PubMed Health⌕ Search

Biomedical subjects

O Avallet

Publications and source records attributed to O Avallet.

At least 19 recordsLinked to original sources
PubMed1999

Transcriptional and translational regulation of angiotensin II type 2 receptor by angiotensin II and growth factors.

The regulatory effects of angiotensin-II (AngII) and several growth factors, including insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor beta1 (TGFbeta1) on the AngII subtype 2 (AT2) receptor were studied using R3T3 cells, a mouse fibroblast cell line that expresses only AT2 receptors. AngII increased (in a time- and dose-dependent manner) AT2 binding sites but had no effects on AT2 messenger RNA (mRNA) levels. At maximal concentration (10(-7) M) AngII caused a 4-fold increase of AT2 receptor number. In contrast, IGF-1 increased (3- to 4-fold), whereas bFGF and TGFbeta1 decreased (by about 90% and 80%, respectively) AT2 receptor and mRNA levels. Moreover, AngII potentiated the effect of IGF-1 on receptor number, but not on AT2 mRNA levels, and significantly reduced the inhibitory action of bFGF and TGFbeta1 on AT2 binding sites but not on AT2 mRNA levels. None of these factors modified AT2 mRNA half-life. The potential effects of these factors on transcription of the AT2 gene were measured by means of nuclear run-on assays. IGF-1 increased the rate of transcription by about 2.5-fold, whereas bFGF and TGFbeta1 reduced it by 90 and 80%, respectively. In contrast, AngII did not modify either the basal or IGF-1-stimulated transcription rate. Finally, AngII alone or together with IGF-1, but not IGF-1 alone, increased the attachment of AT2 mRNA to polysomal fractions. The present findings demonstrate that the main mechanism by which AngII regulates the AT2 receptor is by increasing the rate of AT2 mRNA translation, whereas the stimulatory (IGF-1) or inhibitory (bFGF and TGFbeta1) effects of these growth factors on AT2 expression are exerted at the transcriptional level.

Angiotensin II↗
PubMed1998

Effects of growth factors on cell proliferation and angiotensin II type 2 receptor number and mRNA in PC12W and R3T3 cells.

Previous studies have suggested that the expression of angiotensin type 2 receptor was inversely related to cell proliferation. We examined the effects of insulin-like growth factor (IGF-1), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGFbeta1) and fetal calf serum (FCS) on cell proliferation and AT2 binding sites and mRNA level in PC12W (rat pheochromocytoma cell line) and R3T3 (mouse fibroblast cell line) which express abundant AT2 receptors. In both cell lines, serum deprivation markedly increased both AT2 receptor number and mRNA. However, in the absence of serum cell proliferation continued in PC12W and R3T3 at late passages (R3T3 LP) but not at early passages (R3T3 EP). In PC12W, none of the three growth factors studied stimulated cell proliferation, but TGFbeta1 and more particularly bFGF markedly reduced AT2 expression. In R3T3 LP, IGF-1 and bFGF, but not TGFbeta1, slightly stimulated cell proliferation, but the three factors, specially bFGF, reduced AT2 expression. In contrast, in R3T3 EP, the three growth factors significantly increased cell proliferation, but whereas TGFbeta1 and bFGF markedly reduced AT2 binding sites and mRNA, IGF-1 caused the opposite effects. These results indicate that regulation of AT2 expression is not correlated with cell proliferation and appears to be more complex than initially suspected. In addition, they show that the same factor can have an opposite effect on AT2 expression in the same cell line depending upon the cell passage.

Angiotensin II↗
PubMed1998

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B, the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat.

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III-IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX-X. p19 mRNAs were present in PS from stages III-IV onward and in RS up to stages 1-2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15-16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2.

Animals↗
PubMed1998

Food-dependent Cushing's syndrome: characterization and functional role of gastric inhibitory polypeptide receptor in the adrenals of three patients.

