Immature Sertoli Cells Research Articles

Interactions between immature porcine Leydig and Sertoli cells in vitro. An ultrastructural and biochemical study.

Interactions between Leydig and Sertoli cells, as well as a stimulatory effect of FSH on Leydig cell activity, have been reported in many studies. In order to investigate these interactions, the ultrastructure of immature pig Leydig cells under different culture conditions has been studied. When cultured alone in a chemically defined medium, there is a marked regression of the Leydig cell smooth endoplasmic reticulum and a swelling of the mitochondria. Addition of FSH or hCG does not prevent these phenomena. Co-culturing of Leydig cells with Sertoli cells from the same animal maintains the smooth endoplasmic reticulum at the level seen in vivo and in freshly isolated Leydig cells. The addition of FSH to the co-culture stimulates its development and increases Leydig cell activity, as assessed by an increase in hCG binding sites and an increased steroidogenic response to hCG. These results suggest that Sertoli cells exert a trophic effect on Leydig cells, and that the stimulatory effect of FSH on Leydig cell function is mediated via the Sertoli cells. These results reinforce the concept of a local regulatory control of Leydig cell steroidogenesis.

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.

Autoimmune orchitis can be induced by thymic lymphocytes autosensitized against syngeneic sertoli cells in vitro.

Thymic lymphocytes from normal inbred Lewis/Wistar rats were cocultured with syngeneic Sertoli cell-peritubular cell preparations in the presence of heterologous or allogeneic serum. Thymic cells cultured in this manner bound to Sertoli cells, became autosensitized , and markedly altered syngeneic Sertoli cell surface properties and remodeling functions in vitro. In contrast, control thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not become sensitized. Coculture of autosensitized thymic cells with syngeneic seminiferous tubule segments, or local transfer of such lymphocytes into syngeneic rat testes, resulted in intratubular infiltration by "light cells." Intratesticular injection of autosensitized thymic cells was followed by derangement of the seminiferous epithelium, and by morphologic changes characteristic of experimental autoimmune orchitis. Thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not elicit these changes. Thymic cells incubated with peritubular cells in heterologous or autologous serum behaved like control thymocytes, and were not sensitized. Data presented indicate that thymic cells are potentially capable of recognizing syngeneic Sertoli cell self-antigens. We speculate that factors normally present in serum may inhibit the recognition by thymic lymphocytes of antigenic determinants present on Sertoli cells. We discuss the possibility that the modulation of interactions between immature thymic lymphocytes and Sertoli cells is implicated in the prevention of autoimmune reactions against the testis.

Metabolism of ( 3H) 5α-androstane-3α,17β-diol by cultures of isolated rat sertoli cells and the effect of LH and FSH

Sertoli cells from immature rats metabolized ( 3H) 5α-androstane-3α, 17β-diol to ( 3H) 5α-androstane-3α, 16α, 17β-triol and ( 3H) 3α-hydroxy-5α-androstan-17-one. This is the first report of 16α-hydroxylation of 5α-reduced androgens in the testis. FSH significantly stimulated 16α-hydroxylation while LH significantly decreased this activity. 3α-Hydroxy-5α-androstan-17-one was the major metabolite formed and its production was significantly increased in the presence of both LH and FSH, although FSH stimulation was significantly more than LH. The possible role of 16α-hydroxylase in androgen metabolism by immature rat Sertoli cells is discussed.

Effects of FSH on acidic nuclear protein synthesis in cultured pig Sertoli cells

We studied the effect of FHS on nuclear protein synthesis by a primary culture of immature porcine Sertoli cells. The cells were cultured in a chemically defined medium and treated with FSH (50 ng/ml) for various periods (4, 8, 25 and 64 h). About 65 spots (pH i < 8) were identified by two-dimensional polyacrylamide gel electrophoresis of the radiolabelled nuclear proteins. After 25 h of FHS treatment, we observed an increase of 5 proteins, a slight decrease of two and a disappearance of one. For the short periods of FSH treatment (4 and 8 h), no effect of FHS on nuclear protein synthesis was observed. After 64 h of FSH treatment, the synthesis of all nuclear proteins appears to be decreased.

