Transient postnatal over nutrition induces long-term alterations in cardiac NLRP3-inflammasome pathway.

BACKGROUND AND AIMS: The prevalence of obesity is increasing worldwide at an alarming rate. Altered early nutrition, in particular postnatal overfeeding (PNOF), is a risk factor for impaired cardiac function in adulthood. In the understanding of the initiation or progression of heart diseases, NLRP3 inflammasome and non-coding RNAs have been proposed as key players. In this context, the aim of this study was to decipher the role of NLRP3 inflammasome and its post transcriptional control by micro-RNAs in the regulation of cardiac metabolic function induced by PNOF in mice. METHODS AND RESULTS: Based on a model of mice exposed to PNOF through litter size reduction, we observed increased cardiac protein expression levels of NLRP3 and ETS-1 associated with alterations in insulin signaling. Additionally, miR-193b levels were down-regulated in the adult hearts of overfed animals. In a cardiomyocyte cell line, transfection with miR-193b induced down-regulation of ETS-1 and NLRP3 and improved insulin signaling. CONCLUSIONS: These findings suggest that the miR-193b could be involved in cardiac phenotypic changes observed in adulthood induced by PNOF likely through the regulation of ETS-1 and NLRP3 expression, and through this of insulin signaling.

Tumor necrosis factor-308 polymorphism increases the embryo implantation rate in women undergoing in vitro fertilization.

STUDY QUESTION: Do TNF-308 and -238 polymorphisms impact the embryo implantation rate after in vitro fertilization (IVF) in women without female infertility factor? SUMMARY ANSWER: The presence of the TNF-308A allele is associated with high implantation and multiple pregnancy rates in women without known infertility factors after ovarian hyperstimulation with exogenous FSH. WHAT IS ALREADY KNOWN: Multiple pregnancies are frequent after the use of Assisted Reproductive Technologies. Single embryo transfer (SET) has been proposed as a simple way to prevent these risks. However, the extension of SET indications to patients not selected based on specific criteria is controversial because of reduced pregnancy rates. To date, the predictive value of the parameters used for SET (age, gynecological history of the patient and uterine characteristics) allows a pregnancy rate of ~30%. STUDY DESIGN, SIZE, DURATION: The potential predictive value of TNF polymorphisms (-308, rs1800629 and -238, rs361525) on implantation rate was evaluated in 424 women requiring IVF due to male fertility factors. This cohort retrospective study was conducted over 4 years in University-affiliated hospitals. PARTICIPANTS, SETTING, METHODS: The entire patient group included 424 women undergoing intracytoplasmic sperm injection (ICSI) due to male fertility factors without the contribution of any female factor. From among this group, a selected patient group included 120 women with a normal karyotype, age under 38 years, serum follicle-stimulating hormone (Day-3 FSH) levels below 10 IU/l, a long agonist desensitization protocol associated with recombinant FSH treatment and a Caucasian background. MAIN RESULTS AND THE ROLE OF CHANCE: The TNF-238 polymorphism was not associated with implantation rate. In contrast, the presence of the TNF-308A allele was associated with increased Day 3-E2 levels as well as higher implantation and multiple pregnancy rates after fresh embryo transfer in women from the entire and selected patient groups. Moreover, in the selected patient group, the presence of the TNF-308A allele was also associated with a decrease in the miscarriage rate. The benefit of the TNF-308A allele in predicting implantation rates was not observed after the use of frozen embryos. LIMITATIONS, REASONS FOR CAUTION: Future studies are needed to evaluate whether the TNF-308A allele might also be a biomarker in women with infertility factors. WIDER IMPLICATIONS OF THE FINDING: The TNF-308A allele may represent a good candidate for a potential predictive, non-invasive biomarker in the SET strategy. However, its impact should be evaluated in prospective studies. STUDY FUNDING/COMPETING INTEREST: This study was conducted with financial support from the French Institute for Health and Medical Research (INSERM), Organon France for a FARO (Fond d'Aide à la Recherche Organon) fellowship (to V.T.) and CHU Nice PHRC (PHRC 09-279).There are no competing interests.

Tumor necrosis factor-alpha -308 polymorphism in infertile men with altered sperm production or motility.