In the present work, the presence of gastric inhibitory polypeptide (GIP) receptors and their functional role in the adrenal cells of three patients with food-dependent Cushing's syndrome were studied. RT-PCR and in situ hybridization studies demonstrated the presence of GIP receptor in the adrenals of the three patients. The presence of this receptor was also demonstrated in two human fetal adrenals, but not in two normal adult human adrenals or in the adrenals of one patient with nonfood-dependent Cushing's syndrome. Freshly isolated cells from patient adrenals responded in a dose-dependent manner to the steroidogenic action of both ACTH and GIP, whereas cells from normal adrenals responded only to ACTH. Treatment of cultured normal adrenal cells with ACTH, but not with GIP, increased the messenger ribonucleic acid (mRNA) levels of cholesterol side-chain cleavage cytochrome P-450, P450c17, and 3beta-hydroxysteroid dehydrogenase, whereas both hormones enhanced these mRNAs in patients' adrenal cells, although the effects of ACTH were greater than those of GIP. Moreover, pretreatment with ACTH enhanced the steroidogenic responsiveness of both normal and patient adrenal cells, whereas GIP caused homologous desensitization, and this was associated with a marked reduction of GIP receptor mRNA levels, as demonstrated by RT-PCR and in situ hybridization. Finally, both ACTH and GIP inhibited DNA synthesis in one patient's adrenal cells, whereas in normal adrenal cells only ACTH had this effect. In conclusion, the present data demonstrate that ectopic expression of functional GIP receptors is the main cause of food-dependent Cushing's syndrome.

Adrenal Gland Neoplasms↗
PubMed1997

Sertoli cell-germ cell interactions and TGF beta 1 expression and secretion in vitro.

Transforming growth factor beta 1 (TGF beta 1) has been reported to be secreted and to act within the somatic cells of the testis. We examined whether the TGF beta 1 expression is present at mRNA and protein levels in purified rat Sertoli cells (SC), purified pachytene spermatocytes (SPC), and early spermatids (SPT) cultured alone or together. SC expressed a single TGF beta 1 transcript of 2.5 kb, but no TGF beta 1 protein could be detected in SC conditioned medium indicating that, if at all, SC secreted less than 10 pg/10(6) cells/24 h. Neither TGF beta 1 mRNA nor protein could be detected in either SPC or SPT. Coculture of SC with either SPC or SPT resulted in a 2-fold increase of TGF beta 1 mRNA and more importantly in the secretion of TGF beta 1 protein. These findings demonstrate that Sertoli cell-germ cell interactions regulate TGF beta 1 expression and secretion and indicate that TGF beta 1 may be involved in spermatogenesis.

Animals↗
PubMed1997

Pre- and postmeiotic expression of male germ cell-specific genes throughout 2-week cocultures of rat germinal and Sertoli cells.

The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2B were specifically expressed in PS whereas those encoding the transition proteins TP1 and TP2 were specific to RS. This resulted in p19:TP1 and TH2B:TP2 ratios that were much higher in PS fractions than in RS fractions prepared by elutriation. When PS or RS were seeded on Sertoli cell monolayers in bicameral chambers, both the number and the viability of the cells decreased during the coculture. However, both parameters were equal to, or higher than, 60% after 2 wk. In PS-Sertoli cell cocultures, the ratios of p19:TP1 and TH2B:TP2 decreased dramatically during the second week of culture; this was due not only to a decrease in the levels of p19 and TH2B mRNAs but also to an enhancement in the relative amounts of TP1 and TP2 as compared to the amounts present on the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the levels of the four mRNAs studied during the coculture period. DNA flow cytometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decrease in the tetraploid cell population (4C). No such changes were observed in Sertoli cell-only cultures. By contrast, the haploid population decreased 3-fold during the first week in RS-Sertoli cell cocultures. Immunocytochemical studies demonstrated further that 5-bromo-2'-deoxyuridine-labeled PS of stages V-VIII were able to differentiate into RS under the present coculture conditions. Hence, although clearly imperfect, the present coculture system should help to clarify the local regulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.

Animals↗
PubMed1996

Transcriptional regulation of the lutropin/human choriogonadotropin receptor and three enzymes of steroidogenesis by growth factors in cultured pig Leydig cells.

