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.     

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.     

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.     

The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats

Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function. Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism. Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.     

A filter disc assay for the measurement and characterization of androgen-binding protein in unconcentrated media from Sertoli cell-enriched cultures

As easy, rapid and sensitive assay which permits measurements of androgen binding protein (ABP) in unconcentrated spent media from Sertoli cell cultures is described. The method is based on the adsorption of the 5 alpha-dihydrotestosterone-ABP complex onto DEAE-cellulose filter paper discs at pH 8.5. It is demonstrated that this method permits the characterisation of ABP (ligand specificity, kinetic properties) and represents a useful tool for the study of the effects of various hormones on ABP production by cultured Sertoli cells. When added on day 5 of culture, FSH and other agents that raise intracellular cAMP increase ABP secretion up to 3 times. Natural and synthetic androgens provoke a 1.5-1.8-fold increase. The effects of androgens and FSH are additive and cyproterone acetate blocks the effects of androgens in the presence as well as in the absence of FSH.     

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.     

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.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.     

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.     

Structural and functional modifications of sertoli cells in the testis of adult follicle-stimulating hormone receptor knockout mice

Follicle stimulating hormone (FSH) plays important roles during testicular development and in the maintenance of spermatogenesis in the adult. However, the cellular events or pathways that FSH regulates to achieve these effects in Sertoli cells, where the FSH receptors (FSH-R) are located, is still not fully elucidated. The development of FSH-R knockout (FORKO) mice provides a model to examine alterations in testicular structure and function in its absence. To this end, light (LM) and electron microscopic (EM) analyses of perfusion-fixed testes of wild-type and FORKO mice of different ages were performed. Under the LM, a significant reduction was noted in the profile area of seminiferous tubules of FORKO mice compared with their wild-type counterparts at different ages. In addition, FORKO testes revealed large irregularly shaped spaces within the seminiferous epithelium, extending from the base to the lumen. Such spaces were often separated by anastomotic cords of spherical germ cells or completely surrounded elongating spermatids. This phenotype was restricted to half or less of the circumference of only some tubules, but was seen at all stages. EM analyses revealed that the spaces corresponded to an apparent accumulation of fluid in the Sertoli cell cytoplasm, coincident with an absence of the fine flocculent ground substance seen in wild-type mice. However, the Sertoli organelles, while less prominent, appeared intact and to be floating in the enlarged fluid-filled cytoplasm. Functionally, androgen-binding protein (ABP), a major secretory protein of Sertoli cells, was dramatically reduced in FORKO mice. These results suggest that FSH-R signaling normally maintains water balance in Sertoli cells in addition to regulating ABP production.     

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.     

A specific androgen-binding protein (ABP) in Necturus testis and its zonal distribution

The urodele amphibian Necturus maculosus has a zoned testis, which is advantageous for separating Leydig cells from germinal elements and for studying stage-dependent biochemical changes. Using [3H]testosterone (T) in a standard binding assay and dextran-coated charcoal (DCC) or Sephadex LH-20 to separate free and bound steroids, we identified an androgen-binding protein (ABP) in Necturus testis cytosols. This protein was of high affinity (Kd = 10(-9) M) and was saturable (Bmax = 10(-9) M) and specific for androgen (T; 5 alpha-dihydrotestosterone, DHT) but could be distinguished from the androgen receptor of Necturus testis by its relative abundance (300-550 fmol/mg protein), short half-time of dissociation (3 min at 22 degrees C), inability to adhere to DNA-cellulose, and absence from nuclear extracts. Additionally, when analyzed on sucrose gradients, the ABP of Necturus testis sedimented at 6-7 S in both low or high ionic strength buffers. In that estradiol (E2) is a poor competitor for T-binding, this protein resembles a sex steroid-binding protein previously identified in urodele serum but differs from the ABP and testosterone-estradiol-binding globulin (TEBG) of rodents, humans, goldfish, and sharks. It is differentially distributed within the testis, with the highest levels in immature lobular regions composed of Sertoli cells and germ cells in premeiotic stages and lower levels in regions composed primarily of Leydig cells. The cellular source and function of this protein in Necturus testis remain to be determined.     

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.     

Dicyclohexane derivatives that bind to androgen-binding protein (ABP) also inhibit hormone stimulated aromatase activity in rat Sertoli cells

Dicyclohexane derivatives are known to inhibit testosterone binding to rat androgen-binding protein (ABP) a secretory product of Sertoli cells. In this paper we show that these compounds also inhibit the aromatization of testosterone by Sertoli cells in response to cyclic AMP and to hormones that act via this nucleotide. The inhibitory activity of the nonsteroidal androgen analogues is dose-dependent and roughly parallels their ability to interfere with the aromatase activity in human placental microsomes and their affinity for ABP. Diethylstilbestrol and mesohexestrol--two nonsteroidal estrogens which resemble the dicyclohexane derivatives--also inhibit aromatase activity in Sertoli cells and placental microsomes. The effects of the synthetic estrogens on Sertoli cells, however, are less specific. Unlike the dicyclohexane derivatives they also block hormone induced activation of the adenylate cyclase. We conclude that dicyclohexane derivatives are representative of a novel series of inhibitors of aromatase activity.