Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

Hormone interactions in the Sertoli cells.

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.     

Human Sertoli cells in vitro: morphological features and androgen-binding protein secretion.

Sertoli cells play a pivotal role in the regulation of spermatogenesis as they provide the anatomical basis of the blood-testis barrier. In the present paper we report some results of our studies on the ultrastructural features, the responsiveness to FSH, and the ability to secrete androgen-binding protein (ABP) of human Sertoli cells in vitro. The nucleus showed the characteristic foldings of the nuclear membrane, scattered chromatin, and a fibrillar nucleolus. In the cytoplasm Charcot-Boettcher crystals were present and active phagocytic activity was documented by the presence of vacuoles containing lipids and cellular debris. Human Sertoli cells in culture responded to FSH with a maximal rise in cAMP that was approx. 3-fold. This response to FSH is comparable to that reported for the adult rat but lower than that of the immature rat, and suggests that human as well as rat Sertoli cells could have a reduced response to FSH since sexual maturation was achieved. As no evidence has been reported on ABP secretion by human Sertoli cells in culture we evaluated the concentration of this protein in the Sertoli cell spent media. Human Sertoli cells in culture produced ABP and the response to FSH was dose-related. The Kd value of human ABP (hABP) was approx. 7.5 nM, being slightly higher than that of the rat ABP and an order of magnitude different from that of sex hormone-binding globulin (SHBG) present in human plasma. We also measured the association and dissociation rates of dihydrotestosterone-hABP complexes and the Kd/Ka ratio was very close to the value of Kd of the Scatchard analysis. The differences between hABP and SHBG may open the way to the selective measurement of ABP in many conditions of male infertility.     

The role of cell-cell interactions in androgen action

Androgen-regulated mesenchymal-epithelial interactions play an important role during embryonic development of the male urogenital tractus. Studies on the effects of androgens on cultured testicular cells derived from the immature rat testis indicate that, even during postnatal life, similar interactions may be instrumental for normal androgen action. Androgen receptors are found in epithelial Sertoli cells as well as in mesenchymal peritubular cells. The effects of androgens on isolated Sertoli cells, however, are limited. Coculture with peritubular cells increases the sensitivity and/or the responsiveness of a number of Sertoli cell parameters (transferrin, ABP, aromatase activity) to androgens. This effect is at least in part mediated by the secretion of one or more diffusible factors (P-Mod-S) by the peritubular cells. We investigated whether such indirect effects of androgens, relying on mesenchymal—epithelial interactions are also observed in other androgen target tissues. To this end stromal cells were isolated and cultured from the immature rat ventral prostate and the production of factors with P-Mod-S activity was monitored using Sertoli cells as the test system.Under coculuture conditions these stromal cells stimulate Sertoli cell transferrin secretion in an androgen-regulated fashion, exactly as peritubular cells. This stimulatory effect is related in part to the collaborative (and androgen-independent) deposition of an extracellular matrix and in part to the secretion of an androgen-regulated diffusible mediator. This mediator has the same physicochemical characteristics as P-Mod-S and it affects other Sertoli cell parameters (ABP, aromatase activity, inhibin, cGMP) in the same way as P-Mod-S. Cultured stromal and peritubular cells look very similar and stain positive after immunostaining for -smooth muscle isoactin. Tissue sections suggest that these cells may be derived from myoid peritubular cells in the testis and similar periacinar cells in the prostate. The hypothesis is advanced that P-Mod-S may be a more universal mediator of indirect effects of androgens in diverse target tissues and that this factor is derived from myoid cells closely associated with the epithelial component.     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures.

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.     

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).     

Changes in androgen binding protein (ABP) production following continuous low dose gamma irradiation (IR) of adult rat

Continuous low dose gamma irradiation induces a progressive degeneration of germ cells with a concomittant increase in blood FSH; however, the Sertoli cell function is not too much altered since serum ABP level is normal and it is likely that the decrease of epididymal ABP content is the consequence of a reduction in seminiferous tubule fluid excretion. Obviously, spermatids seems to be involved in the regulation of Sertoli cell ABP synthesis.     

Regulation of seminiferous tubular function by FSH and androgen.