Regulation of hormone-responsive Sertoli cell adenylyl cyclase in a cell-free system

Homologous desensitization of either FSH- or isoproterenol-responsive adenylyl cyclases in Sertoli cell membranes can be achieved in a cell-free system. Incubation of membrane particles from cultured immature Sertoli cells with either FSH or isoproterenol resulted in a time- and concentration-dependent loss of subsequent adenylyl cyclase response to the homologous hormone. Half-maximal refractoriness was achieved within 20–30 min of incubation. The concentration of FSH required to obtain half-maximal loss of adenylyl cyclase response (400 ng/ml) was in the same order of magnitude as the apparent K m for FSH-stimulated adenylyl cyclase activity (300 ng/ml). Hormone-induced homologous desensitization was dependent on the presence of both ATP and Mg 2+. Increasing concentrations of ATP in the presence of FSH caused a concentration-dependent loss of subsequent response to the homologous hormone. Halfmaximal desensitization was achieved at an ATP concentration of 0.2 mM. Increasing concentrations of Mg 2+ in the presence of either FSH or isoproterenol caused desensitization for the homologous hormone. The concentration of Mg 2+ giving half-maximal effect was approximately 5 mM in excess of ATP and EDTA. However, higher concentrations of free Mg 2+, in the absence of hormone, caused desensitization of both FSH- and isoproterenol-sensitive adenylyl cyclase with half-maximal effect at approximately 30 mM. Hormone-specific desensitization was obtained in the presence of GTP. However, when GTP was substituted with the non-hydrolysable analogue GMPP(NH)P the hormonal activation remained constant throughout 90 min of incubation. The hormone-induced refractoriness was probably not due to cAMP, since all incubations were performed in the presence of an excess of cAMP (1 mM) to block phosphodiesterase (PDE) activity. However, the finding that both ATP and Mg 2+ were required for hormone-induced desensitization suggests that the process may be mediated via a cAMP-independent phosphorylation reaction. Furthermore, the hormone appears to accelerate desensitization of the adenylyl cyclase system by increasing its sensitivity to the actions of Mg 2+.

Extracellular matrix promotes rat Sertoli cell histotypic expression in vitro.

We describe procedures for the preparation of a cell-free seminiferous tubule biomatrix, and provide evidence demonstrating that this material constitutes a useful substratum for maintaining the normal architecture of Sertoli cells in primary culture. Seminiferous tubule biomatrix, which has the morphological appearance of a fibrillar network rich in filaments and amorphous substance, is shown to consist of about 50% protein, most of which is collagen and glycoproteins. Fibronectin and laminin are also present in the seminiferous tubule biomatrix, as judged by immunofluorescence microscopy. Sertoli cell aggregates plated on this substratum retain a cuboidal to columnar shape, spread very slowly to form a monolayer, and survive for at least 3 weeks when cultured in a hormone-free, serum-free, chemically defined medium. In contrast, Sertoli cells plated onto uncoated polystyrene readily spread to form a monolayer of flat squamous cells which do not survive as well. Other morphological and ultrastructural characteristics are described which indicate that cells cultured on the seminiferous tubule biomatrix more closely resemble those of Sertoli cells in vivo than do cells plated on uncoated plastic. These differences in cell structure, including the maintenance of normal polarity as indicated by the presence of basolateral tight junctional complexes, remain evident for periods of 10 to 14 days after plating Sertoli cells onto biomatrix substratum. Rates of DNA synthesis by immature Sertoli cells plated onto biomatrix are less than rates by cells plated onto uncoated plastic. The data are discussed in relation to the role of substratum in the preservation of normal functions and histotype of Sertoli cells.

Cellular processing of follicle-stimulating hormone by Sertoli cells in serum-free culture

Specific binding of radioiodinated human follicle-stimulating hormone ([ 125I]hFSH) to receptors on the cell surface of immature rat Sertoli cells in serum-free culture (4–7 days) was concentration-, time- and temperature-dependent with an apparent steady state of binding stained after 2 h at 4, 18 or 32°C. Analysis of [ 125I]hFSH binding data indicated an apparent K a of 3× 10 9 M −1 with approximately 670 fmoles binding sites/mg protein. The radioiodinated hFSH (60–70 μCi/μg) bound to receptor and stimulated estradiol production, thus confirming its biological activity and Sertoli cell viability. Approximately 50% of the specifically bound radioactivity was lost from the cell surface after 0.25 h at 32°C, 1 h at 18°C or 4 h at 4°C. Most (> 90%) of the receptor-bound hormone was removed from the cell surface and degraded to [ 125I]mono- and diiodotyrosine within 4 h at 32°C. FSH degradation was inhibited at low temperatures (4°C) and by the lysosomotropic agents chloroquine, NH 4Cl or concanavalin A. Bacitracin and methylamine, inhibitors of internalization, were effective in blocking degradation but not loss of membrane-bound [ 125I]hFSH. Thus it appears that, as with small peptide and simple protein hormones, complex glycoprotein hormone-receptor complexes, such as FSH Sertoli cell receptors, are endocytosed by normal target cells and subsequently degraded, most probably within the lysosomes.

