Biomedical subjects

M Benahmed

Publications and source records attributed to M Benahmed.

At least 55 records · Page 3Linked to original sources

PubMed1997

Tumor necrosis factor-alpha regulates plasminogen activator inhibitor-1 in rat testicular peritubular cells.

We examined the regulation by tumor necrosis factor-alpha (TNF alpha) of plasminogen activator inhibitor-1 (PAI-1) in cultured peritubular cells recovered from 20-day-old rat testes. We demonstrated that TNF alpha in a nanomolar dose range stimulated PAI-1 messenger RNA (mRNA; Northern blots) as well as immunoreactive (Western blots) and bioactive (Stachrom) PAI-1 protein. Induction of PAI-1 mRNA started 4 h after the addition of TNF alpha (2.5-fold increase) and peaked (7-fold increase) after 24 h of treatment. Actinomycin D and cycloheximide inhibited the effects of TNF alpha on PAI-1 mRNA, suggesting that ongoing RNA and protein syntheses were required. The combined actions of transforming growth factor-alpha (TGF alpha), a potent inducer of PAI-1, and TNF alpha on PAI-1 were less than additive, suggesting the activation of some common pathway. TNF alpha action on PAI-1, like that of TGF alpha demonstrated previously, was masked by a preexposure to phorbol myristate acetate (a stimulator of protein kinase C) and strongly reduced by staurosporine (an inhibitor of the protein kinase C). Furthermore, using genistein to inhibit tyrosine kinase activity, we not only blocked the action of TGF alpha on PAI-1 [initiated upon binding to the tyrosine kinase epidermal growth factor/TGF alpha receptor (EGFR)], but also markedly reduced that of TNF alpha. Finally, TNF alpha, at a dose range that stimulated PAI-1, enhanced EGFR mRNA levels and EGF binding. Together, the present findings suggest that some of the biological effects of TNF alpha on PAI-1 might be secondary to de novo synthesis of EGFR. Because TNF alpha probably originates from testicular macrophages, such a regulation of PAI-1 by TNF alpha may occur in the context of physiological interactions between the testis and the immune system.

Animals↗

PubMed1997

Tumor necrosis factor-alpha-stimulated lactate production is linked to lactate dehydrogenase A expression and activity increase in porcine cultured Sertoli cells.

By using, as a model, cultured testicular immature Sertoli cells, the action of tumor necrosis factor-alpha (TNF alpha) and the site of action of the cytokine on lactate production were studied. TNF alpha stimulated in a time- and dose-dependent manner (with an ED50 of 0.1 nM) Sertoli cell lactate production. Two major sites involved in TNF alpha action were identified. Firstly, TNF alpha was shown to increase the uptake of glucose substrate in a time- and dose-dependent manner. The maximal effect was observed after 24 h of treatment, with an ED50 of 0.1 nM. Secondly, TNF alpha increased the activity of lactate dehydrogenase (LDH) A isoform, which is involved in the conversion of pyruvate into lactate. This increase in LDH-A activity was detected at 12 h and was maximal after 24 h of treatment with TNF alpha. The stimulatory effect of the cytokine on the LDH-A isoform was observed with an ED50 of 0.05 nM. Such an increase in LDH-A activity was related to an increase in LDH-A expression, because TNF alpha stimulated LDH-A messenger RNA (size, 1.5 kilobases, determined by Northern blotting analysis). Together, assuming that in the seminiferous tubules, TNF alpha is produced by spermatids that use lactate for their energetic metabolism, we suggest that the cytokine may potentially represent a signal used by germ cells to enhance lactate production in Sertoli cells through, at least, a redistribution of LDH isoforms in favor of LDH-A.

Animals↗

PubMed1996

Cellular distribution of EGF, TGFalpha and their receptor during postnatal development and spermatogenesis of the boar testis.

