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G Verhoeven

Publications and source records attributed to G Verhoeven.

At least 91 records · Page 5Linked to original sources
PubMed1994

Different cells and cell lines produce factors that modulate Sertoli cell function.

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-beta, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45-50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

Animals↗
PubMed1994

Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements.

Comparable fragments of the androgen receptor (AR) (amino acids 540-607) and of the glucocorticoid receptor (GR) (amino acids 412-515) were expressed in E. coli as fusion proteins with protein A. Both fusion proteins, denoted ARF1 and GRF1, contain the DNA-binding domain and some flanking amino acids. In vitro binding assays have shown that both fusion proteins interact with androgen/glucocorticoid response elements (ARE/GREs) in an intron fragment of the C3(1) gene of the androgen-regulated rat prostatic binding protein and in the typically glucocorticoid-responsive long terminal repeat (LTR) promoter of mouse mammary tumour virus. Present results indicate that the interaction of both ARF1 and GRF1 with the C3(1) as well as the LTR fragments is enhanced in the presence of nuclear extract. The factor that gives rise to this enhancement appears to be ubiquitous and sensitive to trypsin and temperature treatment. In the C3(1) fragment, the enhancing effect requires the presence of an intact functional ARE/GRE (Core II) as well as a region spanning the ARE/GRE half-site Core I.

Androgens↗
PubMed1994

Androgen-dependent expression of cystatin-related protein (CRP) in the exorbital lacrimal gland of the rat.

Cystatin-related protein (CRP), also known as 20 (22)-kDa glycoprotein is expressed not only in the ventral prostate, but also in the lacrimal gland of adult male rats. In this study the expression of CRP in androgen-treated female animals is studied. CRP mRNA is absent in the lacrimal gland of untreated adult female rats, but can be induced by androgens, although this induction is slower than in castrated male rats. Estradiol, progesterone or glucocorticoids have no effect. In testicular feminized rats, however, CRP mRNA is not induced in the lacrimal gland by androgens. At the protein level, the presence of CRP in tears of adult male rats is demonstrated. In female animals or castrated male animals CRP can be induced by androgens in a dose-dependent way. Here also the induction is slower in female rats, even during secondary induction after previous full stimulation by androgens. These results indicate that androgens and a functionally normal androgen receptor are essential for the expression of CRP in the lacrimal gland. The time course of induction depends on the dose of androgens, the previous contact with androgens, the duration of the androgen-free interval and the sex of the animals.

Androgen-Insensitivity Syndrome↗
PubMed1994

Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines.

The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.

Adenylyl Cyclases↗
PubMed1994

Contribution of the fetal adrenal to circulating immunoactive inhibin in the chicken embryo.

High concentrations of immunoactive inhibin are observed in the plasma of male and female chicken embryos. The exact nature and the source of this material remain obscure. In a previous study we presented evidence that the fetal gonads are unlikely to be the main source of circulating inhibin (Biol Reprod 1993; 49:549-554). Here we demonstrate that the fetal adrenal may account for the major portion of circulating immunoactive inhibin in the chicken embryo. A comparison of the inhibin content of different fetal organs shows that, expressed per milligram of tissue, the adrenal ranks second. Only the testis has a higher inhibin content, while the inhibin content of the fetal ovary is considerably lower than that of the fetal adrenal. Suppression of endogenous ACTH secretion by administration of dexamethasone results in a marked decrease of plasma inhibin. Maximal suppression (down to 24% and 25% of the control values in male and female embryos, respectively) was observed with the lowest dose of dexamethasone tested (1 microgram/egg). Dexamethasone (100 microgram/egg) reduced testicular weight and testicular inhibin content to approximately 50% of the control value. It is unlikely, however, that this contributed significantly to the fall in circulating immunoactive inhibin. In fact, ovarian inhibin content was unaffected, but even so, a comparable drop in circulating inhibin was observed in female embryos. Dexamethasone caused only marginal changes in plasma FSH. Administration of a synthetic peptide with ACTH activity (Synacthen) in vivo did not produce measurable changes in circulating inhibin. Isolated and cultured fetal adrenal cells, however, produced immunoactive inhibin, and this production was increased fivefold after stimulation with synthetic ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1994

Peritubular myoid cells from immature rat testes secrete activin-A and express activin receptor type II in vitro.

