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PubMed1986

Cell-cell interaction between rat Sertoli cells and mouse germ cells in vitro.

An antiserum against rat germ cell membranes was prepared, and after absorption with protein extracts of rat liver and kidney and mouse testis, this antiserum reacted only with rat germ cell membranes and juxtanuclear vesicles in rat Sertoli cells. Germ cell-free rat Sertoli cell monolayers were cultured in vitro. Freshly isolated mouse germ cells adhered to these monolayers within 1 h. After a minimum of 3 days of such a co-culture, immunofluorescence and immunoblotting revealed that the mouse germ cells had obtained rat-antigenic determinants in their membranes. Our results indicate that this appearance of rat-specific antigens on mouse germ cells is specific and inducible.

Animals↗
PubMed1996

Neonatal administration of FSH increases Sertoli cell numbers and spermatogenesis in gonadotropin-deficient (hpg) mice.

We previously demonstrated that androgens alone, in the complete absence of gonadotropins, initiated qualitatively complete spermatogenesis in hypogonadal (hpg) mice. Although germ cell to Sertoli cell ratios were normal in hpg mice with androgen-induced spermatogenesis, testicular size. Sertoli cell and germ cell numbers only reached 40% of those of non-hpg mice, and Sertoli cell numbers were unaffected by androgen treatment started at 21 days of age. We postulated that these observations were due to diminished gonadotropin-dependent. Sertoli cell proliferation during perinatal life while the Sertoli cells still exhibited normal carrying capacity for mature germ cells. In order to test this hypothesis, we examined the effects of administering androgens and gonadotropins to hpg mice during the first 2 weeks of postnatal life when Sertoli cells normally continue to proliferate. The study end-points were Sertoli and germ cell numbers in hpg mice following induction of spermatogenesis by 8 weeks treatment with 1 cm subdermal silastic testosterone implants. Newborn pups (postnatal day 0-1) were injected s.c. with recombinant human FSH (rhFSH) (0.5 IU/20 microliters) or saline once daily for 14 days, with or without a single dose of testosterone propionate (TP) (100 micrograms/20 microliters arachis oil) or human chorionic gonadotropin (hCG) (1 IU/20 microliters). Untreated hpg and phenotypically normal littermates were studied as concurrent controls. At 21 days of age, all treated weaning mice received a 1 cm silastic subdermal testosterone implant and, finally, 8 weeks after testosterone implantation, all mice were killed. As expected, qualitatively complete spermatogenesis was induced in all groups by testosterone despite undetectable circulating FSH levels. Exogenous rhFSH increased testis size by 43% (P < 0.002) but a single neonatal dose of either TP or hCG reduced the FSH effect although neither TP nor hCG had any effect alone. In contrast, a single neonatal dose of TP or hCG increased final seminal vesicle size whereas FSH had no effect. FSH and TP treatment significantly increased absolute numbers of testicular spermatids compared with saline treatment, whereas hCG and TP significantly increased testicular sperm when expressed relative to testis size. Stereological evaluation of Sertoli and germ cell numbers demonstrated a rise in the absolute numbers of Sertoli and all germ cell populations induced by neonatal administration of hormones. When expressed per Sertoli cells the numbers of germ cells in the treated mice were between 85 and 90% of non-hpg controls. We conclude that exogenous FSH treatment during the first 2 weeks of postnatal life, coinciding with the natural time of Sertoli cell proliferation, increases Sertoli cell numbers and thereby the ultimate size of the mature testis and its germ cell production. Thus neonatal gonadotropin secretion may be a critical determinant of the sperm-producing capacity of the mature testis. In addition, neonatal exposure to androgens could be important for the imprinting of sex accessory organs in hpg mice, with the long-term effects of altering the sensitivity of the accessory organs to exogenous testosterone later in life.

Animals↗
PubMed2006

Human sex hormone-binding globulin is expressed in testicular germ cells and not in sertoli cells.

The human sex hormone-binding globulin ( SHBG) gene contains at least two transcription units. A 4.3 kb human SHBG transcription unit encodes the precursor polypeptide, which is processed and secreted by hepatocytes as plasma SHBG. The proximal promoter of this transcription unit differs from the corresponding sequence in other mammals, in which it is also expressed in Sertoli cells. In particular, its proximal promoter sequence contains a binding site for USF transcription factors that represses its activity in Sertoli cells. Although human SHBG is not expressed in Sertoli cells, human SHBG transcripts containing an alternative exon 1 sequence are present in testicular germ cells. These are the products of an approximately 8 kb human SHBG transcription unit, and they appear to encode an SHBG isoform that is 4 - 5 kDa smaller than plasma SHBG. This sperm SHBG isoform accumulates between the outer acrosomal membrane and the sperm plasma membrane, and it is released during the capacitation reaction. These remarkable differences in human SHBG expression in the testis, when compared to other mammals, force us to reconsider the functional significance of SHBG expression in the testis in relation to male reproduction.