The epidermal growth factor (EGF), the transforming growth factor alpha (TGFalpha) and the epidermal growth factor receptor (EGFr) have been immunolocalized, (i) during the testicular postnatal development (i.e. at the perinatal, prepubertal and adult periods), and (ii) during the seminiferous epithelium cycle in the different germ cell types. While TGFalpha was essentially observed in somatic cells, specifically in perinatal Leydig cells and in mature Sertoli cells, EGF was localized both in germ cells and in somatic cells with a preferential tubular expression. Furthermore, identification of EGFr in different testicular cell types indicates that during postnatal development and spermatogenesis, testicular cells are potentially responsive to EGF in that they express EGFr. Indeed, in the course of the gonadal development, the EGFr distribution was evidenced both in somatic and germ cells with a specific germ cell pattern depending upon the seminiferous epithelium cycle. A predominant EGFr staining was evidenced during the meiotic process and the spermiogenesis. Together, the present data are in favor of the involvement of the TGFalpha/EGF system in the local control of testicular cells during development and particularly of its potential direct implication in crucial steps of spermatogenesis such as meiosis and spermiogenesis.

Aging↗

PubMed1996

Sertoli cells as potential targets of prolactin action in the testis.

Using primary cultures of porcine Sertoli cells as a model, the effects of ovine prolactin (oPRL) on Sertoli cell function were investigated through FSH binding. PRL treatment (0.3-5 ng/ml) induced a dose-dependent increase (ED50 = 5.10(-11) M) of 125I-FSH binding to Sertoli cells to a maximal stimulation (about 2.5-fold increase). This effect was time-dependent, being detected within 2 h (P < 0.02) after oPRL treatment and was maximal after 24 h. The effect of oPRL is probably mediated via specific PRL receptors identified by different approaches such as immunohistochemistry, binding assays and cross-linking experiments. Immunohistochemical experiments were performed using two antibodies directed against the PRL receptor. Immunoreactivity was detected both in the Sertoli cell cytoplasm and in the perinuclear area. Scatchard plots of binding studies revealed the presence of specific binding sites for PRL both in the Sertoli cell membranes and nuclear fractions with high affinity constants (Kd = 0.8 and 1.4 nM, respectively). Affinity labeling of these receptors by covalently binding to 125I-oPRL and subsequent electrophoretic analysis of the labeled complexes revealed for the cell membranes, two major labeled bands of 74 and 64 kDa and three other faintly labeled bands of 190, 150 and 140 kDa. For the nuclear fractions, three major labeled bands with high molecular weights of 190, 150 and 140 kDa were observed. Taken together, the present findings suggest that Sertoli cells are potential targets for prolactin action in the porcine testis.

Animals↗

PubMed1996

Tumor necrosis factor alpha receptor p55 is under hormonal (follicle-stimulating hormone) control in testicular Sertoli cells.

Identification of TNF alpha receptors and their regulation by the endocrine system were examined in testicular Sertoli cells. By using cross-linking experiments as well as northern blotting analysis, we detected the p55 receptor type (MW: 55 kDa, mRNA: 2.3 kb), but not the p75 TNF alpha receptor type. Furthermore, we report that Sertoli cell TNF alpha receptors are under the control of FSH, but not of other hormones which affect Sertoli cell activity. FSH enhances (ED50: 2 ng/ml) TNF alpha binding to Sertoli cells. This stimulating effect of FSH occurs at the level of p55 TNF alpha receptor, as indicated by the increase in p55 mRNA after FSH treatment. Together, these observations show that the action of TNF alpha (which originates in the testis from germ cells and/or interstitial macrophages) on Sertoli cells remains under the control of the endocrine system as shown by the regulatory effect of FSH on TNF alpha p55 receptors.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

European collaborative study of LH assay: 3. relationship of immunological reactivity, biological activity and charge of human luteinizing hormone.

This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.

Animals↗

PubMed1996

Biodegradation of synthetic biphasic calcium phosphate by human monocytes in vitro: a morphological study.

Biodegradation processes (both intra- and extracellular) occur immediately after implantation of calcium phosphate (CaP) ceramics. Monocytes and macrophages, among the first cells to appear in wound healing, are largely implicated in phagocytosis and may be involved in CaP degradation because of their sensitivity to secreted cytokines. We tested the behaviour of human monocytes placed on the surface of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) tablets in the presence of vitamin D3 (VD3) and interferon gamma (INF gamma). After short-term culture (6 days), morphological events were observed in histological and scanning electron microscopy studies, and degradation lacunae were characterized. There were cell prints but no pits on the HA surface, but pits appeared near cells on the BCP surface. Preincubation of biomaterial in culture medium was essential. Variations in cell morphology were observed in different culture types. In the presence of VD3, degradation was greater than in the control, and cells were more polarized and rounded. With INF gamma, cells were extensively spread out on the sample surface, and the biomaterial seemed to be extracted from the surface by cells. Thus, monocytes are clearly influenced by soluble factors (vitamins, cytokines) and could be key cells in initiating the degradation of biomaterial.

