Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells.

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic variation of human sex hormone-binding globulin: evidence for a worldwide bi-allelic gene.

Genetic variation of human sex hormone-binding globulin (SHBG) has been investigated on 1690 unrelated neuraminidase-treated serum samples using isoelectric focusing followed by transfer to nitrocellulose membranes and immunostaining. Three clearly distinct isoelectric focusing patterns, consistent with the expression of an autosomal genetic system, were identified. Using allele frequencies, calculated on the basis of a bi-allelic gene, an excellent agreement between observed and expected phenotype numbers was obtained in every examined population sample. Family data along with the observed distribution of the three SHBG phenotypes among racially different groups and sexes indicate that SHBG is worldwide encoded by two autosomal codominant alleles. Compared with healthy Belgian blood donors no statistically significant differences were noted for the allele frequencies among 399 patients and 70 hirsute women of Belgian origin. Evidence is also presented that the subunit produced by the variant allele (SHBG2) has a higher molecular mass than the one produced by the regular allele (SHBG1) and that the three SHBG genotypes have identical binding characteristics for 5 alpha-dihydrotestosterone.

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 coculture 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 alpha-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.

Rat tumor Leydig cells as a test system for the study of Sertoli cell factors that stimulate steroidogenesis.

Transplantable rat Leydig cell tumor H-540 was used to study the interactions between Leydig and Sertoli cells. These tumor cells maintain their steroidogenic capacity when cultured. Their responsiveness to a number of agonists that affect normal Leydig cells is markedly changed. Steroidogenesis can no longer be stimulated by luteinizing hormone (LH), but the cells remain responsive to dibutyrylcyclic adenosine monophosphate (dbcAMP) and cholera toxin. Cultured cells mainly produce C21-steroids, but the ability to produce androgens can be restored by pretreatment with dbcAMP. Coculture with Sertoli cells increases steroidogenesis in H-540 cells, and this effect is enhanced by follicle-stimulating hormone (FSH). Experiments with a two-chamber culture system show that these effects are mediated by one or more diffusible factors, some of which may be short-acting. SCF, a Sertoli cell-derived factor that stimulates normal Leydig cells, is not the mediator in this system since it is unable to stimulate steroidogenesis in Leydig tumor cells. Immunoneutralization experiments show that insulin-like growth factor I (IGF-I) is a permissive factor required to maintain steroidogenesis in Leydig tumor monocultures and in cocultures with Sertoli cells, but addition of IGF-I does not mimic the stimulatory effect of coculture. It was concluded that factors other than SCF and IGF-I must be involved in the stimulatory effects of coculture, and that H-540 cells may be a useful tool for the study of these factors.

Morphological and functional similarities between cultured prostatic stromal cells and testicular peritubular myoid cells.

A number of androgen effects on epithelial cells may be mediated by androgen-regulated paracrine factors produced by underlying mesenchymal cells. In previous studies we demonstrated that prostatic stromal cells and testicular peritubular cells, derived from immature rats, produce mediators of androgen action with identical effects on Sertoli cells. In the present paper we further compared the morphological and functional characteristics of both mesenchymal cell types. Cultured prostatic stromal cells and testicular peritubular cells look identical under phase-contrast microscopy, share the ability to form tubular structures and "balls" when cocultured with Sertoli cells, and contain proteins immunoreactive with an antiserum against alpha-smooth muscle isoactin. Two-dimensional gel electrophoresis shows that the pattern of proteins produced by both cell types is nearly identical. Conditioned media from stromal and peritubular cells contain a factor that stimulates transferrin and cGMP production in Sertoli cells. The behavior of the active principle in the media from both cell types is comparable. On reverse-phase HPLC the elution profile of this factor is comparable for media from both cell types. In conclusion, these data point to a striking similarity in the morphological and functional characteristics of mesenchymal cells cultured from the prostate and testis.

Influence of coculture with Sertoli cells on steroidogenesis in immature rat Leydig cells.

