Jean Cailleau

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.

A factor in spent media from Sertoli-cell-enriched cultures that stimulates steroidogenesis in Leydig cells

We investigated whether Sertoli cell spent media (SCM) contain a factor (or factors) which influences steroidogenesis in Leydig cells. Freshly prepared prepubertal interstitial cells or Percoll-purified Leydig cells and similar cells cultured in the presence or absence of LH were incubated for 24 h in the presence of a 5 alpha-reductase inhibitor and in the presence or absence of SCM. The accumulation of C19-steroids (testosterone and androstenedione), C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-4-en-3-one) and cyclic AMP was measured by radioimmunoassay. It could be demonstrated that SCM contains a factor that stimulates an early step in the steroidogenic pathway but at the same time hampers the conversion of C21-precursors into androgens. In freshly prepared Leydig cells the final effect is a stimulation of the androgen output. In Leydig cells cultured in the absence of LH, mainly C21-steroid output is increased. These biological effects resemble those observed with LHRH and its agonists. The activity of the Sertoli cell factor is not affected by an LHRH antagonist, however, and maximally effective concentrations of the factor and LHRH have additive effects, suggesting that they act by distinct receptor systems. Preliminary characterization shows that the factor in SCM is a thermolabile protein with a MW greater than 10 000. The production of the factor decreases during prolonged culture in serum-free medium. Addition of fetal calf serum causes a marked and dose-dependent increase in the production or activity of the factor. Several permanent cell lines (B16, Bowes, BHK, Ratec, RK13, Vero) produce a factor with comparable biological effects on Leydig cells. Nonetheless, the observation that the production of this factor by Sertoli cell cultures is stimulated by FSH and dbcAMP suggests that, in the testis, it may play a role in the paracrine control of Leydig cell function.

Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells.

We studied the effects of epidermal growth factor (EGF) on steroidogenesis in freshly prepared Percoll-purified Leydig cells from prepubertal and adult rats and mice, and in interstitial cells from immature rats cultured in the presence or absence of LH. It is demonstrated that EGF directly stimulates the output of C19-steroids (testosterone and androstenedione) as well as C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-4-en-3-one) in all systems studied. When combined with LH, EGF has little effect on freshly isolated cells but still stimulates steroidogenesis in cells cultured in the presence of LH. The strong inhibitory effects of EGF on androgen production that have been reported previously are only observed when cells that have lost part of their steroidogenic potential by prolonged culture in the absence of LH are acutely challenged with LH (or cholera toxin or dbcAMP) and EGF. Under the latter conditions EGF blocks the conversion of C21-steroids into C19-steroids. The stimulatory effects of EGF on androgen production are evident within the first hours of incubation and occur at ED50 values of 0.3 up to 2.5 ng/ml. They are not accompanied by any measurable change in the production of cAMP. The effects of EGF are compared to those of LHRH and those of SCF, a putative paracrine factor produced by Sertoli cells. Although there are many similarities between the effects of these three polypeptides on steroidogenesis in Leydig cells, our data indicate that they must act by different mechanisms. Moreover, SCF is the only one of these agonists that markedly stimulates androgen production in the presence of LH.

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.

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.

Specificity and partial purification of a factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells

There is increasing evidence that factors derived from the seminiferous tubules influence Leydig cell function in a paracrine way. In previous experiments we demonstrated that conditioned media from Sertoli cell-enriched cultures contain a protein with stimulatory activity on prepubertal rat Leydig cells. In this paper we further studied the specificity of this factor. In addition we describe a simple but efficient partial purification procedure. It is demonstrated that Sertoli cell conditioned media contain a factor that stimulates the testosterone output from prepubertal and adult Leydig cells. The effects are evident within the first hour of incubation and can be observed in the presence as well as in the absence of LH. Peritubular cells do not produce a similar factor but enhance the production of the Leydig cell stimulating factor when cocultured with Sertoli cells. The Sertoli cell factor acts on rat as well as on mouse Leydig cells. It barely influences the adrenostenedione output of ovarian stromal cells or the corticosterone output of adrenal cells. The production of this factor is enhanced by dbcAMP, FSH, L-isoproterenol and glucagon but is not affected by androgens. The characteristics of the Sertoli cell factor have been compared with those of a Leydig cell stimulating factor in the medium from an established rabbit kidney cell line: RK13. It is shown that the active principle in RK13 conditioned medium is also a thermolabile trypsin-sensitive protein with a mol. wt of more than 10,000. Nonetheless, the RK13 and Sertoli cell derived factors act by different mechanisms since at maximally effective concentrations their effects are additive. Finally it is demonstrated that molecular weight fractionation of Sertoli cell conditioned medium using an Amicon ultrafiltration system results in a 50- to 130-fold increase in Sertoli cell factor activity in a fraction corresponding to a mol. wt of 10,000 up to 30,000.

Desensitization of cultured rat sertoli cells by follicle-stimulating hormone and by l-isoproterenol

When Sertoli cell-enriched cultures derived from 19-day-old Wistar rats are exposed to FSH or L-isoproterenol, cAMP accumulates in the medium. Previous contact of cultured cells with either one of these agents results in a reversible state of desensitization. During secondary stimulation up to 20 times less cAMP accumulates in the medium and in the cells. The extent of desensitization depends on the concentration of FSH or L-isoproterenol to which the cells have been exposed and on the duration of this previous contact. Pre-incubation with FSH results in cross-desensitization for L-isoproterenol. Comparable heterologous desensitization is not observed after pre-incubation with L-isoproterenol. This suggests that down-regulation of the respective receptors cannot be the only explanation. Refractoriness can also be induced by dbcAMP. This opens the possibility that cAMP itself contributes to the homologous and heterologous desensitization process.

Hormonal control of phosphodiesterase activity in cultured rat Sertoli cells.

Phosphodiesterase activity was studied in cultures derived from 19-day-old rats and enriched with Sertoli cells. Pretreatment of such cultures with follicle-stimulating hormone or L-isoproterenol increased cAMP-phosphodiesterase activity 5.2 and 2.0 times, respectively. cGMP-phosphodiesterase activity was not affected. Similar effects were observed in freshly isolated cells. The stimulatory effect was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was mimicked by cholera toxin and dbcAMP. Increased activity was observed after a latent period of 1 h. Stimulation was blocked by cycloheximide and actinomycin D. The enzyme had an apparent Km for cAMP of 1.4 micro M. Its activity was enhanced by Mg2+ but not by Ca2+. It is concluded that phosphodiesterases play an important role in the hormonal control of Sertoli-cell function and may contribute to the refractory state of these cells after stimulation with various agonists.

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.

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.