Estrogen synthesis by immature rat sertoli cells in vitro.

Past research has implicated both the Leydig and Sertoli cells as sources of testicular estrogens. To study this issue further, we investigated the ability of Sertoli cells isolated from the testes of immature (10‐day‐old) rats to synthesize estrogens in response to FSH in vitro. The aromatase activity of Sertoli cells was examined by comparing simultaneously two different methods: 1. the conversion of 19‐(6,7‐3H)‐hydroxyandrostenedione to (3H)estrone and (3H)estradiol, measured by silica gel thin layer chromatography; and 2. the conversion of unlabeled 19‐hydroxyandrostenedione to total estrogen (El + E2), measured by RIA.Sertoli cells, cultured in the presence of substrate, exhibited aromatase activity in response to FSH. Values obtained for total estrogen synthesis, measured by direct tracer methodology, at FSH dosages of 0.5 μg/ml and 1.0 μg/ml, during a 24‐hour period of incubation, were 620 pg/mg and 580 pg/mg, respectively; and after a 48‐hour incubation, values obtained were 570 pg/ml and 560 pg/mg, respectively. When measured by RIA, similar results were obtained for estrogen synthesis at identical FSH dosages and times of incubation. A dose response of FSH‐stimulated aromatization was also obtained by RIA. Neither control nor LH‐treated cells, however, in the presence of substrate, produced detectable levels of labeled or immunoassayable estrogens.Aromatase activity was also studied in the presence of a cold estrogen trap and measured by direct tracer methodology. In this case, estrogen synthesis was more than doubled. These data may suggest the further metabolism of estrogen by Sertoli cells. Finally, our results are discussed with respect to the role of the immature Sertoli cell in the synthesis of testicular estrogens.

Histochemical location of 17 beta-hydroxysteroid oxidoreductase in the adult and infantile human testis.

17 beta-Hydroxysteroid oxidoreductase in the human testis was investigated histochemically using tissues obtained from seven patients with undescended testis or varicocele at the time of orchiopexy or high ligation of spermatic vein. Formazan precipitates were formed from nitro-blue tetrazolium in the tissue utilizing hydrogen released by oxidation of testosterone, which is catalyzed by the reductase function of 17 beta-hydroxysteroid oxidoreductase. The precipitates were formed specifically in the presence of 17 beta-hydroxy-C19-steroids under the conditions employed in the present study. In infantile testes, the precipitates were formed in cytoplasm of immature Sertoli cells, while in pubertal or adult testes, marked formazan precipitates were found in cytoplasm of both Sertoli and Leydig cells. The results indicate the presence of two distinct 17 beta-hydroxysteroid oxidoreductases in the human testis; one in Sertoli cells and detectable independent of age and the other only in functional Leydig cells.

Regulation of follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate of a phosphodiesterase isoenzyme of the Sertoli cell.

The regulatory role of FSH on phosphodiesterase was studied in immature Sertoli cells in culture. Cells were prepared from 15-day-old immature rats, cultured for 3 days in defined medium, and then stimulated for 24 h with gonadotropin or with other factors known to regulate Sertoli cell cAMP. All agents that increased cAMP intracellular levels also had an effect on the total phosphodiesterase activity of cell homogenates, FSH and dibutyryl cAMP being the most potent stimulators. Further, stimulation was more evident when phosphodiesterase was measured at cAMP concentrations below micromolar. With 1 microM cGMP as substrate, no modification of the activity could be detected. Reversal of phosphodiesterase activation was observed at 24 or 40 h when dibutyryl cAMP was removed from the incubation medium. Separation of the isoenzymes present in the soluble fraction of Sertoli cell made it possible to demonstrate a selective stimulation of one isoenzyme. FSH and dibutyryl cAMP increased 10- and 50-fold, respectively, the activity of a high affinity cAMP phosphodiesterase, while the Ca2+-dependent cGMP hydrolyzing enzymes were not apparently affected. The enzyme regulated by FSH and dibutyryl cAMP had an apparent Km for cAMP of 2 microM, was Ca2+ and calmodulin insensitive, and migrated on sucrose density gradients with a sedimentation coefficient of 5.5S. These results indicate that FSH, after stimulation of intracellular cAMP, induces an increase in a high affinity phosphodiesterase and, therefore, an increased cAMP turnover in the Sertoli cell. This, in turn, might be a relevant phenomenon in the control of the responsiveness of this cell to gonadotropin.

