Claire Mauduit

Tumor Necrosis Factor-α Inhibits Leydig Cell Steroidogenesis through a Decrease in Steroidogenic Acute Regulatory Protein Expression1

The aim of the present study was to identify the sites of the inhibitory action of TNFα (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFα 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 TNFα 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 TNFα, respectively. Maximal inhibitory effects of TNFα on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of ...

Tumor Necrosis Factor-α-Stimulated Lactate Production Is Linked to Lactate Dehydrogenase A Expression and Activity Increase in Porcine Cultured Sertoli Cells1

By using, as a model, cultured testicular immature Sertoli cells, the action of tumor necrosis factor-α (TNFα) and the site of action of the cytokine on lactate production were studied. TNFα stimulated in a time- and dose-dependent manner (with an ED50 of 0.1 nm) Sertoli cell lactate production. Two major sites involved in TNFα action were identified. Firstly, TNFα 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, TNFα 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 TNFα. 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 TNFα stimulated LDH-A messenger RNA (size, 1...

Up-regulation of mitochondrial peripheral benzodiazepine receptor expression by tumor necrosis factor alpha in testicular Leydig cells: Possible involvement in cell survival

Porcine Leydig cells in primary cultures are resistant to tumor necrosis factor alpha (TNFalpha) cytotoxicity. Here we report that these cells can be rendered sensitive to TNFalpha killing by treatment with the translational inhibitor cycloheximide, suggesting the existence of proteins that can suppress the death stimulus induced by the cytokine. In search of these cytoprotective proteins, we focused on the constituents of the mitochondrial permeability transition pore (PT pore), whose opening has been shown to play a critical role in the TNFalpha-mediated death pathway. We found that TNFalpha up-regulated mRNA and protein expression of the mitochondrial peripheral benzodiazepine receptor (PBR), an outer membrane-derived constituent of the pore. A strong correlation was established between the resistance of the cells to TNFalpha killing and the density of PBR-binding sites. Concomitantly, TNFalpha down-regulated Bcl-2 mRNA and protein expression. As Bcl-2 has been shown to be an endogenous inhibitor of the PT pore, we hypothesize that the TNFalpha-induced up-regulation of PBR expression may compensate for the decrease in Bcl-2 levels to prevent the opening of the PT pore.

SIRNA-Directed In Vivo Silencing of Androgen Receptor Inhibits the Growth of Castration-Resistant Prostate Carcinomas

Background Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. Methodology/Principal Findings To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. Conclusions/Significance Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

Alteration of lactate production and transport in the adult rat testis exposed in utero to flutamide

Although it is established that in utero exposure to the antiandrogen flutamide induces alteration of spermatogenesis in the adult rat testis offspring, the cellular and molecular mechanisms involved in such an effect remain to be investigated. In the present paper, by using as model adult rats exposed in utero to flutamide (0, 2, 10 mg/kg per day), we have investigated the hypothesis that germ cell alterations could be related to defects of energy metabolism and particularly to defects of the production and transport of lactate. Lactate is a preferential energy substrate produced by Sertoli cells and transported to germ cells by monocarboxylate transporters (MCT). A significant decrease (60%, P<0.001) in lactate production was observed in cultured Sertoli cells from rat testes exposed in utero to flutamide from the dose of 2 mg/kg per day. Such a decrease is concurrent to a decrease in lactate dehydrogenase A (LDHA) mRNA levels (evaluated through semiquantitative RT-PCR) and LDHA4 activity. The decrease in LDHA mRNA levels (to 64 +/- 9% of the control, P<0.05) was observed with the lowest dose (2 mg/kg per day) of flutamide tested. The decrease in LDHA mRNA levels was observed in both the whole testis and in isolated Sertoli cells, suggesting that such a decrease in LDHA expression occurred also in the (Sertoli) cells producing lactate. Lactate is transported from Sertoli cells to germ cells via MCT1 and MCT2. We immunolocalized MCT1 to all the different germ cell types and MCT2 exclusively to elongated spermatids. In the adult testis exposed in utero to flutamide, MCT1 (53 +/- 8%, P<0.02) and MCT2 (52 +/- 9%, P<0.02) mRNA levels were significantly reduced indicating that lactate transport to germ cells could be also altered. Together, these data support (i) the existence of a relationship between the antiandrogen activity and the energy metabolism in the testis and (ii) the concept of an androgen-dependent programming, occurring early in the fetal life in relation to the expression of some of the key genes involved in the production and transport of lactate in the seminiferous tubules.

Cellular distribution of EGF, TGFα 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.

Developmental and Hormonal Regulation of the Monocarboxylate Transporter 2 (MCT2) Expression in the Mouse Germ Cells

Abstract During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFα and TGFβ also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFβ and TNFα, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell–germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.

Growth regulatory factors and signalling proteins in testicular germ cell tumours.