The hypothesis has been advanced that Sertoli cells produce one or more follicle-stimulating hormone (FSH)-dependent paracrine factors which stimulate Leydig cell maturation and steroidogenesis. In an attempt to identify these factors we studied the effect of coculture with Sertoli cells on the steroidogenic capacity of immature Leydig cells. It is demonstrated that coculture, during a period of 6 days, markedly increases the capacity of the Leydig cells to secrete C21-steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-4-en-3-one) and C19-steroids (testosterone, androst-4-ene-3,17-dione) in response to stimulation with luteinizing hormone (LH). Pretreatment of the cocultures on days 4, 5 and 6 with low concentrations of gonadotropins further enhances the steroidogenic response to LH. This pretreatment results in an overall increase in steroid output. At low concentrations of Sertoli cells and when short incubation times are used, pretreatment with FSH is clearly more effective than pretreatment with LH. Pretreatment with gonadotropins also results in a disproportionate increase in C19 output caused by increased conversion of C21 precursors into C19-steroids. This effect is also observed in Leydig cell monocultures and is mainly due to LH action on Leydig cells. Finally pretreated cocultures display a selective increase in testosterone output. The latter effect is caused by FSH-dependent conversion of androstenedione into testosterone in Sertoli cells. Pretreated cocultures can be maintained for at least 28 days. During this entire period their basal steroid output increases. Using a two-chamber culture system it is demonstrated that direct cell-cell interactions are not required to observe the stimulatory effects of coculture. One or more diffusible factors are involved and continuous contact with these factors is required to maintain the effect. Immunoneutralization experiments using an insulin-like growth factor (IGF-I) antiserum show that IGF-I is an important permissive factor to maintain steroidogenesis in isolated Leydig cells and in cocultures. Under none of the conditions studied, however, does the antiserum neutralize the stimulatory effect of coculture. It is concluded that the stimulatory effects of coculture on the testosterone output of Leydig cells are complex and that important diffusible mediators still remain to be identified.

Transferrin receptor expression in rat liver: immunohistochemical and biochemical analysis of the effect of age and iron storage.

Hepatic transferrin receptors were studied in normal male rats at 1 to 59 wk after weaning, using immunohistochemical and biochemical techniques. The number of transferrin receptors measured and the intensity of the staining in situ decreased rapidly during the first 10 wk of life and more slowly thereafter. Immunohistochemistry further demonstrated changes in the topographical and (sub)cellular localization of the transferrin receptor. In the young rat livers, staining was almost exclusively present on hepatocytes in acinar zone 2 + 3 in a honeycomb to sinusoidal pattern. With aging, a panacinar heterogeneous and mainly sinusoidal staining of hepatocytes was more frequent. Kupffer cell positivity was more obvious as compared with the young rat livers. The observed changes in transferrin receptor expression may partly be explained by age-dependent alterations in DNA synthesis and proliferative potential of the liver cells. A series of rats were iron loaded with carbonyl iron up to 39 wk and "unloaded" by administration of a normal diet during 20 wk. In these animals, serial histochemical studies showed predominantly parenchymal (7 to 14 wk), mixed parenchymal and reticuloendothelial (39 wk) and almost exclusive reticuloendothelial siderosis (59 wk). In the siderotic livers transferrin receptor numbers tended to be lower than in the controls with significant differences after 14 and 39 wk. Immunohistochemistry showed decreased parenchymal but increased reticuloendothelial transferrin receptor expression with iron load. After the period of unloading, parenchymal transferrin receptors were virtually absent despite the negligible siderosis of these cells. In contrast, siderotic reticuloendothelial cells were intensely positive. These findings support down-regulation of parenchymal transferrin receptor resulting from iron storage. However, the positivity of siderotic reticuloendothelial cells and the absence of re-emergence of parenchymal receptors in conditions of minimal parenchymal and prominent reticuloendothelial siderosis need further elucidation.

Independent control of the production of insulin-like growth factor I and its binding protein by cultured testicular cells.

The production of insulin-like growth factor I (IGF-I) and IGF-I binding protein (BP) was investigated in Sertoli, Leydig and peritubular cells derived from the immature rat testis and cultured in vitro. It is demonstrated that all these cells secrete not only IGF-I but also IGF-I BP. In Sertoli cells follitropin (FSH) and other agonists which increase intracellular cAMP stimulate IGF-I secretion but inhibit IGF-I BP release. The response of the BP is pronounced and very sensitive which makes it a new and useful parameter of FSH action. The calcium ionophore A23187 markedly decreases IGF-I BP production in Sertoli cells without noticeable effect on IGF-I itself. This effect can only partially be mimicked by a phorbol ester suggesting that intracellular calcium itself may play a major role in the control of IGF-I BP secretion. Peritubular cells produce high amounts of IGF-I and low amounts of IGF-I BP. Androgens do not affect the production of IGF-I or its BP neither by monocultures nor by cocultures of peritubular and Sertoli cells. In Leydig cells, lutropin (LH) and cAMP stimulate both IGF-I and IGF-I BP secretion. The production of IGF-I by Leydig-Sertoli cocultures clearly exceeds that expected from the monocultures suggesting that cell-cell interactions may also play a role in the control of testicular IGF-I production. The observation that the production of IGF-I and its activity are tightly and independently controlled supports the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular function. Whether IGF-I BP increases or decreases the effects of IGF-I in the testis remains to be investigated.