Hyperplasia and the immature appearance of sertoli cells in primary testicular disorders

Testicular biopsy specimens from adult patients affected with cryptorchidism, Klinefelter's syndrome, and Del Castillo's syndrome were examined by light and electron microscopy. The study revealed a high proportion of testes showing seminiferous tubules with hyperplasia of Sertoli cells (from 25 to 45 cells per transverse tubular section). These cells had an immature appearance and showed a pseudostratified distribution. The nucleus was round to ovoid and regular in outline, with a smaller nucleolus than that of mature Sertoli cells. The cytoplasm showed less development of the endoplasmic reticulum as well as of the secondary lysosomes and lipid droplets than that in mature Sertoli cells. Characteristic features of these immature Sertoli cells were abundant cytoplasmic microfilaments, elaborate interdigitations between adjacent cells, and extensive tight junctions, from basement membrane to lumen. In the cryptorchid testes, a more immature Sertoli cell was found to constitute the majority of the cells in hypoplastic zones. In Klinefelter's and Del Castillo's syndromes as well as in cryptorchid testes to a lesser degree, a transitional type of cell-from immature to mature-was also observed. These observations suggest that Sertoli cells in these primary testicular disorders reflect a congenital deficiency producing abnormal development.

Desensitization of FSH-responsive adenylyl cyclase in cultured immature Sertoli cells by homologous hormone.

Sertoli cell monolayers were prepared from 19-day-old rat testes. On day 7 of culture cells were incubated for 24 hr in the presence or absence of ovine follicle stimulating hormone (oFSH). Cells were harvested, and adenylyl cyclase responses of the membrane particles to FSH, human chorionic gonadotropin (hCG), isoproterenol, and fluoride (F-) were examined in the presence of either GTP or the nonhydrolyzable guanylyl nucleotide GMP-P(NH)P. Culturing the cells in presence of FSH caused a hormone specific desensitization of FSH-responsive adenylyl cyclase, whereas responses to isoproterenol and fluoride were unaffected. Activation of Sertoli cell adenylyl cyclase by GTP and GMP-P(NH)P showed no difference between cells preincubated with or without FSH, indicating that FSH did not change the activity of the G/F (or N) component or its interaction with the catalytic subunit of the adenylyl cyclase. FSH-responsive adenylyl cyclase in cultured Sertoli cells has been shown to be selectively desensitized by homologous hormone. The mechanism may involve alteration or loss of the FSH receptor or changes in the "coupling" of the FSH receptors to the G/F component of the adenylyl cyclase, since there was no alteration in the guanylyl nucleotide and fluoride activation.

An improved sensitive and specific in vitro bioassay for FSH

In recent years, it has become increasingly clear that immunological reactivity does not always reflect biological activity of gonadotropins. The present in vitro bioassay for human FSH is based on the FSH dependent aromatase activity of immature rat Sertoli cells. Testes from 10 day old rats are minced and dispersed by enzymatic digestion, and plated in tissue culture micro-wells. After an initial 24 h culture, the FSH containing sample, a phosphodiesterase inhibitor and the substrate (19-hydroxyandrostendione) is added to the cells in a defined medium, and the accumulation of 17β-estradiol during the following 24 h is measured. Recent modifications have increased the sensitivity of the method to 0.1 mIU per sample (0.4 mIU/ml), and the specificity is such that hLH, hCG, hTSH showed <0.6% of the activity of highly purified hFSH, while ACTH, hGH, hPRL, and LH-RH preparations showed no detectable FSH activity. All FSH preparations studied showed a dose-response curve that was parallel to the 1st IRP of human gonadotropins for bioassay (69/104). Serum and urine normally contain inhibitory proteins, that must be removed before the assay.

FSH Stimulation of cAMP Accumulation in Hamster Sertoli Cells: Effect of Age and Optic Enucleation

Sertoli cells isolated from immature, adult, and adult optic‐enucleated hamsters respond to FSH, but not to LH, with an increase in cAMP accumulation. There is an age‐related decline in responsiveness of Sertoli cells to FSH. Sertoli cells cultured from optic‐enucleated adult hamsters with regressed testes respond to FSH with an Increased accumulation of cAMP similar in magnitude to that of immature Sertoli cells. These data indicate that optic enucleatlon results in a reversal of the sensitivity of Sertoll cells to FSH, an effect which may be important for the reinitiation of spermatogenesis, which naturally follows the regressed state.

