Biomedical subjects

G Verhoeven

Publications and source records attributed to G Verhoeven.

At least 127 records · Page 7Linked to original sources

PubMed1989

Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein.

We demonstrate that the 204 bp intronic gene fragment of C3(1), which has a specific in vitro affinity for the androgen receptor, is able to confer androgen responsiveness to a heterologous promoter. This characteristic is completely destroyed by a single G----T substitution, affecting a 5'-TGTTCT-3' element that closely resembles the consensus sequence of the glucocorticoid and progesterone response elements (GRE/PRE). In fact we could show that this androgen response element (ARE) also acts as a similarly weak GRE or PRE in T-47D cells.

Androgen-Binding Protein↗

PubMed1989

Kallikrein-related protease in the rat ventral prostate: cDNA cloning and androgen regulation.

A kallikrein-related protease was purified from rat ventral prostate cytosol by means of DEAE-Sepharose chromatography, followed by gel filtration on Sephadex G-100 and CM-cellulose chromatography. Antibodies raised in rabbits against the purified protease recognize two bands on immunoblots of prostatic cytosol: a 31,000 Da band and an 18,000 Da band, which constitutes a proteolytic breakdown product of the former. The corresponding cDNA was isolated from a prostatic cDNA library, inserted in a lambda gt11 vector, using immunodetection for screening and identified as encoding a kallikrein- and tonin-related protease. Castration resulted in a marked decrease of the level of the protease and its mRNA, whereas administration of androgens to castrated animals resulted in marked stimulation. These data support the hypothesis that this protease is a member of a cluster of proteins, that are regulated in parallel by androgens in prostatic epithelial cells.

Amino Acid Sequence↗

PubMed1989

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.

Androgens↗

PubMed1989

Binding of androgen-receptor complexes to alpha 2u-globulin genes and to the long terminal repeat of mouse mammary tumor virus.

The binding of androgen-receptor complexes to fragments derived from two alpha 2u-globulin genes (RAP 01 and RAO 01) was studied using a DNA-cellulose competition assay. Rat prostate cytosol labelled with [3H]mibolerone was used as a source of the androgen receptor. Two controls were included in these studies: the long terminal repeat (LTR) of mouse mammary tumor virus which has previously been shown to act as an androgen response element and a fragment of the C3 gene of prostatic binding protein which has been demonstrated to bind androgen-receptor complexes. Our experiments indicate that androgen-receptor complexes bind specifically and with comparable affinity to the C3 gene fragment, the LTR and a fragment of RAP 01 located in the 5'-upstream region (bp -642 up to -584). No specific interaction was observed with fragments derived from RAO 01. The region of RAP 01 which binds androgen-receptor complexes has previously been shown to interact with glucocorticoid receptors and contains a 17 bp sequence homologous with the consensus sequence for glucocorticoid-receptor binding. A mutation in this sequence in RAO 01 may be responsible for the loss of glucocorticoid and androgen-receptor binding. It is concluded that at least one member of the alpha 2u-globulin gene family interacts directly with androgen-receptor complexes with an affinity comparable to that observed for other androgen-dependent genes. The binding is observed in a region displaying also affinity for the glucocorticoid receptor.

Alpha-Globulins↗

PubMed1989

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.

Alkanesulfonates↗

PubMed1989

Influence of development, estrogens, and food intake on apolipoprotein A-I, A-II, and E mRNA in rat liver and intestine.

UNLABELLED: The influence of development and ethinylestradiol (EE) on apolipoprotein (apo) A-I, A-II, and E mRNA in rat liver and intestine was studied by dot blot hybridization and Northern blot analysis. ApoA-I mRNA levels were maximal in the perinatal period and declined after day 15. An opposite trend was noted for the apoA-II mRNA levels, whereas apoE mRNA remained fairly constant. Liver apoA-I mRNA levels increased after ovariectomy (OVX). A further rise was observed when EE was given at 2000 micrograms/day. When the influence of OVX and EE was controlled for food intake by pair-feeding, OVX still increased hepatic apoA-I mRNA. The rise in liver apoA-I mRNA after EE, however, was no longer significant. Under the same conditions OVX slightly increased intestinal apoA-I mRNA. EE (2000 micrograms/day) decreased intestinal apoA-I mRNA to 80% of the pair-fed controls. Liver apoA-II mRNA levels did not change after OVX when the animals were fed ad libitum, but decreased slightly when the rats were pair-fed. EE caused a dose-dependent decrease in liver apoA-II mRNA, irrespective of food intake. None of these treatments caused any change in liver apoE mRNA levels. Serum apoA-I levels increased upon OVX, while serum apoE did not change. EE provoked a dose-dependent decrease of both apolipoproteins in serum. IN CONCLUSION: 1) Changes in food intake play an important role in the in vivo effects of estrogens on apolipoprotein mRNA levels. 2) The stimulatory effect of OVX on hepatic apoA-I mRNA as well as the inhibitory effect of EE on hepatic apoA-II mRNA are independent of food intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

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.

Adenosine↗

PubMed1988

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.

17-alpha-Hydroxyprogesterone↗

PubMed1988

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.

Androgens↗

PubMed1988

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.