Alternative Splicing↗
PubMed2001

Follicle-stimulating hormone and leukemia inhibitory factor regulate Sertoli cell retinol metabolism.

Sertoli cells, the somatic epithelial cells of the seminiferous tubules, provide both structural and biochemical support for developing male germ cells. The Sertoli cells are targets of retinoid action in the testis. We have found that FSH, (Bu)(2)cAMP, and leukemia inhibitory factor elicit substantial changes in the metabolism of [(3)H]retinol (vitamin A) in primary cultures of purified rat Sertoli cells. Addition of (Bu)(2)cAMP for 2 h or FSH for 6 h results in a 3-fold increase in the metabolism of [(3)H]retinol to [(3)H]retinoic acid ([(3)H]RA); the esterification of [(3)H]retinol to [(3)H]retinyl esters, especially [(3)H]retinyl palmitate, is also increased by approximately 5-fold. The addition of 1 microM all-trans-RA also elicits changes in [(3)H]retinol metabolism, but in this case the metabolism of [(3)H]retinol to [(3)H]RA is inhibited, whereas the metabolism of [(3)H]retinol to [(3)H]retinyl esters is increased by over 50-fold. Leukemia inhibitory factor increases the esterification of [(3)H]retinol by 2- to 3-fold. FSH leads to a reduction in the level of cellular retinol binding protein I transcripts, whereas RA increases the cellular retinol binding protein I messenger RNA level by about 2-fold at approximately 24 h. Levels of AHD-2 (aldehyde dehydrogenase-2) and RALDH-2 (retinaldehyde dehydrogenase-2) messenger RNAs, which encode enzymes that convert [(3)H]retinaldehyde to [(3)H]RA, are increased by about 2-fold by FSH, whereas no change in CYP26 (RA hydroxylase) expression is seen. Our results suggest that one function of FSH (and/or (Bu)(2)cAMP) in Sertoli cells is to increase the metabolism of retinol to the biologically active metabolite RA and to retinyl esters.

Animals↗
PubMed1994

In vitro Sertoli cell bioassay of follicle-stimulating hormone (FSH)-serum from different animal species alters the morphology of rat Sertoli cells without affecting their response to FSH.

Follicle-stimulating hormone (FSH) stimulates aromatase activity of immature rat Sertoli cells cultured in vitro in the presence of an androgen substrate, a phenomenon that can be used to measure FSH bioactivity. This paper analyzes the effects of sera from various animal species on both aromatase activity and morphological aspects of cultured rat Sertoli cells. Treatment of Sertoli cells with increasing concentrations of serum from humans, monkeys, rats, hamsters, and mice gave a dose-dependent stimulation of estradiol production parallel to the response obtained with FSH standard. Serum from these species and from rabbits also had a characteristic morphological effect on the cells, to produce a fibroblast-like aspect and clumping. This effect was dose-dependent, was increased by the addition of FSH, and was eliminated by heating the sera at 56 degrees for 30 min prior to incubation with the cells. Clumping did not interfere with the aromatase activity of Sertoli cells and did not cause the cellular response to deviate from parallelism with the standard curve. Heat treatment of FSH standard and serum samples did not significantly change the aromatase activity. FSH bioactivity could be measured accurately in intact and castrated monkeys. It is concluded that the Sertoli cell aromatase bioassay can be applied directly to the measurement of serum bioactive FSH in several animal species without any preliminary treatment of the serum samples.

Animals↗
PubMed1998

Stage- and subcellular-specific expression of Id proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis, and Sertoli cell function.