Prostatic stromal cells and testicular peritubular cells produce similar paracrine mediators of androgen action.

The role of mesenchymal-epithelial interactions in androgen action was explored using Sertoli cells as the epithelial cells and testicular peritubular cells or prostatic stromal cells as mesenchymal cells. Footsole fibroblasts served as a control. The secretion of transferrin was used as an androgen-regulated parameter of Sertoli cell function. It is demonstrated that coculture of peritubular or stromal cells with Sertoli cells markedly increases the production of transferrin. This effect requires a 4-day latent period and is maximal with low concentrations (10%) of mesenchymal cells. Stimulatory effects of androgens can only be demonstrated at suboptimal concentrations of the latter cells. Fibroblasts are inactive. At least two mechanisms contribute to these stimulatory effects. Peritubular cells and stromal cells share the ability to promote the deposition of an extracellular matrix when cocultured with Sertoli cells. When Sertoli cells are seeded on this matrix, the production of transferrin is increased. This effect requires no latent period and is independent of the presence of androgens during the period of matrix deposition. In addition, peritubular cells and stromal cells produce diffusible mediators which increase transferrin production by Sertoli cells. In both cell types, the production of these mediators is controlled by androgens, and their action is preceded by a 4-day latency period. The mediators have a comparable mol wt (45,000) and resemble P Mod-S, known to be secreted by peritubular cells. These data suggest that mesenchymal-epithelial interactions play a role in androgen-supported maintenance of adult function and that mesenchymal tissue from different androgen target tissues produces similar or identical mediators of androgen action.

Cholera and pertussis exotoxins protect mice against the lethal Schwartzman reaction and antagonize the effects of lipopolysaccharide on second messenger systems.

Pertussis toxin, and also cholera toxin are capable of inhibiting the effects of LPS in the elicitation of the generalized Schwartzman reaction. This is a potentially lethal generalized thrombo-haemorrhagic hypersensitivity and inflammatory-type response that occurs after two consecutive injections of LPS. The two exotoxins furnish significant protection against the lethal outcome of this reaction. It is known that the acute haematological and haemodynamic changes are accompanied by alterations in the levels of various endogenous mediators: glucocorticoid hormones, prostaglandins, arachidonic acid metabolites, cytokines and proteases. In vitro effects of LPS on murine leukocyte cell lines can be antagonized by pertussis toxin, implicating a Gi-like regulatory protein in the mediation of these effects. Experiments designed to study the involvement of particular second messenger systems (cAMP and phosphatidylinositol) used by LPS in vivo, revealed that the protective effects conferred by these exotoxins are associated with the antagonization of alterations caused by LPS. No correlation was found between the levels of IL-6 and the mortality rate in this experimental mouse model. The results indicate that G proteins play a role in the generation of the Schwartzman reaction and open a new approach for pharmacological intervention in endotoxemia and in clinical settings with Gram-negative sepsis.

Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production.

The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17 alpha-hydroxylase inhibitor and epostane (WIN-32729), a 3 beta-hydroxysteroid dehydrogenase inhibitor, were studied. Metabolism of [14C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 microM SU-10603 and 5 microM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5 alpha-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22 R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher respectively, than testosterone production. The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.

Inhibitory effects of alkane sulphonates on the function of immature rat Leydig, Sertoli and peritubular cells cultured in vitro.

Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40-160 micrograms/ml) blocked the conversion of C21 and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C21 steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.

Stromal cells from the rat prostate secrete androgen-regulated factors which modulate Sertoli cell function.

Testicular peritubular cells produce paracrine mediators which modulate Sertoli cell function. The production of these mediators (P Mod-S) is controlled by androgens suggesting that mesenchymal-epithelial interactions play an important role in androgen action in the testis. We investigated whether mesenchymal cells from the prostate, another androgen target tissue, produce analogous mediators. To this end rat Sertoli cell cultures were exposed to dialyzed spent media derived from testicular peritubular cells, prostatic stromal cells or footsole fibroblasts. It is demonstrated that the effects of spent media from peritubular cells and stromal cells are nearly identical: they stimulate the production of androgen binding protein and transferrin and they inhibit FSH-inducible aromatase activity. The active principle (or principles) involved is non-dialyzable, heat sensitive and trypsin sensitive. Its production is markedly stimulated by androgens. Fibroblast spent media are inactive. It is concluded that mesenchymal tissue derived from different androgen target tissues may produce identical or similar mediators of androgen action acting on epithelial cells.