The expression of activin type II and IIB receptors and inhibin alpha-, beta A-, and beta B-subunit messenger RNAs (mRNAs), and the secretion of immunoreactive and bioactive activin during culture of testicular peritubular myoid cells and peritubular myoid cell lines were studied. Cultured peritubular myoid cells and cell lines expressed high levels of inhibin beta A-subunit mRNA and some inhibin alpha- and beta B-subunit mRNA. Activin receptor type II mRNA was also detected, whereas activin receptor type IIB mRNA expression was not found. Expression of the beta A-subunit mRNA was present immediately after isolation of the cells and increased during culture in Eagle's Minimum Essential Medium containing 10% fetal calf serum. beta A-Subunit mRNA expression was not regulated by the synthetic androgen R1881. Western blotting of peritubular myoid cell- and peritubular cell line-conditioned media with a polyclonal antiserum against recombinant activin-A revealed the presence of 25-kilodalton activin-A, whereas activin bioactivity was detected using the animal cap assay. Because of the secretion of activin-A by peritubular myoid cells, the effects of recombinant activin-A on Sertoli cell inhibin and transferrin secretion were examined. Activin-A stimulated both basal and FSH-stimulated inhibin and transferrin production by Sertoli cells after 72 h of culture. These effects resemble the effects of the testicular paracrine factor PmodS on Sertoli cell function. It is concluded that activin-A is secreted by peritubular cells in vitro and that activin-A shares a number of effects on Sertoli cell function with PmodS.

Activin Receptors↗
PubMed1993

A Fos-Jun element in the first intron of an alpha 2u-globulin gene.

The hepatic expression of the alpha-2u-globulin gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect alpha 2u-globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an alpha 2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M2 peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG3 hepatoma cells treated with TPA. Co-transfection with fos and jun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.

Alpha-Globulins↗
PubMed1993

Developmental extinction of liver lipoprotein lipase mRNA expression might be regulated by an NF-1-like site.

The molecular mechanism underlying the extinction of lipoprotein lipase (LPL) expression in rat liver during development was investigated. A mouse (BWTG3) and a rat (7777) hepatoma, both of which exhibit characteristics of fetal hepatocytes, were found to contain LPL mRNA, whereas the more differentiated human (Hep G2 and Hep 3B) or rat (Fa32) hepatoma cell lines did not. Somatic cell hybrids between LPL-producing hepatoma cells and non-LPL-producing cells, such as adult rat hepatocytes or fibroblasts, exhibited extinction of LPL gene expression. Assay of expression of nested deletions in the 5' regulatory sequences of the LPL gene in the Hep G2 cell line and in BWTG3 cells localized sequences involved in the suppression of LPL production to a region between -591 and -288 relative to the transcription initiation site. A site with sequence homology to a glucocorticoid responsive element (GRE) was shown not to play an important role in the extinction process. A novel transcription factor, termed RF-1-LPL, was shown to bind to an NF-1-like site in this region. In contrast to neonatal animals, in adult animals an additional protein complex (RF-2-LPL), was formed on the NF-1-like site, suggesting that this sequence might recruit a trans-acting factor involved in the extinction of LPL gene expression in adult rat liver.

Animals↗
PubMed1993

Intronic androgen response elements of prostatic binding protein genes.

Transcription of the genes that code for the different components of Prostatic Binding Protein is dependent on androgens. DNA-cellulose competition assays have demonstrated the presence of regions with affinity for the androgen receptor located immediately upstream and in the first intron of these genes. DNase I footprinting in vitro now reveals the exact binding site of a receptor dimer in the first intron of the C3(1), C3(2) and C1 genes. Gene transfer experiments resulted in the description of functional androgen response elements in the C3 genes. The C1 intronic binding site, however, has a much weaker affinity for the DNA-binding domain of the androgen receptor, a characteristic that might explain its inability to function as an androgen response element in chimeric gene constructs.

