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REBalpha.

Animals↗
PubMed1988

Isolation and characterization of rat fetal Sertoli cells.

Sertoli cells were prepared from fetal rats, aged 15, 18, and 21 days. Cultures of these cells can be prepared at a purity of 85% by a method that is widely used to prepare the same cells from postnatal rats. The fetal cells are identical in appearance to postnatal Sertoli cells. Fetal Sertoli cells round up in response to (Bu)2cAMP, but not in response to FSH. Protein synthesis in the cells is not stimulated by (Bu)2cAMP or FSH. However, incorporation of [35S]methionine into secreted proteins appearing in the incubation medium is stimulated by the cyclic nucleotide, but not by FSH. Reproducible patterns for the incorporation of [35S]methionine into cellular and secreted proteins are presented as autoradiograms of one- and two-dimensional electrophoretograms. These autoradiograms show that the response of secreted proteins to (Bu)2cAMP is a general one; most or all proteins participate in the response. No clear differences were observed in the nature of the proteins synthesized when cells from fetal rats of 15, 18, and 21 days of age were compared. Fetal Sertoli cells produced approximately 2 nmol lactate/10(6) cells.h, which is approximately one fifth of the amount produced by postnatal cells. Lactate production was increased by the addition of FSH or (Bu)2cAMP to the medium. Fetal Sertoli cells also synthesize androgen-binding protein, and this activity is not increased by either FSH or (Bu)2cAMP.

Androgen-Binding Protein↗
PubMed1991

Partial characterization of a unique mitogenic activity secreted by rat Sertoli cells.

Sertoli cell conditioned medium (SCCM) contains a potent mitogen, Sertoli cell secreted growth factor (SCSGF). A431 cells, derived from a human epidermoid carcinoma have provided an excellent model cell line for the study of this apparently unique activity secreted by rat Sertoli cells in vitro. Previously, it was shown that SCCM contained an epidermal growth factor (EGF)-like activity which was thought to be the mitogen for A431 cells. The present study showed that these two factors are distinct entities. The secretion of the EGF-like activity decreased with increasing number of culture days, while that of SCSGF and of another Sertoli cell specific protein, transferrin remained constant. The addition of SCCM stimulated whereas 2.5 ng/ml EGF inhibited the A431 cell growth. The proliferative response of A431 cells to a wide variety of growth factors and known Sertoli cell secretions was investigated. SCSGF was the only growth factor of known Sertoli cell secretions tested (transforming growth factors (TGF alpha, TGF beta), EGF, bombesin, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), prostaglandins E-1 and E-2, insulin, transferrin and lactate) which stimulated A431 cell proliferation. SCSGF was mitogenic for A431 cells even in the presence of serum in the culture medium. The partially purified SCSGF was heat- and acid-stable, protease-sensitive with a molecular weight of 14,000. It did not bind to heparin or concanavalin A-Sepharose. The secretion of a mitogenic activity by the Sertoli cell which is different from other previously identified growth factors and which coincides with active spermatogenesis could have important implications in the regulation of spermatogenesis.

Animals↗
PubMed1988

Age-related and testicular regression-induced changes in adenosine 3',5'-monophosphate responses in cultured hamster Sertoli cells.

Sertoli cells prepared from adult hamsters with maximally regressed testes responded to FSH with an increased accumulation of intracellular cAMP similar to that of Sertoli cells from an immature animal. That is, the age-dependent decline in responsiveness of Sertoli cells to FSH was reversed during testicular regression. To determine whether this testicular regression-induced restoration of FSH responsiveness was mechanistically a reversal of the age-dependent decline in response, the ability of cholera toxin, forskolin, catecholamines, and pertussis toxin to stimulate cAMP accumulation in Sertoli cells cultured from hamsters undergoing testicular regression was assessed and compared to the responses of Sertoli cells from 18- and 36-day-old hamsters. The age-related decline in responsiveness was evident not only with FSH but also when cells were stimulated with isoproterenol, cholera toxin, forskolin, or pertussis toxin singly or in combination. While the FSH response of Sertoli cells from regressed testes was restored to values indicative of an immature Sertoli cell, the response of the cultured cells to cholera toxin, forskolin, catecholamines, or pertussis toxin either singly or in combination suggested that the adenylate cyclase system of Sertoli cells from regressed testes was unchanged from its "adult" activity. Thus, our original hypothesis that testicular regression/recrudescence was a mirror image of sexual maturation was an oversimplification. However, since FSH is the physiological regulator of Sertoli cell function, and its response is restored to levels found in the immature animal during testicular regression and declines to adult levels during testicular recrudescence, our original hypothesis that testicular recrudescence mimics sexual maturation (i.e. the animal goes through "puberty" each time it goes through a regression/recrudescence cycle) is still tenable. However, at the molecular level, differences in mechanism exist.

Adenosine Triphosphate↗
PubMed1999

E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the presence of a number of conserved response elements that have previously been shown to regulate cell specific expression of the human transferrin (hTf) promoter. One of these elements is the human PRII region, which is a cAMP response element (CRE)-like element that is more than 80% conserved in the mTf promoter. The activation of the hTf promoter by FSH and cAMP in rat Sertoli cells has been shown to be mediated in part through the CRE-like PRII region and binding of the CRE binding protein (CREB). The present study investigates the role of PRII in the activation of mTf promoter by FSH and cAMP in rat Sertoli cells. Mutations in the PRII of the mTf promoter reduced FSH activation by only 50% and cAMP activation by more than 90%. In contrast, the mutant PRII mTf promoter construct was fully activated by a partially purified testicular paracrine activity PModS(S300). Gel shift experiments demonstrated that proteins that can bind a consensus CRE oligonucleotide also bind the PRII region of the mTf promoter. An immunoblot confirmed that CREB binds the PRII and promotes the gel shift observed. The hypothesis developed was that another cis-acting element in addition to the CRE-like PRII is also involved in FSH actions. A conserved response element in both the mTf and hTf promoters is the basic helix-loop-helix (bHLH) responsive E-box sequence. Both FSH and PModS (S300) activity were found to promote a mTf E-box gel shift that contained the E2A gene product the bHLH protein E47. Interestingly, mutations in the E-box of the mTf promoter completely abolished the PModS(S300) activation and partially (52%) inhibited the activation by FSH. In contrast, the mutant E-box mTf promoter construct was fully activated by cAMP. Finally a double mutation of both the PRII and the E-box completely abolished FSH activation of the mTf promoter. These results suggest that optimal activation of the mouse transferrin promoter by FSH requires both CREB binding to the CRE-like PRII region and bHLH binding to the E-box. Information is provided that indicates a number of Sertoli cell promoters contain a close association of E-box and CRE-like elements. Observations are discussed in regards to the potential interactions of the CRE and E-box response elements in mediating FSH actions in Sertoli cells.

Animals↗
PubMed1986

Production of components of extracellular matrix by cultured rat Sertoli cells.

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.

Animals↗