Testicular peritubular cells secrete a protein under androgen control that inhibits induction of aromatase activity in Sertoli cells.

We investigated the role of peritubular cell-Sertoli cell interactions in the control of Sertoli cell function by androgens. Decreased FSH-inducible aromatase activity and increased secretion of androgen-binding protein (ABP) were used as parameters of androgen action on Sertoli cells. It is demonstrated that coculturing Sertoli cells with limited amounts of peritubular cells (20%) has only marginal effects on inducible aromatase activity or ABP secretion, but markedly increases the response of these parameters to androgens. Conditioned media derived from peritubular cells pretreated with androgens mimick the effects observed in the coculture system. Evidence is presented that androgen action on peritubular cells is mediated by an androgen receptor and that the concentration of this receptor is increased up to 3-fold by androgens. Preliminary experiments suggest some analogy between the peritubular cell factors that stimulate ABP production and those that inhibit aromatase induction. The active principles responsible for both activities are thermolabile trypsine-sensitive proteins with a mol wt between 50,000-100,000, and androgen induction by both activities shows an identical time course, characterized by a 4-day latent period. Nonetheless, much higher concentrations of peritubular cell secretion products seem to be required to inhibit aromatase induction than to stimulate ABP production, indicating that the active principles are not necessarily identical. It is concluded that peritubular cell secretion products mimick and mediate not only stimulatory but also inhibitory effects of androgens on Sertoli cells.

Stimulatory effects of antiviral adenosine analogs on steroidogenesis in Leydig cells.

We studied the effects of 12 adenosine analogs which are active as antiviral agents on basal and LH-stimulated steroidogenesis in Leydig cells. It is shown that several of these analogs markedly stimulate the production of androgens and androgen precursors in the absence of LH. These effects are observed in interstitial cell cultures derived from immature rats as well as in freshly prepared Percoll-purified Leydig cells derived from adult mice. Some compounds (neplanocin A, S-isobutyladenosine) are active from a concentration of 10(-6) M on. In the presence of maximally effective concentrations of LH or dbcAMP the stimulatory effects disappear and some compounds even become inhibitory. Only within the neplanocin series of derivatives did we observe a correlation between antiviral and steroidogenic activity. Four representative test compounds were studied in more detail: neplanocin A, 7-deazaadenosine, 4'-thioadenosine and S-isobutyladenosine. The first three significantly inhibit phospholipid N-methyltransferase activity in intact Leydig cells. However, our data do not suggest a close link between phospholipid methylation and the stimulatory or inhibitory effects of these test compounds on steroidogenesis. In cultured rat interstitial cells neplanocin A, S-isobutyladenosine and in particular 4'-thioadenosine markedly stimulate the production of cAMP. This effect is probably mediated via adenosine (A2) receptors which are known to appear in such cultures. Comparable effects are not observed in freshly prepared mouse Leydig cells. Again, however, there is no obvious correlation between the ability of the test compounds to stimulate cAMP production and their effects on steroidogenesis. It is concluded that compounds to stimulate cAMP production and their effects on steroidogenesis. It is concluded that antiviral adenosine analogs have complex effects on Leydig cell steroidogenesis. There may not be a unifying mechanism of action underlying the various biological effects of these agonists.

Interleukin-1 stimulates steroidogenesis in cultured rat Leydig cells.

We have studied the effects of human interleukin-1 beta on steroidogenesis in cultured immature rat Leydig cells. In the presence of low concentrations of LH or in its absence interleukin-1 beta markedly stimulates the production of C19-steroids (testosterone and androstenedione) and C21-steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-4-en-3-one). In the presence of maximally effective concentrations of LH, on the contrary, interleukin-1 beta inhibits C19-steroid production by provoking a block at the level of the 17,20-desmolase. These actions were observed with similar low doses of interleukin-1 beta (ED50 = 1 U/ml), but the stimulatory effects are evident within the first 2 h of incubation whereas the inhibitory actions appeared after a latent period of 6 h. None of the effects of interleukin-1 beta is accompanied by measurable changes in cAMP output, and the effects are much less pronounced in freshly isolated Leydig cells than in cultured cells. At maximally effective doses the effects of interleukin-1 beta are additive with those of a number of other Leydig cell agonists: LHRH, epidermal growth factor, arginine vasopressin and Sertoli cell-derived factor(s), suggesting that these agonists act by mechanisms different from that of interleukin-1 beta. The possibility is considered that Leydig cells may act as target cells for interleukin-1 beta derived from testicular macrophages or for interleukin-1-like factors derived from testicular tubules.