Recent data have shown that Leydig-cell-specific functions, and therefore steroidogenic capacity, can be regulated by lutropin/human choriogonadotropin collectively termed gonadotropin and by several growth factors that are produced by and act within the testis. However, the molecular mechanisms by which these factors regulate Leydig cells are not understood. In the present study, we have investigated the effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on mRNA for the gonadotropin receptor and three steroidogenic enzymes: cytochrome P-450scc, cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (17 alpha-hydroxylase), and 3 beta-hydroxysteroid dehydrogenase. IGF-1, which can enhance testosterone production, increased gonadotropin-receptor density after an increase in receptor mRNA levels, and it increased the level of mRNA for cytochrome P-450scc and 17 alpha-hydrolyase. Micromolar concentrations of insulin had similar effects to those of IGF-I. Moreover, the three factors that decreased testosterone production (EGF, bFGF and TGF beta 1) decreased gonadotropin receptor density, receptor mRNA levels and the mRNA levels for 17 alpha-hydroxylase. The potential effects of these growth factors on the transcription on the gonadotropin genes for the receptor and these three steroidogenic enzymes were measured by means of nuclear run-on assays. We demonstrated that the long-term inhibitory (EGF, bFGF, TGF beta 1) or stimulatory (IGF-I) effects of these growth factors are primarily due to a variation in the transcription rates of genes for the gonadotropin receptor, cytochrome P-450scc and 17 alpha-hydroxylase. Moreover, since previous studies have shown than some of these growth factors are expressed within the testis, they may play a physiological role in the regulation of differentiated testicular functions.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

[Paracrine regulation of Leydig cells].

In addition to the endocrine control of Leydig cell functions by LH, paracrine control of Leydig cell functions has been suspected from the indirect stimulatory effect of FSH on Leydig cells. Coculture experiments of Leydig and Sertoli cells and the effect of Sertoli cell conditioned media on Leydig cells confirmed the production by Sertoli cells of acute steroideogenic factor(s) and factors involved in the positive or negative control of Leydig cell differenciated functions. Characterization and purification of these paracrine factors has been until recently unsuccessful. Another approach has been to investigate whether compounds of known biological activities in other systems, were produced within the testis and act on leydig cells. IGF-I is produced by Sertoli and Leydig cells under the control of their respective gonadotropin, LH and FSH. IGF-I enhanced hCG responsiveness of Leydig cells by increasing both LH receptor and steroidogenic enzymes. On the contrary TGF-beta which is also produced by Sertoli and Leydig cells is a potent inhibitor of Leydig cell functions. Its production by Sertoli cells is inhibited by FSH. Inhibin enhanced Leydig cell differentiated cell functions. Activin has, conversely to what has been published in the rat, a stimulatory effect on Leydig cell functions in the immature porcine Leydig cell model. The effects of these growth factors or related molecules mainly consist in positive (IGF-I, Inhibin, Activin) or negative (TGF-beta, TGF-alpha/EGF, bFGF) trophic effects regulating LH/hCG receptor number and mRNAs and steroidogenic enzyme mRNAs and activites, allowing regulation of the responsiveness of Leydig cells to LH. Thus both gonadotropins contribute, directly for LH and indirectly, through paracrine mecanisms, for FSH, to testosterone production.

Activins↗
PubMed1995

Transcription and post-transcriptional regulation of luteotropin/chorionic gonadotropin receptor by the agonist in Leydig cells.

