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G Verhoeven

Publications and source records attributed to G Verhoeven.

At least 19 recordsLinked to original sources
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↗
PubMed1992

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.

Animals↗
PubMed1992

Local control systems within the testis.

A number of physiological and pathological observations cannot readily be explained unless one accepts that there exists within the testis some sort of local control system. This local network of regulatory interactions offers not only an additional level of fine regulation for individual testicular functions, but also creates an opportunity for co-ordination and integration of distinct activities such as germ cell development and androgen production. There is an overwhelming amount of data indicating that the testis produces a variety of regulatory molecules and that many of these agonists have marked effects on the function of testicular cells in vitro. Some of these molecules are identical with or are at least related to known hormonal and humoral agonists. Others are novel and require further characterization. The exact cellular origin of many of these regulatory factors remains unknown. This overview has been limited to regulatory interactions between somatic testicular cells. Particular attention has been paid to communications between the interstitial and the tubular compartment. It should be evident that the nature and the significance of these interactions is only beginning to emerge. The major difficulty remains to distinguish effects that are restricted to the specific and often artificial conditions of in vitro systems from phenomena that are relevant to testicular control in vivo. Further progress in this field will rely on the development of appropriate systems to study local interactions in vivo. Valuable attempts have been made in this direction: vitamin A induced synchronization of spermatogenesis may offer a model to study stage dependent alterations in the interstitial compartment (Morales and Griswold, 1987; Bartlett et al, 1989); destruction of Leydig cells followed by substitution with androgens might clarify the role of non-steroidal Leydig cell mediators on tubular function (Shape et al, 1988). Up to now these approaches have failed to demonstrate an important role for local regulatory interactions. It is obvious that both models are relatively crude, however, and that subtle changes may have been missed under the experimental conditions used. It should be stressed that some of the observed complexities may be inherent to local regulatory networks. In fact, such networks tend to display a certain level of redundancy. It is evident, for example, that a number of locally produced mediators can also reach the testis via the circulation. In this setting the relative contribution of circulating and locally produced factors may vary depending on developmental stages, physiological or pathological conditions. A relative redundancy may exist for distinct locally produced mediators.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗
PubMed1992

Immunoreactive inhibin in plasma, amniotic fluid, and gonadal tissue of male and female chick embryos.

Immunoreactive inhibin was measured in plasma, amniotic fluid, gonads, and Wolffian bodies (mesonephros) of male and female chick embryos during the last week of their 21-day incubation period. The antiserum used was raised against bovine 31-kDa inhibin and was validated for RIA of inhibin in the chicken. Amniotic fluid concentrations of immunoreactive inhibin were relatively low and remained constant between Days 14 and 19. Plasma concentrations, in contrast, were high on Day 14 but declined steeply thereafter. Significantly higher plasma concentrations were noted in male than in female embryos and an even more pronounced sex difference was observed for the gonadal inhibin content. On Day 21, testes contained approximately 35 times more immunoreactive inhibin than ovaries. Surprisingly, inhibin contents in testes and male Wolffian bodies increased rather than decreased towards the end of the incubation period, indicating that gonadal and plasma inhibin concentrations are regulated, at least in part, independently. It is concluded that the chick embryo presents a convenient model for study of the secretion, the control, and the role of inhibin from fetal origin. The sex difference in plasma and gonadal inhibin suggests a differential role of inhibin in the development of the reproductive system of both sexes.

Amniotic Fluid↗
PubMed1992

Source of immunoreactive inhibin in the chicken ovary.

High concentrations of immunoreactive inhibin were detected in the plasma of the laying domestic hen using a heterologous RIA validated for use in the chicken. Cessation of egg production induced by restricting the intake of nutrients decreased circulating inhibin to approximately 20% of its original concentration within 8 days, indicating that the ovary is the major source of the measured material. Dissection of ovarian follicles revealed that inhibin is nearly exclusively produced in the granulosa cell layer. When expressed per milligram cell protein the concentration of inhibin decreased significantly in granulosa layers of follicles of succeeding order in the hierarchy (F4 to F1). The concentration of progesterone increased in the granulosa layers of the same follicles whereas oestradiol in the surrounding theca layers decreased. In vitro culture of granulosa cells derived from follicles at different stages of development confirmed the decrease in inhibin secretion as a function of follicular growth observed in vivo. The granulosa cell inhibin secretion is stimulated by LH as well as by FSH, the former being the most effective one. The physiological significance of these changes in inhibin concentration during follicular maturation requires further investigation. It may be concluded, however, that the chicken presents a useful model for the study of the endocrine as well as the paracrine function of ovarian inhibin.