Androgen-Binding Protein↗
PubMed1993

Interaction of androgen and glucocorticoid receptor DNA-binding domains with their response elements.

Fusion proteins containing the glucocorticoid and the androgen receptor DNA-binding domain (ARF1 and GRF1) were produced in Escherichia coli. DNAse I footprinting was used to compare the interaction of these proteins with responsive elements (REs) in a typically glucocorticoid-responsive gene (mouse mammary tumour virus (MMTV)) and in an androgen-responsive gene (the C3(1) gene of rat prostatic binding protein). It is demonstrated that response elements which most closely resemble the consensus sequence show identical footprinting patterns for ARF1 and GRF1. The protected regions suggest that these sequences are occupied by two DNA-binding domains (DBDs) forming a dimer. Regions that constitute imperfect RE sequences, however, are apparently recognized by only one DBD, which mainly protects the TGTTCT motif. At these REs, the protection patterns produced by ARF1 and GRF1 are not identical. In the long terminal repeat (LTR) of MMTV but not in C3(1), a mechanism other than classical dimer formation seems to increase the affinity of ARF1 and GFR1 for these imperfect REs.

Androgen-Binding Protein↗
PubMed1993

Proteins interacting with an androgen-responsive unit in the C3(1) gene intron.

The expression of the three genes encoding the components C1, C2 and C3 of prostatic binding protein (PBP) is under androgen control and restricted to the rat ventral prostate. The SstI-PvuII fragment of the first intron of the C3(1) gene displays two binding sites for ubiquitous transcription factors and one for a tissue-specific factor in a 80-bp region upstream of its androgen response element (ARE). The octamer transcription factor 1 (OTF-1) binds to the most distal element (site 1) while a member of the nuclear factor I (NF-I) family recognizes site 2. A third unidentified prostate-specific factor, which also occurs in castrated rats, interacts with the proximal element (site 3). In T-47D cells, both the OTF-1 and the NF-I-like factor can modulate the androgen response of the promoter in a reporter gene construct containing the C3(1) intronic fragment.

Androgen-Binding Protein↗
PubMed1993

Multiple binding sites for nuclear factors in the 5'-upstream region of two alpha 2u-globulin genes: implications for hormone-regulated and tissue-specific control.

In order to understand the tissue- and hormone-specific control of alpha 2u-globulin synthesis we isolated the 5'-upstream putative regulatory region of two alpha 2u-globulin genes: RAP 01 and RAO 01. Both clones seem to be expressed in rat liver. DNAseI footprinting analysis after incubation with rat liver nuclear extracts was used to identify regions of potential interest. Particular attention was paid to protected regions located in the neighbourhood of domains which, according to our previous studies, interact specifically with androgen- and glucocorticoid-receptor complexes. Fifteen DNAseI footprints could be mapped in clone RAO 01 (bp -758 up to the cap site). Nineteen footprints were observed in the corresponding region of RAP 01. Differences in the footprinting patterns were mainly observed in the more distal regions. Our data confirm the presence in both clones of two binding sites for the liver enriched factor pseudo-NF1 and one site for C/EBP previously observed in other alpha 2u-globulin genes. In addition we have been able to demonstrate, in RAP 01 only, a binding site for transferrin-liver factor 1. No differences in footprinting patterns could be demonstrated using liver nuclear extracts derived from animals with a high hepatic expression of alpha 2u-globulins (normal male rats) and animals with low to absent expression (prepubertal rats, female rats, rats with the testicular feminization syndrome, diabetic rats and hypophysectomized animals). Transfection experiments indicate that a fragment of RAP 01 (bp -643 up to -617) is able to act as a glucocorticoid and as an androgen response element. Larger fragments of RAP 01 and fragments of RAO 01 are ineffective. It is concluded that the expression of individual alpha 2u-globulin genes is probably the result of combinatorial interactions of several trans-acting factors with appropriate cis-acting elements. Moreover, important sites for tissue-specific and hormone-regulated expression may be situated outside the regions investigated.

