Interaction of rat sertoli cells with a collagen lattice in vitro

Summary Sertoli cells from rats aged 15, 20, and 25 d were subcultured onto collagen-coated, plastic dishes. If the collagen was released from the plastic surface by rimming, the floating rats of collagen showed uniform shrinkage. If the collagen was allowed to remain attached to the plastic, holes appeared in the collagen with cells from rats aged 25 d but not with those of 15 d. Cells from rats aged 20 d caused fewer and smaller holes to appear. The holes were associated with the formation of clumps of spherical cells from which elongated Sertoli cells extended into the surrounding collagen to end near holes. Rhodamine-phalloidin revealed diffusely distributed actin in the spherical cells in contrast to well-developed microfilaments in the peripheral elongated cells. Addition of cytochalasin B (5 μg/ml) to the medium prevented contraction of the floating rats and the development of holes in the attached collagen. In addition, cytochalasin B caused the peripheral cells to become spherical and to separate from the clumps. Moreover, rhodamine-phalloidin revealed that actin in the peripheral cells occurred as clumps without microfilaments when cytochalasin B was present. When Sertoli cells were subcultured onto silicone rubber films, the cells produced wrinkling of the rubber surface within 4 h of plating. These observations were interpreted to mean that Sertoli cells exert local tractional forces on various substrata. These forces require actin, presumably acting by a contractile mechanism. When the collagen is attached to plastic and the cells are organized into clumps with radiating elongated cells (cells from rats aged 25 d), the tractional forces in the elongated cells reorganize the collagen fibers to produce holes. When cells are uniformly distributed (cells from rats aged 15 d), holes are not formed. When the collagen is released from the plastic surface, tractional forces cause the floating rafts to shrink. These interactions of the cells with collagen are likely to be important in determining the shape of the Sertoli cell in vivo, the polarity of the cell, and its biochemical differentiation. This investigation was supported by grants HD 16525, AM 32236, and GM 32705 from the National Institutes of Health, and from the Shriners of North America.     

Primary rat sertoli and interstitial cells exhibit a differential response to cadmium

Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of ¹⁰⁹Cd, reduction of the vital tetrazolium dye MTT, incorporation of ³ H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl₂, were less affected. The 72-hr LC₅₀'s for Sertoli cells and interstitial cells were 4.1 and 19.6 M CdCl₂, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.Abbreviations CBF cadmium-binding factor - DMEM Dulbecco's modified Eagle's medium - FSH follicle stimulating hormone - ICC interstitial cell culture - IGF-I insulin-like growth factor-I - LC(50) 50% lethal concentration - MTT 3-(4,5-dimethylthiazol-2-yl) - SCC Sertoli cell culture This work was supported by NIEHS ESO4141 and NIH HD-08358 (AHP).Preliminary results of some of this work were reported at the 26th Annual Meeting of the Society of Toxicology (1987), Washington, DC.     

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle‐stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low‐voltage‐activated calcium channel blocker verapamil on both [³⁵S]‐sulfate and [³H]‐glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20‐day‐old rats. Verapamil induced a dose‐ and time‐dependent decrease in labeling of both secreted and cell‐associated proteoglycans, as determined by quantitative solid‐phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse‐chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP‐dependent syntheses such as estradiol production. J. Cell. Biochem. 76:322–331, 1999. © 1999 Wiley‐Liss, Inc.     

In vitro models of differentiated sertoli cell structure and function

Summary Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testic, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model. Paper presented a the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation. This work was supported by grant HD-16260 from the National Institutes of Health, Bethesda, MD, and a grant from the Mellon Foundation.     

In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken sertoli cells

To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes.     

