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International audience; The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

International audience; p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here, we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells by using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l(-/Y) mice, the weight of Taf7l(-/Y) testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology, including folded tails. Sperm motility was significantly reduced, and Taf7l(-/Y) males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l(-/Y) testes decreased more than twofold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ cell differentiation. Our mouse studies suggest that mutations in the human TAF7L gene might be implicated in X-linked oligozoospermia in men.

International audience; The present study examines whether and to what extent the profiles of adipose-derived factors are altered in epididymal and subcutaneous adipose tissues of long-term fasted/refed and of fasted rats treated by recombinant leptin. Fasting was characterized by three successive metabolic phases. Minor differences in the time-course and magnitude of response were detected between the two adipose sites. Leptin, adiponectin, resistin, adiponutrin, and insulin-like growth factor-1 (IGF-1) gene expressions differentially decreased according to the fasting duration. mRNA levels reached a minimum in late fasting for these secreted factors, being decreased by 60-90% for adiponectin, resistin, and IGF-1, 95-98% for leptin and by 100% for adiponutrin. Refeeding partially or totally restored their mRNA expression in epididymal adipose. Expression levels of apolipoprotein E (ApoE), angiotensinogen (AGT), adipsin and macrophage migration inhibitory factor (MIF) were either unchanged or slightly affected. In leptin-treated rats, leptin mRNA concentrations were significantly decreased in phase 2 of fasting (by 85%) from levels in control phosphate-buffered saline (PBS)-treated rats in both tissues. Leptin treatment also decreased resistin mRNA levels (by 78% in P2L and 63% in P3L relative to control groups) in subcutaneous adipose. These data suggest that adiponectin, resistin, adiponutrin, and IGF-1 could be involved in overall energy homeostasis during prolonged fasting, as leptin is. The mechanisms that underlie the expressions of these adipose-secreted factors remain to be determined.

National audience; BACKGROUND: The molecular mechanisms implicated in regionalized gene expression in the human epididymis have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis and could be involved in the regulation of mRNA stability and post-transcriptional expression in this organ. METHODS: Using a miRNA microarray approach, we investigated the correlation between miRNA signatures and gene expression profiles found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed using TargetScan and Miranda software's. FHCE1 immortalized epididymal cell lines were cotransfected with mimic microRNAs and plasmid constructs containing the 3'UTR of predicted target genes downstream of the luciferase gene. RESULTS: We identified 35 miRNAs differentially expressed in the distinct segments of the epididymis (fold change ≥2, P-value ≤ 0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same epididymis-enriched cluster located on the X chromosome, are significantly more expressed in the corpus and cauda regions than in the caput. Interestingly, a strong negative correlation (r = -0,89, P-value ≤ 0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma) and with miR-145 and Cldn10 mRNA (r = -0,92, P-value ≤ 0.001). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3' UTRs, respectively. CONCLUSION: Our study shows that the expression of miRNAs is segmented along the human epididymis and correlates with the pattern of target gene expression in different regions. Therefore, epididymal miRNAs may be in control of the maintenance of gene expression profile in the epididymis, which dictates segment-specific secretion of proteins and establishes physiological compartments that directly or indirectly affect sperm maturation and fertility.

International audience; Phthalates are abundantly produced plasticizers, and di(ethylhexyl) phthalate (DEHP) is the most widely used derivative in various consumer products and medical devices. Animal studies show that DEHP and various other phthalates cause reproductive and developmental toxicity. Although the evidences are limited, it seems reasonable that DEHP may have a potential for similar adverse effects in humans. Such concerns are increasing, particularly for the developing reproductive system of male infants and children. By taking into account the essentiality of selenium (Se) in testicular structure and functions and the high prevalence of inadequate Se intake in various part of the world, this study was designed to investigate the testicular toxicity of DEHP in Se-deficient male rats and to examine the possible preventive effects of Se supplementation on phthalate toxicity. Se deficiency was generated by feeding 3-week-old Sprague-Dawley rats with a ≤0.05 Se mg/kg diet for 5 weeks. Supplementation groups were on a 1 mg Se/kg diet, and DEHP-treated groups received a 1,000 mg/kg dose by gavage during the last 10 days of the feeding period. Testicular histopathology, sperm count and motility, and sperm morphology were examined, and plasma levels of sex hormones were measured. Toxicity and antiandrogenic effects of DEHP were evidenced by disturbed testicular histology and spermatogenesis, diminished testosterone, leutinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and sperm motility. The effects of DEHP were much more pronounced in Se-deficient rats, whereas Se supplementation was found to be protective, reflecting its regulating role in cellular redox equilibrium.

Adresses actuelles : Dr Maya Belghazi, IFR Jean Roche, Faculté de Médecine, 13344 Marseille Dr Jean-Luc Gatti, UMR IBSV, INRA-CNRS-Université de Nice, 06903 Sophia Antipolis; International audience; Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.

Objectives— This study examines the role of insulin and angiotensin II in high-density lipoprotein (HDL) metabolism by focusing on the regulation and function of scavenger receptor type-BI (SR-BI) in adipose tissue. Methods and Results— Insulin or angiotensin II injection in wild-type mice induced a decrease in circulating HDL and it was associated with the translocation of SR-BI from intracellular sites to the plasma membrane of adipose tissue. Refeeding upregulated adipose HDL selective cholesteryl esters uptake and SR-BI proteins through transcriptional and posttranscriptional mechanisms. This occurred along with a decrease in serum HDL and an increase in adipose cholesterol content. Similar results were obtained with transgenic mice overexpressing locally angiotensinogen in adipose tissue. In adipose 3T3-L1 cell line, HDL induced lipogenesis by increasing liver X receptor binding activity. This mechanism was dependent of insulin and angiotensin II. Conclusions— Our results raise the possibility that adipose tissue SR-BI translocation might be a link between adipose tissue lipid storage and HDL clearance.

International audience; CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1(-/-) mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1(-/-) males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1(-/-) males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.