BACKGROUND: One of the most well-documented cytokines suspected as a hazard to male fertility is tumor necrosis factor-alpha (TNFalpha). Genetic factors such as single-nucleotide polymorphisms (SNPs) in the TNF gene cluster impact TNFalpha levels. Our objective was to establish the potential involvement of -308 TNF SNP in male infertility risk. METHODS: In 684 infertile male patients undergoing an intracytoplasmic sperm injection procedure, we used allele-specific polymerase chain reaction (PCR) and PCR-RFLP to investigate the distribution of the guanine (G)-to-adenosine (A) substitution at position -308 in the promoter region of the TNFalpha gene. RESULTS: An increased frequency of the -308 TNFalpha A allele was found in patients with low sperm count of testicular origin [P = 0.002; odds ratio (OR) = 2.93] or with normal production count but altered sperm motility (P = 0.003; OR = 2.32), compared with a patient group with normal sperm count and quality (morphology and motility). In patients with low sperm count exhibiting TNFalpha A allele, compared with those with G allele, an alteration in hormonal balance was observed with increased inhibin B levels and subsequent reduced FSH plasma levels, leading to an FSH/inhibin B ratio roughly half as high (from 0.07 +/- 0.01 in TNFA versus 0.13 +/- 0.02 in TNFG allele groups, P < 0.0001). CONCLUSION: As the -308 TNFalpha A allele has been associated with an increased expression/production of TNFalpha, the potential use of therapies based on inhibition of TNFalpha activities could represent possible therapeutic opportunities for patients with low sperm count (i.e. primary testicular dysfunction) or with altered sperm motility.

At the heart of programming: the role of micro-RNAs.

Epidemiological and experimental observations tend to prove that environment, lifestyle or nutritional challenges influence heart functions together with genetic factors. Furthermore, when occurring during sensitive windows of heart development, these environmental challenges can induce an 'altered programming' of heart development and shape the future heart disease risk. In the etiology of heart diseases driven by environmental challenges, epigenetics has been highlighted as an underlying mechanism, constituting a bridge between environment and heart health. In particular, micro-RNAs which are involved in each step of heart development and functions seem to play a crucial role in the unfavorable programming of heart diseases. This review describes the latest advances in micro-RNA research in heart diseases driven by early exposure to challenges and discusses the use of micro-RNAs as potential targets in the reversal of the pathophysiology.

Peroxy bleaches Part 1. Background and techniques for hazard evaluation.

Fabric laundering is now a sophisticated chemical process involving a variety of operations including bleaching. The chemistry of peroxy bleaches is described including the use of novel organic compounds to provide effective bleaching at the lower temperatures of modern wash cycles. The instability of peroxy compounds is illustrated using cameo case histories to relate theory and practice. Techniques available for determining their thermochemistry are summarised. A model is provided for hazard and risk assessment of development projects in general (particularly those involving new molecules, processes or formulations) from ideas phase through exploratory laboratory investigations to pilot plant scale-up and eventual manufacture and commercial exploitation. This paper is a prelude to Part 2, which describes the determination of thermodynamic and kinetic properties of peroxy bleaches and discusses the implication of the results in terms of precautions for their safe storage and incorporation into detergent formulations during processing.

Peroxy bleaches Part 2. Determination of the thermodynamic and kinetic chemistry of some peroxy compounds.

The thermodynamic and kinetic properties of a series of inorganic and organic peroxy bleaches were determined using adiabatic rate calorimetry and isothermal microcalorimetry. Results are compared to calculated oxygen balance values. The decomposition of the majority of the compounds is complex. Data indicate the need for cooling during the storage and transport for some materials evaluated. Although no overall structure/activity relationship could be established because of the diversity of molecular architectures studied, a combination of decomposition and activation energy data provides a means for hazard and risk classification.

[Long-term effects of environmental endocrine disruptors on male fertility]. / Effets à long terme des perturbateurs endocriniens environnementaux sur la fertilité masculine.