Hormone interactions in the sertoli cells

Summary The effects of follicle-stimulating hormone (FSH) and testosterone in rat Sertoli cells were investigated in vitro by means of isolated cell populations. The Sertoli cells selectively bind FSH, and respond to FSH stimulation with increased accumulation of endogenous cyclic AMP and secretion of androgen-binding protein (ABP). FSH binding and cyclic AMP response in the Sertoli cells change dramatically during sexual maturation. Cyclic AMP response decreases despite an increase in FSH-binding receptors per cell. Evidence has been provided for the existence of cytoplasmic and nuclear androgen receptors and chromatin acceptor-sites that specifically bind the androgen-receptor complex in the Sertoli cells. A model has been proposed for the hormonal interactions in the seminiferous tubule and the possible role of Sertoli cells in mediating the hormonal effects on spermatogenesis. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This work was supported by Grant P50 HD08338 from the NICHHD. Dr. barbara M. Sanborn is a recipient of Research Career Development Award 1-K04-HD00126 (NIH).     

小鼠睾丸支持细胞体外培养特性

支持细胞是睾丸的重要组成部分,其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞,并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究,发现支持细胞的贴壁时间主要集中在接种后2～4h;当培养液中氨根离子浓度高于2.3mmol/L,渗透压高于326mosm/kg,pH≤6.8时支持细胞生长进入衰亡期;在氨基酸代谢方面,发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低,丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。     

On the morphology of the human sertoli cell

Summary As revealed by light microscopical investigations the human Sertoli cell presents different appearances according to the pattern of infranuclear cytoplasmic inclusions. Although two or three stages of spermatogenesis are seen in a single cross section of a seminiferous tubule the Sertoli cells all show virtually the same features in such a cross sectioned tubule.The different appearances are also evident under the electron microscope. Although no obvious correlation was found with the stages of spermatogenesis in the seminiferous epithelium, the Sertoli cell appearances described here may be assumed to represent different metabolic situations.Other features of Sertoli cell ultrastructure are discussed such as the presence of residual bodies in the apical cytoplasm, glycogen-rich areas protruding towards the tubular lumen or the extracellular space, and membrane bound, round structures, found between the membranes of the smooth endoplasmic reticulum and resembling the microbodies of steroid producing cells.Presented in part at the 69^th Versammlung der Anatomischen Gesellschaft, Kiel, 1974.     

Membrane molecules involved in adhesion properties of cultured sertoli cells

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody-induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS-PAGE), M_r 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.     

Follicle-stimulating hormone increases gap junction communication in sertoli cells from immature rat testis in primary culture

The gap junction communication in Sertoli cells from immature rat testes, cultured either in absence or in presence of follicle-stimulating hormone (FSH), was studied by microinjection of a fluorescent dye and by Fluorescence Recovery After Photobleaching (gapFRAP).The cells cultured for 2–4 days in the absence of FSH showed a flattened epithelial-like appearance. They were poorly coupled, as judged by the low frequency of cell-to-cell spread of microinjected Lucifer Yellow, and by the value of the rate constant of dye transfer (k) estimated in gapFRAP experiments. However, when two different subpopulations of cells were separately analyzed, namely the cells forming small groups contacting over part of their circumference (adjoining cells), and the cells arranged in tight clusters, we found that the value of k in the latter group was much higher, reaching about 75% of that obtained in the presence of FSH.The cells cultured for two days in a medium containing ovine FSH underwent striking morphological changes and presented a rounded, fibroblast-like appearance. They were arranged in networks or in clusters. The frequency of cell-to-cell dye diffusion after microinjection and the rate constant of dye transfer were rapidly increased to the same final level by FSH, although they were initially different in these two groups. A concentration dependence of k, in the range 0.05 to 3 ng/ml, was observed in the cells in networks, contrasting with an all-or-none increase in the cells in clusters.Two days after FSH withdrawal, the dye transfer constant returned to prestimulation control values in the cells in clusters, but not in the cells in networks, which maintained a stable degree of coupling comparable to that of the unstimulated cells in clusters. This observation suggests (i) that an initial promoting effect of FSH already exists in the immature rat testis, which is preserved after enzymatic treatment in the cell clusters, but not in the more dispersed cells, and (ii) that the decreased junctional coupling is re-established in the dispersed cells by FSH, through a synthesis or a membrane insertion of connexin.The effects of FSH were mimicked by a brief exposure to 1 m m dibutyryl-cyclicAMP, but not to 10 n m human chorionic gonadotropin (hCG), indicating that the gap junction communication in Sertoli cells is upregulated by FSH through a specific membrane receptor, with cyclicAMP acting as a second messenger.This work was supported by grants from the CNRS and the DRED du Ministère de l'Education Nationale, and the Fondation Langlois. Frédérique Pluciennik was a recipient of the Dufrenoy scholarship, given by l'Académie d'Agriculture de France.     

