Biomedical subjects

M Benahmed

Publications and source records attributed to M Benahmed.

At least 19 recordsLinked to original sources

PubMed1999

Regulation by pH of the alternative splicing of the stem cell factor pre-mRNA in the testis.

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.

Alternative Splicing↗

PubMed1998

Residual bodies stimulate rat Sertoli cell plasminogen activator activity.

Previous studies suggested indirectly that residual bodies (RB) may play a role in the regulation of the proteolytic system within the testis and in the coordination of the spermatogenetic process. In the present study, we examined the effects of RB recovered from adult rat testes by centrifugal elutriation on Sertoli cell plasminogen activator (PA) levels, by zymography and ELISA procedures. Addition of RB to Sertoli cell cultures prepared from 20-day-old rat testes resulted in a dramatic stimulation of PA. Effects were dose- and time-dependent. Phagocytosis of RB by Sertoli cells leads to a rapid stimulation of Sertoli cell interleukin-1alpha (IL-1alpha), a cytokine potentially involved in the regulation of spermatogenesis; the effects of IL-1alpha were investigated. We found that IL-1alpha augmented PA levels and that immunodepletion of Sertoli cell-RB cocultures with anti-IL-1alpha antibodies abrogated the stimulatory effects of RB on PA. Together, the present findings indicate that RB enhance Sertoli cell PA and that IL-1alpha may be involved in that control.

Animals↗

PubMed1998

Hormone-induced changes in cardiolipin from Leydig cells: possible involvement in intramitochondrial cholesterol translocation.

The rate-limiting and hormonally regulated step in steroid hormone biosynthesis is the delivery of cholesterol from the outer to the inner mitochondrial membrane where cytochrome P450scc resides. Although the exact mechanism of intramitochondrial cholesterol translocation remains unknown, the formation of contact sites between outer and inner mitochondrial membranes appears as a necessary component for cholesterol transfer. Several pieces of evidence suggest that local formation of intermembrane contact is a consequence of a non-bilayer arrangement of polymorphic lipids which are enriched in the junctions. As a step toward clarifying mitochondrial contact sites formation and thus cholesterol translocation in steroidogenic cells, we have undertaken studies to identify the factors which might result in non-bilayer structure to be adopted by mitochondrial phospholipids on stimulation of MA-10 Leydig cells. Our results demonstrate that an increase in the unsaturation of the cardiolipin acyl groups on hormonal stimulation might favor the formation of non-bilayer adhesion points.

Animals↗

PubMed1998

Plasminogen activator inhibitor-1 is expressed in cultured rat Sertoli cells.

Sertoli cells secrete plasminogen activators (PAs) on both sides of the blood-testis barrier, i.e., in the basal and apical compartments of the seminiferous tubules, whereas peritubular cells secrete plasminogen activator inhibitor-1 (PAI-1), a fast-acting and specific PA inhibitor. While it is likely that PAI-1 produced by peritubular cells counteracts the basal secretion of PA, the nature of the PA inhibitor acting in the apical compartment remains to be demonstrated. In the present study, we showed that Sertoli cells recovered from 20-day-old rats and cultured contained a transcript of 3-3.2 kilobases, which hybridized specifically to a PAI-1 cDNA probe (Northern blot). We verified that the observed PAI-1 transcript could not result solely from the peritubular cells (weakly contaminating the Sertoli cell cultures), by comparing PAI-1 mRNA levels of Sertoli and peritubular cells recovered from 20-day-old rats and cultured. We also demonstrated that cultured Sertoli cells secreted a protein that complexed with tissue-type PA (zymography), indicating that it was biologically active. This protein comigrated with purified PAI-1 as a doublet of 46 and 49 kDa (Western blot). The trophic hormone FSH decreased PAI-1 messenger RNA as well as immunoreactive PAI-1 protein (probably via the cAMP protein kinase A pathway), whereas transforming growth factor ss1 and basic fibroblast growth factor (in a nanomolar concentration) increased both of these. These observations support the hypothesis that PAI-1 is expressed by Sertoli cells and is under a complex hormonal (FSH) and paracrine and/or autocrine control exerted at least by basic fibroblast growth factor and transforming growth factor ss1.

Alkaline Phosphatase↗

PubMed1998

Interleukin 1alpha stimulates lactate dehydrogenase A expression and lactate production in cultured porcine sertoli cells.

By using cultured porcine Sertoli cells as a model, the action of interleukin 1alpha (IL-1alpha) on lactate production and the site of this action were studied. IL-1alpha stimulated Sertoli cell lactate production in a time- and dose-dependent manner (with a half-maximal effect [ED50] of 6 pM). Two major sites involved in IL-1alpha action were identified. First, IL-1alpha was shown to increase the uptake of glucose substrate in a time- and dose-dependent manner. The maximal effect, with an ED50 of 10 pM, was observed after 24 h of treatment. Second, IL-1alpha increased the activity of the lactate dehydrogenase (LDH) A4 isoform, which is involved in the conversion of pyruvate into lactate. This increase in LDH A4 activity was detected at 12 h and was maximal, with an ED50 of 9 pM, after 24-h treatment with IL-1alpha. The increase was related to an increase in LDH A4 expression, since IL-1alpha stimulated LDH A mRNA (size: 1.5 kilobases, evidenced through Northern blotting analysis) in a dose- and time-dependent manner. Assuming that IL-1alpha might be produced in the seminiferous tubules by both Sertoli and germ cells, which utilize lactate for their energy metabolism, we suggest that these results together show 1) that the cytokine may represent a signal in the metabolic cooperation existing between Sertoli cells and germ cells, and 2) that a redistribution of LDH isoforms in favor of LDH A4 under IL-1alpha control is a key mechanism(s) in such cooperation used by germ cells to enhance lactate production in Sertoli cells.