Nhibition of rat sertoli cell aromatase by factor(s) secreted specifically at spermatogenic stages VII and VIII

The effect of spent medium recovered from cultured seminiferous tubules on the response of Sertoli cells to FSH was investigated. 4 pools of tubules at different stages of the spermatogenic cycle (VII–VIII, IX–XII, XIII–I, II–VI) were identified by transillumination and cultured for 20 h at 32°C. Then the culture media were added to Sertoli cell cultures and the FSH-dependent secretion of estradiol into the medium after 24-h incubation was measured by radio immunoassay. The spent media from tubules at stages IX–XII, XIII–I and II–VI did not significantly alter the FSH response of the Sertoli cells, while medium from stages VII–VIII inhibited the FSH dependent estradiol secretion in a dose-dependent manner. The inhibiting activity was not destroyed by heating at 60°C for 30 min, and it was not affected by repeated freezing and thawing or 24-h dialysis against fresh medium. The inhibition upon estradiol production was reversible after removal of the inhibitory agent(s) and subsequent culture of Sertoli cells under control conditions. The Sertoli cells had a good viability, as evidenced by trypan-blue exclusion and the undetectable levels of LDH in the medium after culture in the presence of the spent medium from stages VII–VIII. The inhibition of the FSH-induced aromatase activity in cultured immature Sertoli cells does not seem to be due to interference with FSH-receptor interactions or to an inhibition of adenylate cyclase because the dibutyryl-cAMP-induced increase in aromatase activity was also inhibited. These results are discussed in relation to the communication between Sertoli cells and germ cells in different stages of spermatogenesis and the possible physiological significance of the inhibitor(s) on the Sertoli cell activity even in the mature seminiferous tubule.

Role of FSH in the control of testicular function.

Follicle stimulating hormone (FSH) binds to the plasma membranes of Sertoli cells and, apparently, spermatogonia; the hormone may then be taken into the cytoplasm of its target cells and metabolized there. In the immature testis, binding of FSH to Sertoli cells leads to activation of adenylate cyclase, formation of cyclic AMP (cAMP), and activation of a cAMP-dependent protein kinase. There is a subsequent increase in the rates of RNA, protein, and DNA synthesis. Normal adult testis is unresponsive to FSH, one reason being the destruction of cAMP by a phosphodiesterase. After hypophysectomy of adult animals this enzyme disappears and FSH is again able to stimulate RNA and protein synthesis. Other factors contributing to the insen-sitivity of the mature testis are an FSH binding inhibitor, a protein kinase inhibitor, and in-activation of FSH.FSH increases the formation by Sertoli cells of structural proteins and an androgen-binding protein (ABP) that the Sertoli cells secrete into the extracellular fluid surrounding the cells of the germinal epithelium. It is thought that ABP buffers fluctuations in the concentration of androgen in the extracellular fluid of the germinal epithelium as well as transporting androgen to the lumen of the epididymis.In immature animals FSH increases the number of Sertoli cells, which accounts for the testicular hypertrophy that follows hemicastration. FSH is responsible for maturational changes in immature Sertoli cells and for morphological changes associated with in vitro aggregation of Sertoli cells to form colonies. FSH-induced changes in the shape of Sertoli cells are mediated by a calcium-dependent regulator protein, calmodulin. FSH also stimulates the Sertoli cells to secrete a plasminogen activator whose role is not known.FSH has effects on testicular germ cells in immature animals and during the restoration of spermatogenesis in adults, but it is not known whether the Sertoli cells mediate its actions on the germ cells, though this would seem likely. Administration of FSH to immature rats and mice increases the number of spermatogonia by reducing the proportion that degenerate. This may be due to a stimulatory effect of the hormone on DNA synthesis. Androgen and FSH appear to act synergistically to stimulate accumulation of primary spermatocytes and formation of spermatids in immature rats. During the restoration of spermatogenesis in adult rats and mice FSH also acts synergistically with androgen, increasing the proportions of cells passing through meiosis and spermiogenesis. Immunization experiments suggest that FSH is needed for the formation of normal numbers of spermatocytes and spermatids in immature animals, but studies in adults have given conflicting results.FSH appears to influence development of the interstitial tissue, particularly by inducing luteinizing hormone (LH) receptors on Leydig cells. The presence of some LH may be necessary for FSH to have this effect. FSH can stimulate a number of steroid transformations, such as conversion of testosterone to dihydrotestosterone (DHT), in Sertoli cells from immature rats but there is no conclusive evidence that the gonadotropin is capable of stimulating the critical step in steroidogenesis, production of pregnenolone from cholesterol. FSH also stimulates Sertoli cells from immature rats to convert testosterone to a substance cross-reacting with antibodies to estradiol. It has been suggested that loss of the ability of FSH to stimulate estrogen production in maturing Sertoli cells allows the Leydig cells to secrete increased amounts of androgen at puberty.Highly purified preparations of human gonadotropins are not available in sufficient quantity for clinical use. Menopausal gonadotropin, which contains LH as well as FSH activity, stimulates proliferation of immature Sertoli cells and spermatogonia and formation of spermatocytes in prepubertal boys. Treatment with human chorionic gonadotropin (hCG) has similar effects but in addition early spermatids are formed when both preparations are given together. Stimulation of spermatogenesis and, rarely, fertility have occurred in a proportion of eunuchoidal men treated with gonadotropin preparations containing both FSH and LH activity, and combined treatment has also stimulated spermatogenesis after hypophysectomy. Unfortunately gonadotropin treatment has benefited only a small proportion of azoospermic or oligozoospermic men not suffering from androgen deficiency. Isolated deficiency of FSH has been reported in four men but these reports throw little light on the role of FSH in the control of testicular function in normal men.Most studies in immature animals leave little doubt that FSH has an essential role in the stimulation of developmental changes in the testis. At this stage androgen secreted in response to LH may act synergistically with FSH on the germinal tissue. In the normal mature testis it is not clear whether there is a requirement for FSH. If so, its place is subordinate to that of androgen.