Biodegradation, Environmental↗

PubMed1996

Tumor necrosis factor alpha stimulates insulin-like growth factor binding protein 3 expression in cultured porcine Sertoli cells.

The present study was undertaken to analyze how a cytokine, the tumor necrosis factor alpha (TNF alpha), antagonizes the stimulatory action of insulin-like growth factor-I (IGF-I) on FSH receptor levels in testicular Sertoli cells. To achieve this purpose, we measured, in a model of primary culture of porcine Sertoli cells, the effects of TNF alpha on the IGF system that includes IGF-I and IGF-II, IGF binding proteins (IGFBPs), and IGF-I receptor (IGF-I R). We report that while TNF alpha had no consistent effect on the levels of IGF-I, IGF-II, or on IGF-I receptor (protein and messenger RNA (mRNA)], it stimulated IGFBP activity and particularly IGFBP-3. TNF alpha stimulated predominantly IGFBP-3 (about 4-fold) both in terms of mRNA (a 2.6-kilobase transcript, measured by Northern blotting analysis), and protein (a doublet of 40-44 kDa, assessed by ligand blotting analysis). Such a stimulatory effect on IGFBP-3 was detected with a concentration as low as 0.1 ng/ml (5.5 pM) of TNF alpha. The stimulatory action of the cytokine was time dependent and was maximal at 7 h and 48 h, for IGFBP-3 mRNA and protein, respectively. Such an increase in IGFBP-3 in TNF alpha-treated Sertoli cells results probably in a decrease in IGF-1 bioavailability for its receptors and thus in a decrease in IGF-I action. Indeed, addition of recombinant human IGFBP-3 (10 nM) suppressed completely the stimulatory addition of IGF-I (3 nM) on FSH binding to cultured porcine Sertoli cells. Together, the present findings indicate that, in Sertoli cells, TNF alpha antagonizes IGF-I action through the modulation of IGFBPs and particularly through an increase in IGFBP-3. Because of the local production of both TNF alpha and components of the IGF system, such an interaction between the IGF system and the cytokine probably occurs in the context of physiological testicular somatic-germ cell interactions.

Animals↗

PubMed1996

Plasminogen activator inhibitor-1 regulation in cultured rat peritubular cells by basic fibroblast growth factor and transforming growth factor-alpha.

In the present study we examined the in vitro regulation of plasminogen activator inhibitor I (PAI-1) expression in peritubular cells recovered from 20-day-old rat testes. We tested two growth factors, basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha). They are synthesized by Sertoli cells, and peritubular cells exhibit the corresponding high affinity receptors. After exposure to bFGF or TGF alpha (0.1-30 ng/ml), PAI-1 messenger RNA levels, as determined by Northern hybridization analysis, increased in a dose-dependent manner. The first significant effects were noted after 2-h exposure to bFGF or TGF alpha (10 ng/ml), and PAI-1 messenger RNA levels were maximally stimulated approximately 12-fold (bFGF) and 8-fold (TGF alpha) after 4 h. The two growth factors increased the amount of immunoreactive (Western blots) and biologically active (Stachrom) PAI-1 measured in the culture medium. Actinomycin D inhibited the effects of these factors, whereas cycloheximide augmented them. Phorbol myristate acetate, an activator of protein kinase C, mimicked the effects of bFGF and TGF alpha. Interestingly, long term (24-h) pretreatment with phorbol myristate acetate resulted in a severe loss of responsiveness to bFGF or TGF alpha. Staurosporine, an inhibitor of protein kinase C, also significantly reduced the effects of bFGF and TGF alpha. Given that PAI-1 inhibits Sertoli cell plasminogen activator activity and that bFGF and TGF alpha are synthesized by Sertoli cells, these factors are likely to interact to regulate protease activity in localized regions of the seminiferous tubule.

Animals↗

PubMed1995

Transforming growth factor beta receptor expression in cultured porcine granulosa cells.