Alpha-Globulins↗
PubMed1993

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)

Animals↗
PubMed1993

Changes in immunoreactive FSH and inhibin in developing chicken embryos and the effects of estradiol and the aromatase inhibitor R76713.

The interrelationship between immunoreactive inhibin and FSH was measured in chicken embryos from Day 8 to Day 20 of the incubation period. Plasma inhibin was measured by a heterologous RIA and FSH by a recently developed homologous RIA. Male and female inhibin plasma concentrations demonstrated a similar time course: a sharp increase with a peak on Day 13 was followed by a drop towards Day 18, after which inhibin levels stabilized. In male embryos, a similar time course was observed for the FSH concentrations. In sharp contrast, the FSH concentrations in the female embryos remained low and unchanged throughout the incubation period. Accordingly, sex differences were large for FSH and rather small for inhibin, and were noted from Day 10 and Day 12 on, respectively. No obvious inverse relationship was observed between inhibin and FSH. In male embryos, estradiol administration resulted in a dose-related decrease in FSH, accompanied by a less pronounced decrease in inhibin. Treatment with an aromatase inhibitor (R76713) increased FSH in female embryos and inhibin in both sexes. Generally, the experimentally induced changes in FSH were accompanied by comparatively small changes in inhibin. In conclusion, these observations suggest 1) that estradiol is an important inhibitor of pituitary FSH secretion in the chicken embryo, 2) that inhibin is not likely to play a major role in the feedback regulation of the hypothalamo-pituitary-gonadal axis, and 3) that an important fraction of the measured inhibin immunoactivity escapes the control of FSH. In view of the present data, the hypothesis is advanced that the gonads are not the major source of circulating immunoreactive inhibin in the chicken embryo.

Animals↗
PubMed1992

Interaction of estrogen receptor complexes with the promoter region of genes that are negatively regulated by estrogens: the alpha 2u-globulins.

Since estrogens strongly suppress the expression of alpha 2u-globulin genes in the rat liver, we studied the binding of estrogen-receptor complexes to fragments derived from alpha 2u-globulin gene RAO 01 using a DNA-cellulose competition assay. Rat uterus cytosol labelled with [3H]estradiol was used as a source of the estrogen receptor. As a positive control in these experiments we used an oligonucleotide containing the estrogen response element (ERE) cloned into pUC18. Our experiments indicate that estrogen-receptor complexes bind specifically to the ERE and to a fragment of RAO 01 located in the 5'-upstream region (bp -606 up to -575). This fragment is conserved among other members of this gene family. This is the first time that in vitro estrogen receptor binding is observed to gene fragments derived from a gene that is repressed by this steroid in vivo.

Alpha-Globulins↗
PubMed1992

Interaction of the 90-kDa heat shock protein with native and in vitro translated androgen receptor and receptor fragments.

Androgen receptor (AR) from rat ventral prostate and AR synthesized in vitro by translation in rabbit reticulocyte lysate of AR mRNA, transcribed from a pGEM-4Z DNA template were compared by gel permeation chromatography and by sucrose gradient ultracentrifugation. Under non-activating conditions the AR from rat prostate migrated as an 8-9 S complex of approx. 300 kDa. The addition of chicken antibodies against HSP90 shifted this complex to the void volume of the column or to the bottom of the ultracentrifugation gradient. Under activating conditions, on the other hand, the AR migrated as a 110 kDa, 5.2 S protein and was no longer displaced by HSP90 antibodies. Under all these conditions, the behaviour of in vitro synthesized AR was very similar to that of AR from rat prostate. By selective use of restriction enzymes on the template of transcription AR mutants could be prepared from which an increasing part was deleted at their carboxy terminal end. The interaction with HSP90 was conserved for AR1-758 missing the last 145 amino acids, but was lost in AR1-703. Furthermore, a large internal deletion (ARd41-469) of the major part of the amino terminal half of the AR did not result in the loss of HSP90 binding. These results indicate that a specific subregion (amino acids 704-758) of the carboxy terminal half of the AR is required for the interaction with HSP90.

Animals↗