Immunohistological detection of each of the four Id proteins (Id1-Id4) in sections of mouse testis revealed a unique temporal and spatial expression pattern for each Id protein during spermatogenesis. Furthermore, each Id protein displayed a distinctive, dynamic pattern of subcellular distribution. Id1 was uniquely expressed in MI/MII spermatocytes undergoing meiotic division. Id4 protein was detectable in the cytoplasm of type A1 spermatogonia, as well as in late pachytene and in diplotene spermatocytes. Id2 protein, which was most abundant in Sertoli cell nuclei, was also detectable in pachytene and diplotene spermatocytes, but as with Id4, it was absent from MI/MII cells. In postmeiotic spermatids, Id1, Id2, and Id4 proteins were expressed in a stage- and subcellular-specific manner. Expression of Id3 was restricted to Sertoli cell cytoplasm. In malignant seminoma cells, all four Id proteins were abundantly expressed with accompanying changes in their subcellular distribution. The observed expression of Id proteins in postproliferative Sertoli cells and spermatids and during specific stages of meiosis implies novel functional roles for this class of transcriptional regulator during spermatogenesis.

Animals↗
PubMed1995

Liver-regulating protein (LRP) is a plasma membrane protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes.

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene spermatocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and spermatids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interaction-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocultures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte- and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.

Animals↗
PubMed1984

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells.

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.

Animals↗
PubMedDate not supplied

The action of calcitonin on the TM4 Sertoli cell line and on rat Sertoli cell-enriched cultures.

The effects of synthetic salmon calcitonin on primary Sertoli cell-enriched cultures and on an established cell line (TM4 cells, derived from immature mouse Sertoli cells) were studied. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in both cell systems. In addition, this peptide stimulated the secretion of rABP in primary Sertoli cell-enriched cultures prepared from rat testis. Calcitonin also increased the total concentration of both androgen and estrogen receptors in TM4 cells. Because cAMP analogs decreased androgen and estrogen receptor concentrations, the effect of calcitonin on sex steroid receptors may not be mediated by its effect on cyclic AMP in these cells. The possibility that the action of synthetic salmon calcitonin on the receptors might be mediated by a change in cellular Ca2+ was investigated. Lowering extracellular Ca2+ concentrations from 1.5 mM to less than 0.01 mM markedly reduced the concentration of androgen and estrogen receptors; restoration of Ca2+ to 1.5 mM returned receptor levels to normal. When the receptor concentrations were decreased by lowering extracellular Ca2+ concentrations to 0.5 mM, treatment with the calcium ionophore, A23187, restored receptor levels to normal. Although the calcium channel blocker, verapamil, decreased receptor levels, calcitonin partially counteracted its effect. Trifluoperazine, an inhibitor of calmodulin, also diminished androgen and estrogen receptor, levels in the cytosol of TM4 cells. It was concluded that calcitonin stimulates the formation of cyclic AMP and the secretion of rABP by Sertoli cells. This peptide also increases the concentration of androgen and estrogen receptors, possibly by a mechanism that is, in part, Ca2+ -mediated. These results, along with those on Leydig cells, suggest that calcitonin could be a regulator of testicular function.

Androgen-Binding Protein↗
PubMed2004

Endotoxin and proinflammatory cytokines modulate Sertoli cell proliferation in vitro.

Sertoli cells play a key role in testicular function and their final number in the adult testis determines the capacity of germ cell production. Sertoli cell proliferation, stimulated by FSH and paracrine factors, occurs only in fetal and prepubertal life and may be an important target of pathogenic influences affecting testis development. We used a Sertoli cell proliferation assay to address the question whether if bacterial endotoxin (lipopolysaccharide; LPS) and proinflammatory cytokines could influence early postnatal Sertoli cell development. LPS and tumor necrosis factor-alpha (TNF-alpha) dose-dependently stimulated proliferation of primary cultures of isolated Sertoli cells from 8- to 9-day-old rats, assessed by (3)H-thymidine and BrdU incorporation. LPS also significantly increased the number of living cells in culture, measured by supravital staining. Interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) had no direct effect on Sertoli cell growth, but were found to modulate FSH action. IL-6 increased, while IFN-gamma inhibited, FSH-induced Sertoli cell DNA-synthesis. We conclude that endotoxin and TNF-alpha are potent direct stimulators of Sertoli cell proliferation in vitro, and that IL-6 and IFN-gamma can modulate the mitogenic action of FSH on immature Sertoli cells. This may contribute to the pathogenesis of testicular damage after infections and inflammatory diseases in fetal and early postnatal life, with subsequent disturbance of adult germ cell production.

Animals↗
PubMed1993

Culture of intact Sertoli/germ cell units and isolated Sertoli cells from Squalus testis. II. Stimulatory effects of insulin and IGF-I on DNA synthesis in premeiotic stages.