Porcine Leydig, cultured in a chemically defined medium, express luteotropin/human chorionic gonadotropin (LH/hCG) receptor and mRNA transcripts of several sizes (7.6, 6.7, 5.6, 4.7, 4, 2.6 and 1.4 kb). Incubation of these cells with hCG results in a concentration-dependent decrease of both LH/hCG receptor number and of all mRNA transcripts with a half-maximal at 0.01 nM. Time-course analysis of the effects of maximal (1 nM) concentration of hCG on both receptor number and mRNA levels results in a lag period of about 6-8 h. Thereafter, the receptor number progressively declines to reach a low point (20% of control) at 36 h, whereas more than 80% of receptor mRNA were lost between 8-12 h after addition of the hormone. By nuclear run-on assays, we showed that hCG caused a slight reduction (13 +/- 2%) in LH/hCG receptor gene transcription, which could not explain the rapid and pronounced mRNA decline observed between 8-12 h. In fact, we estimated that hCG reduced 10-fold (from < 22 h to 2 h) the half-life of LH/hCG receptor mRNA. Both actinomycin D and cycloheximide blocked the hCG-induced decrease in both receptor number and mRNA levels. These results indicate that the main mechanism by which hCG regulates its own receptor is by inducing a decrease in the stability of its own receptor mRNA and this effect requires induction of transcription and translation, presumably leading to synthesis of a labile factor(s) which favors the degradation of LH/hCG mRNA. Most of the effects of hCG are mediated by cAMP since treatment of cells with its 8-bromo derivative leads to a similar reduction in the level of LH/hCG receptor and mRNA. Finally, the effects of hCG are reversible, since after withdrawal of the hormone there was a recovery of receptor mRNA followed by receptor number.

Animals↗
PubMed1994

Immunohistochemical localization of transforming growth factor-beta 1 in the fetal and neonatal rat testis.

The localization of transforming growth factor-beta 1 in the fetal and neonatal rat testis (from day 13.5 of fetal life to postnatal day 20) was investigated by an immunohistochemical staining method employing a polyclonal anti-TGF-beta 1 antibody that does not cross react with either TGF-beta 2 or TGF-beta 3. In testis and mesonephros tissue, immunostaining for TGF-beta 1 was undetectable on fetal day 13.5 and appeared exclusively in the primordial Sertoli cells on fetal day 14.5. Staining in Sertoli cells was still clearly observed on days 15.5 and 16.5 of fetal life and became faint from fetal day 18.5 onwards. In fetal Leydig cells, a positive reaction for TGF-beta 1 appeared on day 16.5 and became very intense during late fetal life. After birth, fetal-type Leydig cells, which were still observed on postnatal days 4 and 20, also exhibited a very strong immunostaining for TGF-beta 1, whereas adult-type Leydig cells, observed on day 20, showed a slight staining. No immunoreactivity for TGF-beta 1 was found in germ cells and peritubular cells on any day studied. In conclusion, TGF-beta 1 is present very early in the fetal rat testis and its prevailing localization shows age-related changes, which suggests that this factor plays an autocrine/paracrine role in the regulation of testicular function and differentiation, during early development.

Animals↗
PubMed1994

Expression and regulation of transforming growth factor-beta 1 messenger ribonucleic acid and protein in cultured porcine Leydig and Sertoli cells.

In the present work, the expression and secretion of transforming growth factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transcripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were enhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4-beta-phorbol 12-myristate 13-acetate) and significantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl-terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sertoli cells was demonstrated in situ, after cell isolation, and during culture. The immunostaining was more marked in Leydig cells than in Sertoli cells. Western blot analysis of Leydig or Sertoli cell-conditioned medium demonstrated a band of 25 kilodaltons, which was shifted to 12.5 kilodaltons under reducing conditions. Using the mink lung epithelial cell bioassay for TGF beta 1, we could demonstrate the presence of TGF beta 1-like activity in Leydig and Sertoli cell-conditioned media after acid treatment, but not before activation. The inhibitory effects of both pure TGF beta 1 and acidified conditioned medium were almost completely blunted by the TGF beta 1 antibody. The amounts of TGF beta 1 secreted by Sertoli and Leydig cells were not significantly different and varied between 400-800 pg/48 h.10(6) cells. These studies demonstrate for the first time that both pig Leydig and Sertoli cells express TGF beta 1 mRNA, and the TGF beta 1-like activity secreted by these cells corresponds to TGF beta 1. As TGF beta 1 has been demonstrated to have strong effects on testicular cells, in particular on Leydig cell functions, it is suggested that local secreted TGF beta 1 may play a role in the autocrine/paracrine regulation of testicular functions.

Animals↗
PubMed1994

[Functional interactions between somatic cells in the male gonad].