By using primary cultures of porcine granulosa cells as a model, TGF beta receptors have been identified and characterized through three different approaches including cross-linking experiments, Western- and Northern-blotting analysis. In cross-linking experiments, labeled TGF beta was shown to bind to four different molecular species of 300, 168, 72 and 58 kDa. The 300-kDa species may correspond to beta-glycan, while the 72- and 58-kDa correspond to TGF beta type II and I receptors, respectively. The presence of these receptors was further demonstrated by Western-blotting analysis using specific polyclonal antibodies. Finally, both the expression of beta-glycan, type II and type I mRNA, was confirmed through Northern-blotting analysis as shown by the presence of 6.4, 4.6 and 5.8 kb mRNA, respectively. Additionally, we detected another TGF beta binding protein of 168 kDa which remains to be identified. Together, our present data indicate that the regulatory action of TGF beta on cultured granulosa cells previously reported in several laboratories may be mediated through the receptors identified here.

Animals↗

PubMed1995

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation.

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.

Animals↗

PubMed1995

Expression of mRNAs for transforming growth factor-beta receptors in the rat testis.

In this study, we examined by means of northern blots, the mRNA expression of the three transforming growth factor-beta receptors (TGF-beta receptors) during rat testicular development and their cellular localization using fractionated testicular cells. Transcripts for receptors I and II were essentially detected in the immature testis, whereas receptor III mRNAs were present throughout testicular development. Somatic cells contained mRNAs for the three receptor types. In germ cells, transcripts for types I and II were in low abundance compared to type III mRNAs. In addition germ cells expressed three transcripts for the type III. Among the three transcripts, two were expressed exclusively in germ cells. TGF-beta being produced locally, our results are consistent with TGF-beta acting in testis via autocrine and/or paracrine mechanisms.

Animals↗

PubMed1995

In vitro regulation of rat Sertoli cell inhibin messenger RNA levels by transforming growth factor-beta 1 and tumour necrosis factor alpha.

In the present study, we examined the in vitro regulation of 20-day-old rat Sertoli cell inhibin alpha- and beta B-subunits mRNA levels by transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor alpha (TNF alpha), two factors produced in the testis. Addition of TGF-beta 1 to highly purified cultured Sertoli cells resulted in a time- and dose-dependent enhancement in the alpha-subunit mRNA levels (ED50 = 2.4 pM; maximal increase of 2.6-fold after 48 h of treatment), without affecting the beta B-subunit mRNA levels. Similarly, activin A up-regulated the alpha- but did not modulate the beta B-subunit mRNA levels. By contrast, TNF alpha decreased in a time- and dose-dependent fashion the mRNA levels of the two inhibin subunits alpha and beta B (IC50 = 29 pM for both subunits; maximal decrease of 4.4- and of 4-fold after 72 and 24 h of treatment for respectively the alpha- and beta B-subunits). The effects of TGF-beta 1 and TNF alpha on inhibin mRNA levels occurred within a dose range that might be expected under physiological conditions. In addition, TGF-beta 1-treated Sertoli cells responded to FSH or dibutyryl cyclic AMP ((Bu)2cAMP) by a further and significant additive increase of the alpha-subunit mRNA levels. TNF alpha-treated Sertoli cells responded significantly to FSH and to (Bu)2cAMP, thus attenuating the inhibitory action of TNF alpha on the alpha-inhibin mRNA levels. Together, the present findings emphasize the ability of some local growth factors to modulate the effects of FSH on Sertoli cell function.

Activins↗

PubMed1994

Monocyte activity in the presence of calcium phosphate activated by 1,25 (OH)2 VD3 and interferon-gamma.

The monocyte is an essential element in cellular immunity. Consequently, its role in the biocompatibility process is important. A model study of the degradation of bioactive ceramics (calcium phosphate) using human peripheral blood monocytes activated by 1,25 (OH)2 VD3 and interferon (IFN)-gamma was implemented. Activated monocytes cultivated on calcium phosphate tablets led to cellular morphological modifications and to changes in the number of nuclei (from d 2 to 14). IFN-gamma promoted adhesion, the appearance of cytoplasmic extensions and multinuclei. Cavity resorption activity on the material was observed simultaneously.

Biocompatible Materials↗

PubMed1994

Effect of transforming growth factor-beta 1 on the insulin-like growth factor system in cultured porcine Leydig cells.