The molecular basis of testicular germ cell tumourigenesis are not well elucidated. Growth factors regulate cell growth, differentiation and apoptosis. Major families of growth factors are present in the male gonad from early fetal development to adult life. They are involved in germ cell proliferation and differentiation. Growth signalling pathways suffer deregulation in many human malignancies. Given the importance of growth signals in normal testicular development and their acquired deregulation in most human cancers, growth factors and signalling molecules that have been implicated in the genesis of testicular germ cell tumours, are reviewed. We detected a somatic mutation of SMAD4 gene, responsible for loss of protein function in seminomas. This mutational inactivation may affect the activity of several members of TGFβ superfamily (TGFβ, activin, inhibin, BMP). VEGF expression has been shown to predict metastasis in seminomas. A significant association of HST‐1 expression, a member of fibroblast growth factors, with the nonseminomatous phenotype and with tumour stage has been described. In contrast, C‐KIT is expressed by seminomas only, from the preinvasive stage. Despite intense expression in almost all seminomas, activating mutation of C‐KIT gene is seldom reported. Recently, the first animal model of classical testicular seminoma has been identified in transgenic mouse overexpressing GDNF. RET (GDNF receptor) expression is demonstrated in human seminomas, and not in nonseminomatous tumours. However, the exact molecular alterations of GDNF/RET/GFRα1 complex in germ cell tumours are not known. Finally, beside growth factors, other signalling molecules such as peptide hormones may be involved in testicular carcinogenesis. We have demonstrated a specific pattern of somatostatin receptors expression in each type of testicular germ cell tumours, with a loss of sst3 and sst4 in seminomas and loss of sst4 and expression of sst1 in nonseminomas only. These data suggest an antiproliferative action of somatostatin in testicular cancers. In summary, many growth factors and signalling molecules seem to represent specific markers for different histological types of germ cell tumours (seminomas versus nonseminomas) and may play a role in the differentiation of germ cell tumours. Despite a complex signalling pathway involved in the physiological functions of male gonad, little is known about the implication of this signalling network in testicular malignancies. From a practical stand‐point, further studies on the role of growth factors in human germ cell tumours may offer a new therapeutical perspective with the development of specific pharmacological signalling modulators that could be used as therapeutic agents.

Leukemia inhibitory factor antagonizes gonadotropin induced-testosterone synthesis in cultured porcine leydig cells: sites of action.

In the present report, the action of leukemia inhibitory factor (LIF) on testicular steroid hormone formation was studied. For this purpose, the direct effects of LIF were evaluated on basal and human (h)CG-stimulated testosterone synthesis by cultured, purified Leydig cells isolated from porcine testes. LIF reduced (more than 60%) hCG-stimulated testosterone synthesis. This inhibitory effect was exerted in a dose- and time-dependent manner. The maximal and half-maximal effects were obtained with, respectively, 10 ng/ml (0.5 nM ) and 2.5 ng/ml (0.125 nM ) of LIF after a 48-h treatment of the Leydig cells. Such an effect of the cytokine was not a cytotoxic effect, because it was reversible and Leydig cells recovered most of their steroidogenic activity after the removal of LIF. Considering the sites of action of LIF in inhibiting gonadotropin-stimulated testosterone formation, it was shown that LIF significantly (P < 0.002) reduced, in a comparable range (about 60% decrease), testosterone synthesis stimulated with LH/hCG or with pharmacological agents that enhance cAMP levels (cholera toxin, forskolin, and PG E2), and testosterone synthesis stimulated with 8-bromo-cAMP. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step (or steps) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 microg/ml, 2 h), a cholesterol substrate derivative that does not need an assisted process to be delivered to the inner mitochondrial membrane, reversed most of the inhibitory effect of LIF on the steroid hormone formation. Such results indicate that LIF acts by reducing cholesterol substrate availability in the mitochondria. Consequently, LIF action was tested on steroidogenic acute regulatory protein and PBR (peripheral benzodiazepine receptor) shown to be potentially involved in such a cholesterol transfer. LIF reduced, in a dose- and time-dependent manner, LH/hCG-induced steroidogenic acute regulatory protein messenger RNA levels. The maximal inhibitory effect was obtained with 6.6 ng/ml of LIF after 48 h of treatment. In contrast, LIF had no effect on PBR messenger RNA expression or PBR binding. This inhibitory effect of LIF on Leydig cell steroidogenesis is probably exerted via an auto/paracrine action of the cytokine. Indeed, by immunohistochemistry, LIF and LIF receptor proteins were identified in Leydig and Sertoli cells but not in other testicular cell types, except for LIF receptor in spermatogonia. Furthermore, the presence of LIF and its receptor in Leydig cells at the neonatal and adult periods suggests that the inhibitory effect of LIF on androgen formation reported here probably occurs in both the fetal and the adult Leydig cell populations during testicular development.

Chronic crude garlic-feeding modified adult male rat testicular markers: mechanisms of action

BackgroundGarlic or Allium sativum (As) shows therapeutic effects such as reduction of blood pressure or hypercholesterolemia but side-effects on reproductive functions remain poorly investigated. Because of garlics chemical complexity, the processing methods and yield in preparations differ in efficacy and safety. In this context, we clarify the mechanisms of action of crushed crude garlic on testicular markers.MethodsDuring one month of treatment, 24 male rats were fed 5%, 10% and 15% crude garlic.ResultsWe showed that crude garlic-feeding induced apoptosis in testicular germ cells (spermatocytes and spermatids). This cell death process was characterized by increased levels of active CASP3 but not CASP6. Expression of the caspase inhibitors BIRC3 and BIRC2 was increased at all doses of As while expression of XIAP and BIRC5 was unchanged. Moreover, expression of the IAP inhibitor DIABLO was increased at doses 10% and 15% of As. The germ cell death process induced by As might be related to a decrease in testosterone production because of the reduced expression of steroidogenic enzymes (Star, Cyp11a, Hsd3b5 and Hsd17b). Evaluation of Sertoli markers showed that TUBB3 and GSTA2 expression was unchanged. In contrast, AMH, RHOX5 and CDKN1B expression was decreased while GATA4 expression was increased.ConclusionIn summary, we showed that feeding with crude garlic inhibited Leydig steroidogenic enzyme expression and Sertoli cell markers. These alterations might induce apoptosis in testicular germ cells.