Analysis of Variance↗
PubMed1992

Androgens and the testis.

The present survey focuses on some unique features of the testis as an androgen target tissue. Within the testis androgens act in a paracrine rather than in an endocrine fashion. All the available evidence suggests that the concentration of androgens that surrounds testicular target cells is much higher than that observed in peripheral target tissues, but the exact concentration remains unknown. The concentration of androgens required to maintain spermatogenesis considerably exceeds that observed in the peripheral circulation although it is probably lower than that which exists within the testis. The effects of androgens on spermatogenesis are indirect and are mediated by somatic cells. Sertoli cells are the most likely mediators of the effects of androgens on germ cell development. These cells contain androgen receptors which are upregulated by FSH and by androgens and they respond to androgens in vitro. They are not the only androgen-responsive cells in the testis, however, and some effects of androgens on Sertoli cells (epithelial cells) are indirect and are actually mediated by paracrine factors produced by underlying peritubular cells (mesenchymal cells). Androgen-regulated mesenchymal-epithelial interactions may not be limited to the testis but may be a more general feature of androgen action in several target tissues and our data suggest that the mediators involved may be very similar or identical. A final interesting aspect of androgen action in the testis is that the compartment which responds to androgens (the tubular compartment) may locally modulate the activity of the compartment which is responsible for androgen production (the interstitial compartment). A complex network of paracrine mediators is responsible for these interactions.

Androgens↗
PubMed1991

Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli.

A fragment of the rat androgen receptor (amino acids 533-637) containing the DNA-binding domain was produced in Escherichia coli as a fusion product with protein A of Staphylococcus aureus. The fusion protein was purified on IgG-Sepharose, a method that does not involve the use of denaturing agents. Approximately 4 mg of fusion protein was obtained from 500 ml of bacterial culture. In gel shift assays, the recombinant DNA-binding domain displays an affinity for a fragment of the long terminal repeat of mouse mammary tumor virus and for an intronic fragment of the gene coding for the C3 component of the androgen-regulated rat prostatic binding protein. In a DNase I footprinting assay, the fusion protein protects a sequence in the C3 fragment that has previously been shown to act as a functional androgen response element. Interestingly, a single base pair mutation in the response element, which abolishes androgen inducibility, also destroys the ability to interact with the recombinant androgen receptor DNA-binding domain.

Androgens↗
PubMed1991

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.

Animals↗
PubMed1991

Paracrine interactions between the interstitial and the tubular compartment of the testis.

In the present paper we will briefly review the role of testicular peptides in the interactions between the tubular and the interstitial compartment. Two interactions are discussed in some more detail. 1) It is shown that the effects of FSH on Leydig cell function may be mediated by at least three types of diffusible mediators produced by Sertoli cells: SCF (a 10-30 kD protein), IGF-I and one or more other still unidentified factors. 2) Evidence is presented that some of the effects of androgens on Sertoli cells may be mediated by diffusible factors produced by peritubular cells and that similar or identical mediators are also produced by prostatic stromal cells.

Androgens↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Aging↗
PubMed1990

Antibodies against synthetic peptides recognize the human and rat androgen receptor.

Antibodies against two synthetic peptides (aa 299-311 and aa 544-559) selected in different immunogenic domains of the human AR, were induced in rabbits. Antiserum reactivity against the native receptor was investigated by gel permeation chromatography and sucrose density gradient centrifugation using [3H]mibolerone-labeled rat prostate cytosol and [3H]5 alpha-dihydrotestosterone-labeled T-47D cytosol as a source of AR. The absence of cross-reactivity of the antisera with estrogen, progesterone and glucocorticoid receptor was confirmed by density gradient centrifugation of rat uterus cytosol labeled with [3H]E2 or [3H]ORG 2058 and rat liver cytosol labeled with [3H]dexamethasone. After partial proteolytic breakdown of rat prostate AR by endogenous proteases the steroid-labeled receptor was recognized only by the second peptide (aa 544-559) antibody. This proteolytic breakdown could be prevented to a large degree by addition of a high concentration of soybean trypsin inhibitor. The specific AR antibodies provide new tools for the functional analysis of AR, since they interact selectively with specific domains of the receptor.