Seminiferous tubules contain a cytoplasmic androgen receptor similar to the receptors in the epididymis and ventral prostate. The presence of a cytoplasmic receptor indicates that androgens maintain spermatogenesis by a direct action on certain types of cells within the seminiferous tubule. The Sertoli cell appears to be one of the cell types containing androgen receptors and the receptor might also be present in spermatogonia, primary spermatocytes, or peritubular cells. The Sertoli cell is stimulated by FSH to produce an androgen-binding protein which may serve to increase the accumulation of androgen in the seminiferous epithelium and make it available for binding by intracellular androgen receptors. This may be a way in which FSH enhances the action of androgen on spermatogenesis. Androgens act on the Sertoli cell to increase its response to FSH. This action of androgens on the Sertoli cell results in increased production of androgen-binding protein and may enhance the production of other substances which exert trophic effects on spermatogenesis.     

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP

The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.     

Modification of Sertoli cell functions in vitamin A-deficient rats

FSH binding and cAMP responses to FSH in Sertoli cell-enriched testes were not affected by the vitamin A (retinol) status of the animals. These results indicate that changes in Sertoli cell functions during vitamin A deficiency are independent of FSH-Sertoli cell interactions. Concentrations of serum androgen binding protein (ABP) in vitamin A-deficient rats were consistently higher than those of control animals throughout the study period. The accumulation of testicular fluid after efferent duct ligation, an indication of Sertoli cell secretory function, was normal in vitamin A-deficient rats at least until 70 days of age, but declined thereafter. ABP concentrations in seminiferous tubular fluid of vitamin A-deficient rats increased transitorily during the 70-80-day age period but returned to normal by 90 days. The increment of ABP in seminiferous tubular fluid after efferent duct ligation, and ABP concentrations in interstitial fluid were consistently lower in vitamin A-deficient rats. The higher serum ABP in vitamin A-deficient rats therefore cannot be explained by an increase in the permeability of Sertoli-cell tight junctions or basement membrane.     

Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.     

Possible involvement of germ cells in the regulation of oestradiol-17 beta and ABP secretion by immature rat Sertoli cells (in vitro studies)

The effects of germ cells prepared from adult rats and of media conditioned by some of these germ cells have been studied in vitro on both ABP and oestradiol-17 beta secretion by immature rat Sertoli cells. Addition of the germ cells to the Sertoli cell cultures resulted in both a dose-dependent increase of ABP secretion and a dose-dependent inhibition of oestradiol production. These effects were suppressed after removal of germ cells by hypotonic treatment. Furthermore, spent media of highly viable germ cells (SMGC), but not spent media of an epithelial cell line, mimicked the effects of germ cells themselves on ABP and oestradiol levels after FSH or dbcAMP stimulation. These effects were reversible when SMGC were replaced by fresh media and did not result from a change in the conversion of oestradiol to oestrone. SMGC effects were unaltered by heating at 60 degrees C for 30 min, by freezing and thawing and non dialysable (MW greater than 10,000). However, heating at 100 degrees C for 3 min and treatment by trypsin, suppressed the SMGC effects. This indicates that the stimulation of ABP and inhibition of oestradiol levels by germ cells, in vitro, could be mediated by factor(s) of proteinaceous nature.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Delta-9-tetrahydrocannabinol stimulates ABP secretion from rat Sertoli cells in vitro

The effect of delta-9-tetrahydrocannabinol (THC) on rat Sertoli cell function was investigated. THC significantly increased ABP secretion by 1.5- to 2.1-fold but did not consistently enhance the stimulation of ABP induced by FSH, testosterone or dibutyryl cyclic AMP. ABP was measured by steady-state polyacrylamide gel electrophoresis, DEAE Bio-Gel and immunoassay; all three methods gave similar results. The minimal concentration of THC that stimulated ABP was 10 ng/ml; maximal stimulation was observed with 100-200 ng/ml. This effect was specific since THC did not affect gamma glutamyl transpeptidase activity or the secretion of plasminogen activator, lactate and transferrin. This observation that THC affects ABP secretion specifically is the first report of any differential effect of a drug on Sertoli cell secretion.