To investigate growth control mechanisms during spermatogenesis in vitro, [3H]thymidine incorporation into acid-insoluble macromolecules was used to quantify DNA synthesis in cultured spermatocysts (intact Sertoli cell/germ cell clones) derived from premeiotic (PrM), meiotic (M), and postmeiotic (PoM) regions of dogfish (Squalus acanthias) testis. Forty-eight hours after seeding in basal medium, DNA synthesis was > 7-fold higher in PrM cysts than in other stages, thus verifying the staging procedure. In autoradiograms, germ cells of PrM cysts (e.g., spermatogonial and preleptotene stages) were labeled all-or-none, but not all cysts were labeled, and later developmental stages (e.g., cysts with round or elongating spermatids) were never labeled. Fetal bovine serum (FBS, 10%) and insulin-transferrin-selenite (ITS, 10 micrograms-10 ng/ml) doubled DNA synthesis in PrM cyst cultures but had no effect at other stages. Bovine insulin (10 micrograms/ml) and human recombinant insulin-like growth factor-I (IGF-I, 15 ng/ml) also doubled [3H]thymidine uptake in PrM cultures, but lower doses were less effective and estradiol-17 beta, transferrin, adult shark serum, purified shark relaxin, and a variety of other known growth factors were neither stimulatory nor inhibitory at the doses and conditions tested. Sertoli cell monolayers derived from PrM- or M-stage spermatocysts displayed a dose-response increase in DNA synthesis after addition of IGF-I (15-75 ng/ml), with a maximal increment significantly greater than with 10% FBS. Using [3H]thymidine incorporation by PrM cysts as an end-point, stimulatory bioactivity was detected in the < 30,000 kDa fraction of spent media from PrM Sertoli cells, whereas the low molecular weight fraction of M-stage Sertoli cells was inhibitory. Gel electrophoretic analysis of the two fractions revealed qualitative and quantitative differences in protein banding patterns, reinforcing the view that secretory activity of Sertoli cells is stage related. Results of these studies implicate insulin/IGF-I in mechanisms governing proliferation of male germ cells and support the view that Sertoli cells have an autocrine or paracrine role as both targets and sources of growth regulatory factors.

Analysis of Variance↗
PubMed2003

Thyroid hormone regulates the cell cycle inhibitor p27Kip1 in postnatal murine Sertoli cells.

Thyroid hormone regulates early postnatal Sertoli cell proliferation. Transient neonatal hypothyroidism allows prolonged postnatal Sertoli cell mitogenesis and doubles adult Sertoli cell numbers, testis weight, and sperm production. The mechanism of this effect is unknown. Cell proliferation is stimulated by cyclins and cyclin-dependent kinases and inhibited by cyclin-dependent kinase inhibitors (CDKIs). T(3) regulates the CDKI p27(Kip1) in other cell types, and mice lacking p27(Kip1) have increased testis size. To test the hypothesis that T(3) regulates Sertoli cell mitogenesis by acting through p27(Kip1), we compared expression of p27(Kip1) in Sertoli cells of testes from euthyroid, hypothyroid, and hyperthyroid mice. At postnatal d 5-25, testes were collected and immunostained for p27(Kip1) expression, or Sertoli cells were isolated enzymatically and used for p27(Kip1) Western blotting. p27(Kip1) immunostaining was low in rapidly proliferating 5-d-old Sertoli cells but had increased strongly in nonproliferating 25-d-old Sertoli cells. p27(Kip1) immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 d, consistent with increased Sertoli cell proliferation in these mice. Western blotting corroborated the p27(Kip1) immunostaining, and p27(Kip1) expression was greater in Sertoli cells from control compared with hypothyroid mice at postnatal d 10 and 16, but p27(Kip1) expression was comparable by d 25. Hyperthyroidism increased p27(Kip1) immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-d-old hyperthyroid mice expressed more p27(Kip1) than control mice. These results indicate that thyroid hormone status affects p27(Kip1) expression in neonatal Sertoli cells, suggesting that T(3) effects on Sertoli cell proliferation may be mediated through this CDKI.

Age Factors↗
PubMed2003

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation.

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.

Animals↗
PubMed1989

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

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

Androstenedione↗
PubMed1997

Testin is tightly associated with testicular cell membrane upon its secretion by sertoli cells whose steady-state mRNA level in the testis correlates with the turnover and integrity of inter-testicular cell junctions.