Study in vitro of the phagocytic function of Sertoli cells in the rat

Summary Aspects of the interaction between residual bodies/cytoplasts from elongated spermatids (RB/CES) and Sertoli cells were studied in vitro. Highly enriched Sertoli cells (91%; experiment A), very highly enriched Sertoli cells (>96%; experiment B), as well as peritubular cells were isolated from testes of 20-day-old rats by means of hypotonic treatment. Isolated Sertoli cells and peritubular cells were also prepared from 45-day-old rats (experiment C). RB/CES were isolated by centrifugal elutriation from testes of rats aged 90–120 days. The kinetics of adhesion of RB/CES to Sertoli cells were similar in all experiments. FSH accelerated binding of RB/CES but markedly reduced the number of RB/CES phagocytosed. Co-culture of the highly enriched Sertoli cells from experiments A and C with isolated peritubular cells did not change the kinetics of adhesion of RB/CES. However, when the contamination of Sertoli cells by peritubular cells was at a minimum (experiment B), addition of peritubular cells induced a slight but significant stimulation of the binding of RB/CES. Transmission electron microscopy revealed the following events within 24 h of co-culture: adhesion of the RB/CES to microvilli of Sertoli cells; internalization of RB/CES; lysis of the membrane of RB/CES; total digestion. Therefore, FSH and peritubular cells modulate the interaction in vitro between Sertoli cells and RB/CES, and the different steps of residual body disposal can be reproduced in co-culture. The co-culture model described in this study provides a useful system for the study of phagocytic activity by Sertoli cells.     

Sertoli细胞饲养层对小鼠精原干细胞增殖的影响

摘要 目的 以小鼠睾丸支持细胞(Sertoli)为饲养层,小鼠胚胎成纤维细胞(STO) 饲养层做对照,研究它对小鼠精原干细胞增殖的影响。方法 用无血清StemPro-34 SFM培养基培养2～5日龄小鼠精原干细胞,分别用相差显微镜观察,免疫组化法研究Sertoli饲养层对精原干细胞生物学行为的影响。结果 发现精原干细胞在Sertoli及STO两种饲养层上的一周内的生物学行为非常相似,但培养1周后,Sertoli细胞作饲养层的培养体系中保留的精原干细胞要比对照组明显增多,约有30%的精原干细胞能存活下来并能维持存活到60d以上。结论 Sertoli细胞作饲养层明显促进精原干细胞的更新增殖。     

Concomitant sertoli and leydig cell tumor of the testis: a case report

A rare intratubular gonadal stromal tumor was present in the testis of a 45-year-old man who was admitted to our hospital with the chief complaint of gradual enlargement of the left testis. Tumoral markers were negative and no extension was observed. The tumor comprised an intratubular mixture of two types of tumor cells with intercellular junctions: the predominant tumor cells were consistent with a Sertoli cell origin and cells comprising the minor population consistent with a Leydig cell origin. The patient is disease free after 6-month follow-up. The case is considered to be a testicular mixed tubular Sertoli-Leydig cell tumor. It highlights a rare type of primary tumor of the testis that features a good prognosis.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Pachytene spermatocyte protein(s) stimulate sertoli cells grown in bicameral chambers: Dose-dependent secretion of ceruloplasmin,sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin

Summary Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [³⁵S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [³⁵S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 μg/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100 μg/ml of pachytene spermatocyte protein. These results suggest that pachytene spermatocytes modulate Sertoli cell secretory function of at least four proteins in the regulation of spermatogenesis. Supported by grant #DCB-8915930 (D. D.) from the National Science Foundation, Washington, DC.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.