Several epidemiologic studies have demonstrated during the last 50 years an increased incidence in testis cancer, male genital tract malformations (cryptorchidism and hypospadias) and a decrease in sperm quality in men. These three pathologies seem to be linked and to belong to the testicular dysgenesis syndrome (TDS). It was suggested that TDS is a consequence of intra-uterine exposure to environmental compounds that disrupt the metabolism of native hormones. Such substances are so called endocrine disruptors (EDs). EDs are present in our daily environment such as food and water (through the use of pesticides), cosmetics, house-care products etc. Experimental models have been carried out to (i) establish a link between EDs exposure and SDT and (ii) identify the mechanisms that are involved in. After a brief definition of EDs and having underlined the importance of the window of exposure to EDs, several mechanisms will be described such as (i) intergenerational transmission (epigenetic), (ii) programmed cell death of testicular cells, (iii) modification of the androgenic signal and (iv) role of the germ cells-nourishing cells. To conclude, we will try to propose some biomarkers that would be useful to identify the potential link between fetal exposure to anti-androgenic EDs and male testicular pathology.

Leukemia inhibitory factor is a key signal for injury-induced neurogenesis in the adult mouse olfactory epithelium.

The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout adulthood by local, restricted neuronal progenitor cells. The molecular signals that control this neurogenesis in vivo are unknown. Using olfactory bulb ablation (OBX) in adult mice to trigger synchronous mitotic stimulation of neuronal progenitors in the OE, we show the in vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA expression peaks at 8 hr after OBX, and in situ RT-PCR and immunocytochemistry indicate that LIF is upregulated, in part, in the injured neurons themselves. This rise in LIF is necessary for injury-induced neurogenesis, as OBX in the LIF knock-out mouse fails to stimulate cell proliferation in the OE. Moreover, delivery of exogenous LIF to the intact adult OE using an adenoviral vector stimulates BrdU labeling in the apical OE. Taken together, these results suggest that injured OSNs release LIF as a stimulus to initiate their own replacement.

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.

Developmental and hormonal regulation of the expression of oligodendrocyte-specific protein/claudin 11 in mouse testis.

The proliferation and differentiation of testicular progenitor stem cells into highly specialized germ cells (spermatozoa) are largely controlled by the hormonally (FSH and testosterone) regulated adjacent supporting Sertoli cells. However, the factors involved in this control remain largely unknown. In the present study, the technique of differential display PCR was used to identify target transcripts to FSH action in cultured murine Sertoli cells. Among these target transcripts, we identified the oligodendrocyte-specific protein (OSP), also known as claudin 11, which had recently been shown to play a key role in the formation of the hematotesticular barrier. Our data show that the testicular expression of OSP is dependent upon male gonad development and systemic and local signaling molecules. Indeed, OSP is expressed early in fetal development in Sertoli cells, immediately after the peak of SRY (sex-determining region, Y gene) expression, but just before that of the anti-Mullerian hormone. Postnatally, OSP expression starts to increase from day 3 to reach a plateau between days 6 and 16 postnatally. In the prepubertal and adult testes, an apparent decline in OSP messenger RNA (mRNA) levels was found, probably because of the increasing number of germ cells (which do not express OSP). Among the signaling molecules that control testicular OSP expression, we have identified FSH and tumor necrosis factor-alpha (TNFalpha). Indeed, using a model of purified cultured mouse Sertoli cells, we demonstrate that FSH inhibits, in a dose (ED50 = 4 ng/ml)- and time (maximal effect after 24 h)-dependent manner, the levels of OSP mRNA. Such an inhibitory effect was mimicked by 8-bromo-cAMP, suggesting that FSH may use the cAMP/protein kinase A pathway to inhibit OSP mRNA levels. TNFalpha was also shown to inhibit OSP expression in cultured Sertoli cells. The maximal effect was observed after 48 h of TNFalpha treatment with an ED50 of 4.5 ng/ml. Together, our results indicate that OSP expression 1) starts during fetal life at a critical period, probably under SRY control and during testicular formation; and 2) is regulated by hormones (FSH) and cytokines (TNFalpha) in the adult testis, suggesting a critical role for these molecules in the (re)modeling process of the hematotesticular barrier during spermatogenesis.

Direct regulating effects of transforming growth factor-beta 1 on lactate production in cultured porcine Sertoli cells.