The existence of multiple interactions between somatic cells in the testis, has been suspected in vivo, and demonstrated in vitro by cocultures and cultures in the presence of conditioned media. Long term Sertoli-Leydig cell interactions results in a local regulation of the specific differentiated functions of these cells, and more precisely in a control of their responsiveness to their respective gonadotropin. Among the paracrine and/or autocrine factors, growth factors are involved in the control of differentiated functions. It is the case for IGF-I and TGF-beta, which respectively exhibit a stimulatory and inhibitory effect on Leydig cell functions and more specifically on LH responsiveness.

Cell Communication↗
PubMed1993

Enhancement of testosterone secretion by normal adult human Leydig cells by co-culture with enriched preparations of normal adult human Sertoli cells.

An in-vitro method was developed to study Sertoli-Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Possible involvement of transforming growth factor-beta in the inhibition of rat pituitary tumor growth by estradiol.

We have shown that growth of F4Z2 cells and F4Z2 tumors was stimulated by estradiol, that of MtTF4 and F4P tumors was inhibited and that of F4P cells remained insensitive. In the present work we explore the possible role of transforming growth factor-beta (TGF-beta) as a mediator of estradiol action in these pituitary tumors and cell lines. In vivo, estradiol treatment increased the concentration of TGF-beta 1 mRNAs in tumors whose growth was inhibited by estradiol (MtTF4 and F4P) but not in tumors whose growth was stimulated (F4Z2). F4Z2 and F4P cell lines also contained TGF-beta 1 transcripts. These cells and tumors differed by two points: the level of TGF-beta 1 transcript was higher in F4Z2 than in F4P cells while the opposite situation was observed in vivo and the concentration of TGF-beta 1 mRNA in cultured cells was insensitive to estradiol (1 or 100 x 10(-9) M). Moreover, the secretion of TGF-beta like activity assayed by two different methods was estradiol insensitive and the growth of both cell lines was dose-dependently inhibited by TGF-beta 1 (ED50:2 x 10(-11) M). Since estradiol increases TGF-beta 1 mRNA in the tumors MtTF4 and F4P whose growth is inhibited by estradiol and that TGF-beta 1 inhibits the proliferation of F4P cells it is proposed as a working hypothesis that TGF-beta 1 is one of the mediators of the inhibitory effect of estradiol in pituitary tumors. No data favor the hypothesis that estradiol stimulates pituitary tumor proliferation by decreasing TGF-beta production.

Animals↗
PubMed1992

Opposite vectorial secretion of insulin-like growth factor I and its binding proteins by pig Sertoli cells cultured in the bicameral chamber system.

A two-chamber system has been employed to investigate the vectorial secretion of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BPs) by pig Sertoli cells. The kinetics of transport of [3H]insulin as well as the transepithelial electrical resistance of filters coated with reconstituted basement membrane, in the absence of presence of Sertoli cells, indicated the formation of a functional barrier by Sertoli cells. The rates of diffusion of [125I]IGF-I or [125I]human CG were slower from the apical (AC) to the basal (BC) compartment than in the opposite direction. However, the IGF-I content and concentration in the BC was twice that seen in the AC. FSH increased the secretion of IGF-I in the BC, and therefore increased the BC/AC ratio. In contrast, most of the IGF-BPs, with apparent molecular sizes of 39-43, 34, 29 and 24 kDa, were secreted in the AC. FSH increased the secretion of IGF-BP 39-43 kDa (BP3). This is the first report of opposite vectorial secretion of IGF-I and its binding protein by any cell type.

Animals↗
PubMed1991

Regulation by growth factors of Leydig cell differentiated functions.

In this paper the effects of growth factors on the differentiated function of pig Leydig cells and other steroidogenic cells are reviewed. Two types of action have been observed, i.e. positive or negative acute effects on testosterone secretion, and long-term trophic effects of hCG receptor and responsiveness to hCG. Among the growth factors, insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF beta-1) are of particular interest. IGF-I is required for the maintenance and probably the expression of differentiated functions of several steroidogenic cells, including the Leydig cells. TGF beta-1 has effects opposite to IGF-I on Leydig cell functions. When considering effects of growth factors on Leydig cells, caution should be taken in extrapolating results obtained in one species to another.

Animals↗