Amino Acid Sequence↗
PubMed1990

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.

Androgens↗
PubMed1990

Apolipoprotein A-IV messenger ribonucleic acid abundance is regulated in a tissue-specific manner.

The influence of development and estrogen, thyroid hormone, corticosteroid, and fibrate administration on apolipoprotein (apo) A-IV mRNA levels in the liver and intestine and on serum or plasma concentrations of apo A-IV was studied in the rat. Treatment of ovariectomized rats with ethinyl estradiol provoked a dose-dependent decrease in liver apo A-IV mRNA levels, whereas intestinal apo A-IV mRNA did not change. The serum apo A-IV concentration decreased in a dose-dependent manner. Administration of L-T4 increased liver apo A-IV mRNA levels more than 2-fold, while n-propylthiouracil (PTU) decreased these levels more than 4-fold. Intestinal apo A-IV mRNA levels remained constant upon L-T4 treatment, but increased after PTU. Change in thyroid hormone levels caused no significant alteration of plasma apo A-IV levels. Hydrocorticsone increased liver and intestinal apo A-IV mRNA levels 2- and 1.5-fold, respectively, without changing plasma apo A-IV. Liver and intestinal apo A-IV mRNA underwent opposite changes during development. Intestinal apo A-IV mRNA decreased gradually during the period of weaning, while liver apo A-IV mRNA was undetectable before day 20 of life and rose to adult levels thereafter. Both L-T4 and hydrocortisone were able to increase liver apo A-IV mRNA prematurely when rat pups were treated from day 9 on. Hypothyroidism induced by PTU, on the other hand, was able to delay the developmental rise in liver apo A-IV mRNA. The hypolipidemic drug clofibrate reduced liver apo A-IV mRNA more than 10-fold without changing the intestinal levels. Plasma apo A-IV decreased by one third. Ethinyl estradiol, thyroid hormones, and clofibrate regulate apo A-IV mRNA abundance in a tissue-specific manner. Only liver, not intestinal, apo A-IV mRNA levels respond to treatment. Furthermore, opposing changes in liver and intestinal apo A-IV mRNA levels occur during development, and thyroid hormones and glucocorticoids are able to accelerate the developmental changes in liver apo A-IV mRNA.

Animals↗
PubMed1990

Alterations in thyroid status modulate apolipoprotein, hepatic triglyceride lipase, and low density lipoprotein receptor in rats.

The influence of altered thyroid state is investigated on plasma apolipoprotein-A-I (apo-A-I), apo-B, and apo-E levels and on apo-A-I, apo-A-II, apo-B, apo-E, hepatic triglyceride lipase (HTGL), and low density lipoprotein (LDL) receptor mRNA levels in rat liver and intestine. Plasma total cholesterol and triglycerides are unchanged in hyperthyroid rats. Liver apo-A-I mRNA levels increase 3-fold, whereas intestinal apo-A-I mRNA levels remain constant. Plasma apo-A-I levels almost double after L-T4. Liver apo-B and apo-E and intestinal apo-B mRNA levels are not influenced by L-T4, but plasma apo-B and apo-E decrease significantly. In the liver, apo-A-II mRNA levels decrease, whereas LDL receptor mRNA levels increase more than 50%. HTGL mRNA is not influenced by L-T4. N-Propyl-thiouracil-induced hypothyroidism does not influence plasma triglycerides, but plasma cholesterol levels nearly double. Liver and intestinal apo-A-I mRNA levels and plasma apo-A-I concentrations remain constant after propylthiouracil treatment. Accompanying the increase in plasma apo-B, liver and intestinal apo-B mRNA concentrations rise by approximately 100% and 40%, respectively. Plasma apo-E increases nearly 2-fold, but liver, apo-A-II mRNA rises, whereas HTGL and LDL receptor mRNA levels decrease 20% and nearly 50%, respectively. In conclusion, thyroid hormones regulate rat apo-A-I and apo-A-II gene expression in opposite directions. Furthermore, the LDL receptor is regulated at the mRNA level, whereas HTGL gene expression is relatively resistant to alterations in thyroid status.

Animals↗