In the present study we have tested the direct effects of transforming growth factor-beta 1 (TGF beta 1) on lactate production by Sertoli cells isolated from immature porcine testes. In Sertoli cells cultured in a defined medium, TGF beta 1 was shown to stimulate lactate production in a time- and dose-dependent manner. The maximal and half-maximal effects of TGF beta 1 on lactate production were obtained in the picomolar concentration range, respectively 24 and 8 pM TGF beta 1. TGF beta 1 action was found closely related to that of insulin since 1) both TGF beta 1 (40 pM) and insulin (1 microgram/ml) induced the secretion of similar and nonadditive amounts of lactate; and 2) TGF beta 1 and insulin induced comparable increases in lactate production in FSH (1 microgram/ml)-treated Sertoli cells. Because lactate is derived from glucose, 2-deoxy-D-glucose (2-DOG) was used to investigate the hexose transport system of Sertoli cells after insulin, FSH, and TGF beta 1 treatments. Insulin (1 microgram/ml) and FSH (1 microgram/ml) were found to stimulate 2-DOG transport with a similar time course, with an effect detected up to 30 min and maximal at 150 min. In contrast, although TGF beta 1 also enhanced 2-DOG uptake by Sertoli cells, the increase in glucose transport was delayed, since the TGF beta 1 effect was first detected at 150 min and was maximal at 360 min. These effects of TGF beta 1 action on Sertoli cell activity are exerted through specific membrane TGF beta 1 receptors. Scatchard analysis of the binding of TGF beta 1 to cultured Sertoli cells revealed the presence of both a high affinity (Kd, approximately 180 pM) and a low affinity binding site systems for TGF beta 1. Affinity labeling of these receptors by covalent attachment to [125I] TGF beta 1 with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I] TGF beta 1 to three predominant molecules of 260, 130, and 70 kDa. In conclusion, the present study demonstrates that testicular Sertoli cells are targets for TGF beta 1 action. In view of the importance of lactate as a substrate for germ cells, it is suggested that TGF beta 1 might also be involved in the development of normal germinal epithelium.

Tumor necrosis factor-alpha antagonizes follicle-stimulating hormone action in cultured Sertoli cells.

In the present study, we have tested the effect of tumor necrosis factor-alpha (TNF alpha) on FSH action in cultured purified Sertoli cells isolated from immature porcine testes. FSH action was evaluated through three different parameters (aromatase activity, lactate production, and alpha-inhibin production). TNF alpha was shown to reduce (about 40-60% decrease) FSH-stimulated but not basal aromatase activity (evaluated through the conversion of testosterone into estradiol) in a dose- and time-dependent manner. The maximal and half-maximal (IC50) effects were observed with 6 ng/ml (3.5 x 10(-10) M) and 0.6 ng/ml (3.5 x 10(-11) M), respectively, after a long-term (72 h) treatment. TNF alpha (20 ng/ml) also inhibited Sertoli cell aromatase activity when stimulated with 8-bromo-cAMP (0.01-3 mM, 72 h) instead of FSH, suggesting that the antigonadotropin action of the cytokine is probably exerted at a step located beyond cAMP formation. The inhibitory effect of TNF alpha was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action on lactate and inhibin-alpha chain production in Sertoli cells. As for FSH-induced aromatase activity, TNF alpha reduced FSH-stimulated lactate accumulation with an IC50 of 0.6 ng/ml, after a long-term (72 h) treatment. Again, the cytokine reduced lactate production stimulated by 8-bromo-cAMP, suggesting that TNF alpha antagonistic action against FSH is exerted at post-cAMP levels. Finally, TNF alpha exerted a more pronounced inhibitory effect (> 90% inhibition) on alpha-inhibin than on inhibin heterodimer production. These inhibitory effects of TNF alpha on the gonadotropin action are probably exerted directly on Sertoli cells, since TNF alpha high affinity binding sites (dissociation constant approximately 5.3 x 10(-10) M) are present in primary cultures of purified porcine Sertoli cells. Altogether, the present findings show that TNF alpha antagonizes FSH action on Sertoli cell functions such as aromatase activity and lactate and alpha-inhibin production. Such an inhibitory effect is probably exerted at a biochemical step(s) located beyond cAMP generation.

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.

Tumor necrosis factor alpha inhibits gonadotropin action in cultured porcine Leydig cells: site(s) of action.