Animals↗

PubMed1998

Tumor necrosis factor-alpha inhibits leydig cell steroidogenesis through a decrease in steroidogenic acute regulatory protein expression.

The aim of the present study was to identify the sites of the inhibitory action of TNFalpha (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFalpha was shown to inhibit testosterone secretion when testicular cells were stimulated with hCG but not when incubated with 22R-hydroxycholesterol (a cholesterol substrate derivative that readily passes through cell and mitochondrial membranes). Such an observation suggested that the cytokine may affect cholesterol transport and/or availability to cytochrome P450scc in the mitochondria. Specifically, we report here that TNFalpha reduced in a dose- and time-dependent manner hCG-induced StAR (steroidogenic acute regulatory protein) levels. The maximal and half-maximal effects were obtained with 20 ng/ml (1.2 nM) and 1.6 ng/ml (0.09 nM) of TNFalpha, respectively. Maximal inhibitory effects of TNFalpha on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of TNFalpha receptors P55 in terms of protein (identified through cross-linking experiments) and messenger RNA (identified through RT-PCR analysis) suggested that the effects of the cytokine are directly exerted on the testicular steroidogenic cell type.

Animals↗

PubMed1997

Recent evolutionary acquisition of alternative pre-mRNA splicing and 3' processing regulations induced by intronic B2 SINE insertion.

Contrary to the membrane-anchored leukemia inhibitory factor receptor (LIFR), the mouse soluble LIFR is an inhibitor of LIF action, possibly through a ligand titration effect. Two mRNA species encoding the soluble LIFR have been identified. Since the 3'-untranslated end of the shorter form was shown to contain a B2 element, we have examined the possibility that this SINE may be responsible for LIFR mRNA truncation. Transient expression assays, using B2-derived or intron-derived sequences independently or in conjunction, show that the B2 element has fortuitously unmasked a cryptic pre-mRNA 3'processing activity of silent intron sequences. The corresponding locus of the rat genome has been isolated and was shown to be devoid of any retroposon, which may explain why no soluble LIFR has yet been identified in any other species and further indicates that the B2 insertion event in the mouse LIFR gene has occurred recently during evolution. And yet, a tight tissue-specific regulation of alternative synthesis of soluble and membrane-bound LIFR mRNA has already emerged in mice. These results provide striking evidence for the rapid influence of retroposition on genome expression.

Alternative Splicing↗

PubMed1997

LPS increases biomaterial degradation by human monocytes in vitro.

Different cell lines are involved during an immunological reaction, principally lymphocytes and monocytes. Monocyte/macrophage cells, which are among the first to appear in wound-healing and infection sites, are largely implicated in phagocytosis and could be involved in calcium-phosphate degradation. Their role in these processes may relate to cytokine secretions and/or their sensitivity to certain cytokines. We tested the behavior of human monocytes placed on the surface of biphasic calcium-phosphate (BCP) tablets in the presence of two lipopolysaccharide (LPS) concentrations. After short-term culture (48 h), cytokine release (IL-1beta, IL-6) was measured by ELISA, and morphological cell events and biomaterial degradation were observed in scanning electron microscopy. BCP surface pits were noted near cells stimulated by 0.5 microg/mL LPS but were not apparent with 10 microg/mL LPS. The number of lacunae on BCP was increased after LPS treatment of human monocytes. An upmodulation of IL-1beta and IL-6 (in culture medium) released by LPS-activated human monocytes was observed, indicating good cell stimulation. This study demonstrates that LPS-activated human monocytes can degrade the surface of calcium-phosphate ceramic and confirms the role of human monocytes in biomaterial degradation.

Biocompatible Materials↗

PubMed1997

Cellular distribution of transforming growth factor betas 1, 2, and 3 and their types I and II receptors during postnatal development and spermatogenesis in the boar testis.

Transforming growth factor betas (TGF betas) 1, 2, and 3 and their types I and II receptors (TGF betas RI and RII) were immunolocalized 1) during testicular development from the perinatal to the adult period and 2) in maturing germ cell populations at successive seminiferous epithelium stages. In the perinatal testis, TGF beta isoforms and receptors were both preponderant in Leydig cells and in spermatogonia. At prepuberty, their expression appeared in Sertoli cells, while germ cells showed specific TGF beta1 and TGF betaRI staining in the spermatocytes. In the adult testis, TGF beta ligands exhibited a preferential tubular distribution. TGF beta1 was mainly detected in young spermatocytes, TGF beta2 in Sertoli cells, and TGF beta3 in Sertoli and premeiotic germ cells. Although the two receptors were systematically observed together in various cells, our data indicate a predominance of one in comparison with the other depending on the cell type. TGF betaRI was predominant in meiotic and differentiated germ cells and TGF betaRII in somatic cells. Finally, in the adult testis, TGF betas 1, 3, and RI showed a germ-cell pattern that depended upon the stage of the seminiferous epithelium cycle. Specifically, staining for the ligands was predominant before meiosis, and TGF betaRI was present particularly during meiosis and spermiogenesis. Together, the temporal and spatial distribution of the TGF beta system components suggests that these signaling molecules may play a crucial role during specific steps of testicular development and during different waves of seminiferous epithelium maturation leading to spermatogenesis.

Animals↗

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↗