Prolonged exposure to androgens suppresses follicle-stimulating hormone-induced aromatase activity in rat Sertoli cell cultures.

We studied the effects of androgens on basal and FSH-stimulated aromatase activity in Sertoli cell-enriched monolayers. Daily exposure to androgens from the first day of culture results in a time- and dose-dependent decrease in inducible aromatase activity. The inhibition is observed whether FSH, L-isoproterenol or (Bu)2cAMP is used as inducer of the aromatase activity. Basal activity is not affected by preincubation with androgens and the inhibitory effect is not observed after short-term exposure (24 h) to these hormones. The ability of different androgens to decrease inducible aromatase activity does not depend on their ability to serve as a substrate for the aromatase but parallels their ability to stimulate the production of androgen-binding protein. Moreover, the effect of testosterone is neutralized by the antiandrogen cyproterone acetate, suggesting that it is mediated by the androgen receptor. These data suggest that testicular androgens may be responsible for the decrease in FSH-inducible aromatase activity observed in intact rats between days 10 and 30 of life. Similarly, the removal of these androgens during the preparation of Sertoli cell cultures may explain the spontaneous increase in inducible aromatase activity observed when these cultures are maintained in the absence of androgens.

Follicle-stimulating hormone and androgens increase the concentration of the androgen receptor in Sertoli cells.

The influence of FSH and androgens on androgen receptor levels in primary Sertoli cell cultures from immature rats is studied in a monolayer binding assay and by sucrose density gradient centrifugation using the synthetic radiolabeled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) as a ligand. Preincubation of Sertoli cells for 4 days with FSH, testosterone, or 5 alpha-dihydrotestosterone results into a 2- to 3-fold increase in mibolerone binding, as measured 18 h after removal of the agonists. The combination of androgens and FSH has additive effects. The action of FSH can be mimicked by (Bu)2cAMP, and the activity of the androgens can be blocked by the antiandrogen cyproterone acetate. The mibolerone-binding protein has the ligand specificity, affinity, and sedimentation behavior characteristic for an androgen receptor. Using a DEAE-cellulose filter disc assay and 5 alpha-dihydrotestosterone as a ligand, androgen-binding protein (ABP) was measured in the media of the studied Sertoli cell cultures. Despite some similarity in the hormonal control of ABP and the androgen receptor, there are distinct differences in the ligand specificity of the two androgen binding proteins, which exclude that ABP might interfere with the receptor measurements. The effects of androgens and FSH on the androgen receptor are evident at concentrations equal to or lower than those required to provoke a measurable increase in ABP secretion. It is concluded that FSH and androgens control androgen receptor levels in Sertoli cells.

A Leydig cell stimulatory factor produced by human testicular tubules.

It is demonstrated that tubular fragments derived from human testes and cultured in vitro produce a factor that stimulates the production of testosterone by human interstitial cells and by Percoll-purified Leydig cells from rat and mouse origin. The active principle in the conditioned media is a thermo-labile and trypsin-sensitive protein with an MW greater than 10,000. The factor is active in the presence as well as in the absence of maximally effective concentrations of LH and its activity is not accompanied by measurable changes in cAMP production. There are several points of analogy between this factor and a Leydig cell stimulatory protein produced by rat Sertoli cells. Molecular weight fractionation of spent media from human testicular tubules using an Amicon ultrafiltration system results in a 38- to 102-fold increase in Leydig cell stimulatory activity in a fraction corresponding to a molecular weight of 10,000 up to 30,000. These figures are comparable to those observed after molecular weight fractionation of spent media from rat Sertoli cells. Dose-response curves with partially purified preparations from human and rat origin yield parallel dose-response curves. In rat Sertoli cells as well as in human testicular tubules, the production of the active principle is stimulated by FSH and dibutyryl cAMP. Finally, maximally effective concentrations of the active principles of human and rat origin display no additive effects whereas additive effects are clearly evident with other Leydig cell stimulatory factors such as LHRHa and EGF. The hypothesis is advanced that the Leydig cell stimulatory factors from tubular origin may act as paracrine regulatory molecules responsible for the effects of FSH on Leydig cell function.