In the present study, we have tested the direct effects of tumor necrosis factor-alpha (TNF-alpha) on basal and human (h)CG-stimulated testosterone secretion by cultured purified Leydig cells isolated from immature porcine testes. TNF-alpha reduced (as much as 90% decrease) hCG-stimulated, but not basal testosterone secretion in a dose- and time-dependent manner. The maximal and half-maximal effects were, respectively, 3.75 ng/ml (2.2 x 10(-10) M) and 0.66 ng/ml (3.9 x 10(-11) M) of TNF-alpha after 48 h treatment. TNF-alpha antagonizes the gonadotropin hormonal action by affecting at least two types of biochemical steps. First, TNF-alpha reduced LH/hCG binding to a maximal decrease of 45% obtained with 2 ng/ml of TNF-alpha after 48 h of treatment. TNF-alpha also inhibited (44% decrease) hCG-stimulated cAMP production in optimal conditions (20 ng/ml, 72 h). Second, TNF-alpha significantly (P less than 0.001) reduced testosterone secretion stimulated with 8-bromo-cAMP (3 x 10(-3) M) in a similar range (86% decrease) to that observed with the gonadotropin. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step(s) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 micrograms/ml, 2 h) reversed most of the inhibitory effect of TNF-alpha on androgen production. Indeed, the TNF-alpha (20 ng/ml, 72 h) inhibitory effect on testosterone production was limited to about 20% (P less than 0.03) in Leydig cells supplied with 22R-hydroxycholesterol. Such a moderate effect of the cytokine in the presence of 22R-hydroxycholesterol compared with that observed when androgen secretion was stimulated with the gonadotropin (up to 90% inhibition) indicate that TNF-alpha acts by dramatically reducing cholesterol substrate availability in the mitochondria. Such an effect of TNF-alpha is directly exerted on Leydig cells since TNF-alpha receptors (dissociation constant approximately 5.4 x 10(-10) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells TNF-alpha antagonizes the gonadotropin action on testosterone formation predominantly through a decrease in the availability of cholesterol substrate in the mitochondria.

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.

Differential expression of growth factors in irradiated mouse testes.

PURPOSE: By using as an experimental model the male mouse gonad, which contains both radiosensitive (germ) and radioresistant (somatic) cells, we have studied the growth factor (and/or receptor) expression of transforming growth factor-beta receptor (TGFbeta RI), stem cell factor (SCF), c-kit, Fas-L, Fas, tumor necrosis factor receptor (TNF R55), and leukemia inhibiting factor receptor (LIF-R) after local irradiation. METHODS AND MATERIALS: Adult male mice were locally irradiated on the testes. Induction of apoptosis in the different testicular cell types following X-ray radiation was identified by the TdT-mediated dUTP Nick End Labeling (TUNEL) approach. Growth factor expression was evidenced by semiquantitative RT-PCR and Western blot analyses. RESULTS: Apoptosis, identified through the TUNEL approach, occurred in radiosensitive testicular (premeotic) germ cells with the following kinetics: the number of apoptotic cells increased after 24 h (p < 0.001) and was maximal 48 h after a 2-Gy ionizing radiation (p < 0.001). Apoptotic cells were no longer observed 72 h after a 2-Gy irradiation. The number of apoptotic cells increased with the dose of irradiation (1-4 Gy). In the seminiferous tubules, the growth factor expression in premeiotic radiosensitive germ cells was modulated by irradiation. Indeed Fas, c-kit, and LIF-R expression, which occurs in (radiosensitive) germ cells, decreased 24 h after a 2-Gy irradiation, and the maximal decrease was observed with a 4-Gy irradiation. The decrease in Stra8 expression occurred earlier, at 4 h after a 2-Gy irradiation. In addition, a significant (p < 0.03) decrease in Stra8 mRNA levels was observed at the lowest dose used (0.5 Gy, 48 h). Moreover, concerning a growth factor receptor, such as TGFbeta RI, which is expressed both in radiosensitive and radioresistant cells, we observed a differential expression depending on the cell radiosensitivity after irradiation. Indeed, TGFbeta RI expression was increased after irradiation in interstitial radioresistant testicular cells in a dose- and time-dependent manner, while it decreased in seminiferous radiosensitive (germ cells) testicular cells. Such a differential expression between radioresistant and radiosensitive cells in TGFbeta RI levels was observed in terms of both mRNA and protein. In contrast, the growth factors specifically expressed in the somatic radioresistant (Sertoli) cells in the seminiferous tubules (SCF, Fas-L, TNF R55) were not affected by ionizing radiation (up to 4 Gy, 72 h). CONCLUSION: Growth factor expression decreased in the radiosensitive testicular cells after irradiation. Such a decrease occurred before the detection of apoptosis using the TUNEL approach. TGFbeta RI mRNA levels decreased in the radiosensitive cells, whereas it increased in the radioresistant cells, suggesting that TGFbeta RI may represent a biomarker of the intrinsic radiosensitivity of cells.

