Eric Tabone

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.

Glutathione S-Transferase Alpha Expressed in Porcine Sertoli Cells Is under the Control of Follicle-Stimulating Hormone and Testosterone

Abstract Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTα is expressed in somatic testicular Leydig cells and Sertoli cells. GSTα expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTα was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTα was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTα mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTα mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTα protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTα mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTα mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTα protein, we therefore had to determine whether the different hormones were affecting GSTα gene transcriptional activity, or GSTα mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTα mRNA. The effects of FSH and testosterone on GSTα protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTα is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.

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.

Caspase-3 and -6 Expression and Activation Are Targeted by Hormone Action in the Rat Ventral Prostate During the Apoptotic Cell Death Process

Abstract Although the apoptotic cell death process in the prostate is known to be under the control of androgens, the key components targeted by the hormones remain to be investigated. In the present study, we report that the expression and the activation of the effector caspases-3 and -6 are under the control of testosterone in the adult rat ventral prostate. By using a model of adult castrated rats supplemented (or not) with androgens, we observed an increase in caspase-3 (3-fold) and -6 (4-fold) mRNA (P < 0.0001) and procaspase-3 (32 kDa) and -6 (34 kDa) protein levels by 3 days and 1 wk, respectively, after castration in the ventral prostate. Castration also induced an increase in the activation of the procaspases in the ventral prostate, since active (cleaved) caspase-3 (17 kDa) and -6 (12 kDa) forms reached maximal levels by 1 wk after castration. Testosterone administration to castrated adult rats prevented the increase in caspase-3 and -6 mRNA as well as in procaspase-3 and -6 and active caspase-3 and -6 levels in the ventral prostate lobe. In contrast, no changes were observed in the initiator caspase-8 mRNA and protein (procaspase and active) levels after castration. No changes in caspase-3 and -6 expression and activation were observed in the dorsolateral and anterior prostate lobes after castration and testosterone supplementation. Together, the present results show that testosterone inhibits apoptosis in the ventral prostate by potentially targeting the transcriptional activity of effector caspase-3 and -6 genes (but not of casapase-8 gene) as well as the cleavage of procaspase-3 and -6 into active enzymes.

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.

Alterations of Sertoli cell activity in the long-term testicular germ cell death process induced by fetal androgen disruption

Fetal androgen disruption, induced by the administration of anti-androgen flutamide (0.4, 2, and 10 mg/kg day) causes a long-term apoptosis in testicular germ cells in adult male rat offspring. One of the questions raised by this observation is the role of the Sertoli cells in the adult germ cell apoptotic process. It is shown here that Sertoli cells originating from 15-day-old rats treated in utero with the anti-androgen (10 mg/kg d) did no longer protect adult germ cells against apoptosis. Indeed, untreated spermatocytes or spermatids exhibited increased (P<0.0001) active caspase-3 levels when co-cultured with Sertoli cells isolated from rat testes exposed in utero to the anti-androgen. This alteration of Sertoli cell functions was not due to modifications in the androgen signal in the adult (90-day-old) animals, since plasma testosterone and estradiol, androgen receptor expression, and androgen-targeted cell number (e.g., Sertoli cells in the seminiferous tubules) were not affected by the fetal androgen disruption. In contrast, this inability of Sertoli cells to protect germ cells against apoptosis could be accounted for by the potential failure of Sertoli cell functions. Indeed, adult testes exposed in utero to anti-androgens displayed decreased levels of several genes mainly expressed in adult Sertoli cells (anti-Mullerian hormone receptor type II (AMHR2), Cox-1, cyclin D2, cathepsin L, and GSTalpha). In conclusion, fetal androgen disruption may induce alterations of Sertoli cell activity probably related to Sertoli cell maturation, which potentially leads to increased adult germ cell apoptosis.

α6 integrin subunit mediates laminin enhancement of cisplatin-induced apoptosis in testicular tumor germ cells

Our study demonstrates that laminin potentiates cisplatin‐induced apoptosis in NCCIT, a testicular tumor germ cell line. When cultured on laminin, NCCIT cells displayed a significantly higher susceptibility to cisplatin‐induced apoptosis than on plastic or on other ECM components including fibronectin, Type IV collagen and vitronectin. This high cisplatin sensitivity observed on NCCIT cell cultured on laminin was mediated by the α6‐integrin signaling. The knockdown of the α6‐integrin subunit by small interfering RNAs suppressed the higher cisplatin‐sensitivity supporting the existence of a crosstalk between laminin‐α6‐integrin signaling and cisplatin‐induced apoptosis. Our findings indicate that in cisplatin‐treated NCCIT cells, the laminin‐α6‐integrin signaling induces the activation of executioner procaspase‐3 and ‐6 as well as apoptosis‐inducing factor (AIF) transcription and expression. The ability of integrin‐mediated specific stroma–tumor cell interactions to modulate the chemosensitive phenotype of a tumor cell might provide new insights to overcome cisplatin resistance of tumor cells.