Androgen-Binding Protein↗

PubMed1988

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.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Glucocorticoid receptor binding to defined regions of alpha 2u-globulin genes.

A DNA-cellulose competition assay was used to study binding of glucocorticoid receptor complexes to two alpha 2u-globulin genes, RAP 01 and RAO 01. Two binding regions were found in RAP 01, one localized between bp -642 and -452, the other between -252 and -118 from the transcriptional initiation site. Only the second region was found in RAO 01. The binding affinity was comparable to that observed using a long terminal repeat fragment of mouse mammary tumor virus. Both regions contain sequences homologous to a 17-bp consensus proposed for the glucocorticoid receptor binding. Therefore, despite evidence that glucocorticoid induction of alpha 2u-globulin may be indirect, a direct action of the receptor in the 5'-upstream region of the genes cannot be excluded.

Alpha-Globulins↗

PubMed1987

Comparison of the 5' upstream putative regulatory sequences of three members of the alpha 2u-globulin gene family.

We have isolated and characterized seven members of the alpha 2u-globulin gene family from a rat genomic library. The 5' upstream region (up to 1250 base pairs starting from the EcoRI site in exon 2) of three clones was sequenced. The major transcriptional start points were located 25 base pairs downstream from the 'TATA' box. A very high degree of homology was observed over the entire studied region. Two of the examined genes displayed structural features which suggest that their expression may be impeded. A high degree of homology was observed between the promotor regions of alpha 2u-globulin and those of the major urinary protein (MUP) multigene family of the mouse. A remarkable feature is the variable length of an A-rich region between the putative 'CAAT' and the 'TATA' consensus sequences. The size of this region differs markedly between MUP and alpha 2u-globulin and between different members of the alpha 2u-globulin gene family. Comparison of the alpha 2u-globulin promotor with the corresponding region of other androgen-dependent genes (the C1, C2 and C3 subunits of prostatic steroid binding protein) reveals the presence of an A-rich region of homology located approximately 378 base pairs upstream from the cap site in the alpha 2u-globulin genes. This region compares well with a sequence of putative enhancer function previously demonstrated in the alpha-fetoprotein promotor and in the immunoglobulin heavy chain promoter.

Alpha-Globulins↗

PubMed1987

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.

Animals↗

PubMed1987

Feminizing testicular Leydig cell tumor: hormonal profile before and after unilateral orchidectomy.

The effect of chronic hyperestrogenism on gonadal function was studied in three men who had estrogen-secreting Leydig cell tumors before unilateral orchidectomy and for 11-43 months after surgery. All three men had low plasma gonadotropin and testosterone levels and increased estradiol levels. Impairment of testicular steroidogenesis was also suggested by increased progesterone to 17-hydroxyprogesterone and 17-hydroxyprogesterone to androstenedione ratios in both spermatic venous plasma and the medium of Leydig tumor cells from one patient incubated in vitro. Before surgery, spermatogenesis was abnormal in two men. Testicular endocrine function and spermatogenesis did not return to normal after surgery. During the follow-up period, plasma gonadotropin levels were high in all three men, and testosterone was low normal. Estradiol levels decreased to normal immediately after surgery and then returned to the upper normal limit. The response to hCG stimulation in one man was subnormal. We conclude that chronic hyperestrogenism produced hypothalamo-pituitary inhibition as well as direct steroidogenic blockade at the testicular level. Long term impairment of both endocrine and exocrine testicular functions may be secondary to slowly reversible (or irreversible) estrogen-induced damage to tubular and Leydig cells.

Adult↗

PubMed1986

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.

Adrenal Glands↗

PubMed1986

Desensitization of Sertoli cell-enriched cultures by FSH, L-isoproterenol and glucagon. Influence on subsequent stimulation of 17 beta-estradiol production.

Sertoli cell-enriched cultures derived from 19-day old rats were exposed to FSH, L-isoproterenol, glucagon and dbcAMP for 24 up to 96 h. The influence of primary stimulation with these agonists on the response of the cells to subsequent stimulation with the homologous or heterologous agonists was investigated. Particular attention was paid to the response of the aromatase system defined as the ability of the cells to convert testosterone into 17 beta-estradiol. The responsiveness of this system was compared with the responsiveness of two other systems: adenylate cyclase and phosphodiesterase. It could be demonstrated that preincubation with the mentioned agonists results in a decreased responsiveness (maximal response) and a decreased sensitivity (ED50) upon re-stimulation with the homologous agonists. Preincubation with FSH also provokes partial desensitization for glucagon and L-isoproterenol. This heterologous desensitization can be mimicked by dbcAMP. The mentioned desensitization reactions are accompanied by a marked decrease in the accumulation of cAMP in the medium. Whereas desensitization of the adenylate cyclase occurs rapidly, desensitization of the aromatase response requires protracted stimulation for 3-4 days. In contrast with the adenylate cyclase and the aromatase system, the responsiveness of phosphodiesterase to FSH, L-isoproterenol, glucagon and dbcAMP is not affected by repeated stimulation for 96 h. It is concluded that hormonal desensitization affects both early (cAMP) and late (aromatase) responses of the Sertoli cell. However, some responses, such as phosphodiesterase activity seem to escape the desensitization process.

Animals↗

PubMed1986

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.

Animals↗