The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens.

In utero exposure to exogenous anti-androgenic compounds induces a wide range of abnormalities of the reproductive system, including hypospermatogenesis, cryptorchidism and hypospadias. By using rats exposed in utero to the anti-androgenic compound flutamide (0.4, 2 or 10 mg/kg per day), it has been shown that hypospermatogenesis in adult testes could be related to (i) a long-term apoptosis in germ cells but not in somatic Leydig and Sertoli cells as evidenced by the TUNEL approach and (ii) alterations in the mRNA and protein expression of pro- (Bax, Bak, Bid) and anti-apoptotic (Bcl-2, Bcl-w) members of the Bcl-2 family. Indeed, the number of apoptotic germ cells increased with the dose of flutamide administered and the apoptotic germ cells were mainly detected at androgen-dependent stages VII-VIII. Moreover, for the Bcl-2-related proteins that were expressed mainly in the germ cells, a decrease in the levels of anti-apoptotic peptides Bcl-w (60%, P=0.003) and Bcl-2 (90%, P=0.0001) was observed at 2 mg/kg per day flutamide and an increase in levels of the pro-apoptotic Bax (2.3-fold, P=0.0004) was detected at 10 mg/kg per day. In contrast, the levels of pro-apoptotic peptide Bak that was mainly expressed in somatic cells decreased (70%, P=0.0008) at 10 mg/kg per day. Such alterations in Bcl-2-related peptides occurred mainly at the protein level except for Bcl-2 (72%, P=0.0001) and Bak (43%, P=00002) transcripts. Together, these results showed that the apoptosis observed in adult germ cells from rats exposed in utero to flutamide may result from a long-term alteration in the balance between pro- and anti-apoptotic Bcl-2-related molecules in favour of pro-apoptotic proteins. These data further supported the concept of an androgen-dependent fetal programming that is in relation with an alteration of the expression of Bcl-2-related genes/proteins promoting apoptosis in testicular germ cells of adult rats with fetal androgen disruption.

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.

Effect of epidermal growth factor/transforming growth factor alpha on lactate production in porcine Sertoli cells: glucose transport and lactate dehydrogenase isozymes as potential sites of action.

Germ cell development is dependent upon the delivery of essential nutriments such as lactate originating from Sertoli cells. Lactate production is under the systemic control but probably also under a local control exerted via certain growth factors. By using a model of porcine cultured Sertoli cells, we have characterized the action of epidermal growth factor (EGF) on lactate production and further delineated the potential biochemical mechanisms involved in the EGF action. EGF stimulated lactate production in a time and dose dependent manner with a half-maximal (ED50) and maximal effects, respectively with 3.8 (0.6 x 10(-9) M) and 22 ng/ml of EGF. Lactate formation involves several biochemical steps among which the glucose substrate uptake and transport system as well as the lactate dehydrogenase (LDH) activity appear to play key roles. We report here that EGF increased the uptake of glucose evaluated through that of 2-deoxy-D-glucose (2-DOG), a non-metabolizable glucose analog. Such an increase in glucose substrate uptake occurs both after a long term (48 h) and a short term treatment (ED50 = 6.4 ng/ml, 1.1 x 10(-9) M EGF). Moreover, EGF was also able to enhance the activity of the Sertoli cell LDH. The maximal effect of the growth factor on LDH activity was observed after a long term (24 h) treatment with an ED50 of 7 ng/ml (1.2 x 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)