Using as a model system, primary cultures of porcine Leydig cells, we have shown that transforming growth factor-beta 1 (TGF-beta 1) (2 ng/ml, 72 h) antagonizes the stimulatory action of insulin-like growth factor-I (IGF-I) on luteinizing hormone (LH/hCG) receptors. We therefore investigated the action of TGF-beta 1 on the different components of the IGF system, namely, IGF-I, II, IGF binding proteins (IGFBPs) and IGF-I receptor present in testicular Leydig cells. TGF-beta 1 was shown to decrease in a dose and time dependent manner the binding of 125I-IGF-I to Leydig cells. The maximal (40% decrease) effect was obtained with 1.3 ng/ml (0.05 nM) after 72 h of treatment. Such a decrease in IGF-I binding by TGF-beta 1 treatment was shown to be related to the number of receptor but not to their affinity. Affinity labeling of these receptors by covalently binding them to 125I-IGF-I with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complex revealed that the inhibitory action of TGF-beta 1 (2 ng/ml, 72 h) occurs at the level of a 135 kDa protein which represents the classical form of the binding subunit of the IGF-I receptor. Moreover, our study indicates that TGF-beta 1 was unable to affect the other components of the IGF system in cultured porcine Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1994

Fibroblast growth factor receptor type 1 expression during rat testicular development and its regulation in cultured Sertoli cells.

In the present study, we examined the ontogeny of the type 1 receptor for basic fibroblast growth factor (FGFR-1) in whole rat testis, its cellular localization, and its in vitro regulation in 20-day-old rat Sertoli cells. Gene expression of FGFR-1 was developmentally regulated; expression was higher in prepubertal testes and decreased with sexual maturity. The transcript was found to be expressed in Leydig-enriched fractions, peritubular cells, Sertoli cells, and, to a lesser extent, germ cells. FSH as well as (Bu)2cAMP enhanced FGFR-1 messenger RNA (mRNA) levels in cultured Sertoli cells, suggesting an involvement of the protein kinase-A pathway. Addition of basic FGF (bFGF), tumor necrosis factor-alpha (TNF alpha), or interleukin-1 alpha resulted in a dose- and time-related increase in FGFR-1 mRNA levels. The effect of bFGF was specific, because it was neutralized by cotreatment with an anti-bFGF. We tested medium conditioned by germ cells and found a stimulation of the Sertoli cell FGFR-1 mRNA levels, which was abolished by immunodepletion of the conditioned medium with anti-TNF alpha antibodies. It is suggested that in Sertoli cells, bFGF action, when mediated by FGFR-1, is under a complex hormonal (FSH) and paracrine and/or autocrine control exerted at least by bFGF, TNF alpha, and interleukin-1 alpha.

Aging↗

PubMed1994

[TGF beta (transforming growth factor beta) and its related peptides in the local control of the testicle].

Recent data on TGF beta (and related peptides) expression and action in the testis are briefly reviewed. At least, three isoforms of TGF beta are expressed during the male gonadal development. TGF betas are expressed in somatic and germ cells. The expression of the different isoforms varies during gonadal development and is cell specific. Both types I, II and III TGF beta receptors have been localized in somatic cells. TGF beta inhibits gonadotropin action both in Sertoli cells and Leydig cells as well as proliferation of germ cells and Leydig cells. Inhibins and activins (in terms of mRNA and proteins) are expressed in somatic cells and germ cells. Inhibin production in Sertoli cells is under the control of FSH, local factors (ex: EGF, insulin, opiods) and probably of germ cells. Activins and inhibins regulate Leydig and Sertoli cells activities and germ cells development. The existence of activin receptors in somatic and germ cells has been suggested by the presence of type II receptor mRNA (ActRII and ActRIIB).

Activins↗

PubMed1993

Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells.

We have isolated complementary DNA clones coding for a novel member of the insulin-like hormone super-family from a boar testis cDNA library. Northern blot analysis and in situ hybridization revealed that the gene is expressed exclusively in prenatal and postnatal Leydig cells. We have tentatively proposed the name Leydig insulin-like (Ley I-L) for the gene and its encoded protein. The Leydig insulin-like protein is synthesized as a 131-amino acid preproprotein, which contains a 24-amino acid signal peptide. Comparison of the deduced amino acid sequence of pro-Leydig insulin-like protein with members of the insulin-like hormone superfamily predicts that the biologically active protein, after proteolytic processing of the C-peptide, consists of a 32-residue-long B-chain and a 26-residue-long A-chain and has a molecular size of 6.25 kDa.

Amino Acid Sequence↗