Follicle-Stimulating Hormone-Induced, Adenosine 3',5'-Monophosphate-Mediated Movement of Immature Rat Sertoli Cells in Primary Culture*

Sertoli cells dissociated from 10-day-old rat testes form colonies in primary culture in response to FSH. FSH and dibutyryl cAMP stimulated the attachment of Sertoli cells to an equal extent; however, FSH-treated cultures contained a small number of large colonies while dibutyryl cAMP-treated cultures contained a large number of small colonies. This relationship was not altered by the addition of a number of other peptide or steroid hormones. Extracellular cAMP levels and colony density were negatively correlated. Colony size at 24 h of culture was diminished in FSH-treated cultures by the addition of a cAMP antibody at 6 or 12 h of incubation. The addition of cAMP at 24 h to FSH-treated cultures caused a dose-dependent stimulation of colony size but not colony density at 48 h of culture. A point source of cAMP (4 x 10(-3) M in agar) inhibited migration of cells toward the agar spot. An agar spot on the dish substratum containing Sepharose-bound FSH exhibited a halo of cells next to the spot, with a zone lacking cells distal to it. Radioautographs of [125I]iodo-FSH-treated cultures exhibited a nonhomogeneous distribution of silver grains; colony size increased faster than the number of labeled cells. Taken together, the results suggest that 1) FSH is stimulating the attachment of Sertoli cells through an increase in intracellular cAMP, 2) FSH is promoting active aggregation of Sertoli cells in culture through a modulation of extracellular cAMP, and 3) cells with a large amount of bound FSH are acting as centers for aggregation.

Active cell aggregation by immature rat Sertoli cells in primary culture: A role for cell surface glycoproteins

AbstractFollicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10‐day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH‐stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0‐ to 18‐hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose‐specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of 3H‐aminoacid incorporation as well as cell aggregation in 24‐hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease 125I‐FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.

Steroidogenesis by Granulosa and Sertoli Cells

Granulosa and Sertoli cells have been isolated from the gonads of immature rats, and have been maintained in monolayer cultures in a chemically‐defined medium. The hormonal requirements of these cell types for the synthesis of steroids have been studied: Granulosa cells contain cholesterol side‐chain cleavage activity and synthesise progesterone when cultured in the presence of FSH and testosterone (or DHT). Sertoli cells on the other hand cannot synthesise steroids de novo. Granulosa and Sertoli cells are similar in that they are unable to convert significant amounts of progesterone to androgens. In the presence of exogenous testosterone as substrate, granulosa cells and immature Sertoli cells can synthesise estradiol and estrone when stimulated with FSH. In summary, FSH stimulates estrogen synthesis from testosterone in both cell types, and acts synergistically with testosterone (or DHT) to stimulate progesterone synthesis in granulosa cells. FSH is therefore involved in controlling the steroid environment within the gonads.