Testin, a Sertoli cell secretory protein whose mRNA is predominantly expressed in the testis, was shown to become tightly associated with Sertoli cell membrane upon its secretion whose solubilization requires the use of a detergent such as SDS. In the in vitro studies using Sertoli cells cultured at high cell density, where specialized junctions were being formed, the concentration of "soluble" testin in the spent media was greatly reduced versus monolayer cultures at low cell density, where specialized junctions were absent. Conversely, the concentration of "membrane-bound" testin from detergent-solubilized Sertoli cell membrane extract was positively correlated to the existence of specialized junctions in these cultures. In normal rat testes, the level of radioimmunoassayable soluble testin in the cytosol was low. However, when the inter-testicular cell junctions were disrupted either by a drug treatment such as lonidamine in vivo or by a physical treatment in vitro such as exposing Sertoli-germ cell co-cultures where specialized junctions were formed to a hypotonic treatment, a drastic surge in the testin gene expression was noted. Thus, testin can become tightly associated with Sertoli cell membrane upon its secretion when intercellular junctions are formed. It is also a marker to monitor the integrity of inter-testicular cell junctions.

Animals↗
PubMed2005

Expression of androgen and estrogen receptors in sertoli cells: studies using the mouse SK11 cell line.

Sertoli cells (Sc) play a major role in the establishment and maintenance of spermatogenesis. In the adult testis, Sc contain androgen receptor (AR) and estrogen receptor (ER)-beta but exhibit a loss of steroid responsiveness when maintained in primary culture. In the present study, we demonstrated that a transformed murine cell line (SK11) has retained a Sc phenotype and remains steroid responsive. SK11 cells expressed mRNAs found in Sc (aromatase, sulfated glycoprotein-1, sulfated glycoprotein-2, GATA-1, Sry-type high-mobility-group box transcription factor-9, testatin, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) including those for AR and ERbeta but not ERalpha. AR and ERbeta were immunolocalized to cell nuclei, and their ability to activate gene expression was investigated using transient transfections with reporter constructs containing either 3xERE or pem-androgen-responsive element promoters. Expression of the 3xERE reporter was induced after incubation with 17beta-estradiol (E2), 5alpha-androstane-3-beta, 17beta-diol (3betaAdiol), or testosterone (T); up-regulation of the pem-androgen-responsive element reporter was detected only in the presence of T or dihydrotestosterone. Activation of the ERE reporter did not occur after targeted knockdown of ERbeta mRNA. Expression of AR and ERbeta mRNAs was increased after incubation of cells with T or E2, respectively. In conclusion, we have demonstrated that the SK11 Sc cell line contains functional AR and ERbeta and that treatment of the cells with their respective steroids results in an increase in the amount of their mRNAs. Our results suggest that E2 or 3betaAdiol acting via ERbeta might modulate Sc function in vivo and that SK11 cells provide a useful model that can be used to complement studies using Sc selective gene ablation.

Androgens↗
PubMed2005

Method for evaluating quality of cultured neonatal pig Sertoli cells.

BACKGROUND: Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two-step enzymatic digestion with limited assessment of the culture. Here we describe a one-step method, and a series of tests for determining purity, viability, and function of the cultured cells. METHODS: We isolated SC from neonatal pigs using Liberase HI digestion. Viability and apoptosis of cultured cells were measured by flow cytometry with propidium iodide and annexin, respectively. Specific identification of the Sertoli type was made by immunodetection of Sox9, vimentin, and Mullerian inhibiting substance. Moreover, for functionality we were able to detect clusterin in the cultured cells by Western blot. RESULTS: Our isolation method had a yield and purity similar to previous reports measured with two-step methods. Viability was 95.22 +/- 0.57% and apoptotic cells were 10.5 +/- 0.32% after 48 h in culture. At 7 days, practically all cells expressed Sox9, Mullerian inhibiting substance, clusterin, and vimentin. CONCLUSIONS: We describe an alternative strategy for preparing and identifying cultured SC for further assays of metabolic activity or in transplantation models. Establishing a one-step Liberase-digestion method for isolation, evaluating viability and apoptosis by more sensitive methods, and detecting specific markers in culture can help to evaluate the quality of cultured cells. Specific cell markers for identifying SC may be critical when identifying SC outside the testis, in contrast with vimentin which is useful only for in situ cells.

Animals↗
PubMed1991

Detergent-solubilized proteoglycans in rat testicular Sertoli cells.

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.

Animals↗