Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Evaluation of Lipopolysaccharide and Interleukin-6 as Useful Screening Tool for Chronic Endometritis.

Universal diagnostic criteria for chronic endometritis (CE) have not been established due to differences in study design among researchers and a lack of typical clinical cases. Lipopolysaccharides (LPSs) have been reported to cause inflammation in the reproductive systems of several animals. This study aimed to elucidate the influence of LPS in the pathogenesis of CE in humans. We investigated whether LPS affected cytokine production and cell proliferation in the endometrium using in vivo and in vitro experiments. LPS concentrations were analyzed between control and CE patients using endometrial tissues. LPS administration stimulated the proliferation of EM-E6/E7 cells derived from human endometrial cells. High LPS concentrations were detected in CE patients. LPS concentration was found to correlate with IL-6 gene expression in the endometrium. Inflammation signaling evoked by LPS led to the onset of CE, since LPS stimulates inflammatory responses and cell cycles in the endometrium. We identified LPS and IL-6 as suitable candidate markers for the diagnosis of CE.

Seasonal adaptation of Mangalica pigs in terms of muscle morphology and metabolism.

The skeletal muscle plays an important role in maintaining body temperature, which is mediated by thermogenesis and glucose or lipid metabolism. Mangalica is a native Hungarian pig that has cold tolerance and can live in grazing environments throughout the year. We evaluated the morphological and genetic aspects of Mangalica using muscle tissues to elucidate the mechanisms underlying the tolerance to seasonal effects in grazing environments. The muscle tissues in each season were analysed using morphological evaluation and electron microscopy. The cross-sectional area of skeletal muscle cells in summer was significantly larger than that in winter. The thickness of myofibrils in summer was significantly higher than in winter. The thickness of the Z-line in winter was significantly higher than in summer. The expression of MYH4 and GLUT4 was significantly lower in winter than in summer. The result of ATPase staining indicated significantly increase the muscle fibre ratio of type 1 in winter than that in summer. These findings indicate that the muscle fibre in Mangalica shifts from fast-twitch to slow-twitch fibre, and the metabolic physiology of the muscle was adapted to the cold environment. This study demonstrates that Mangalica gained tolerance to both seasonal heat and cold stresses that are caused by significant changes in ambient temperature in a year because of changes in their muscle fibre type and metabolic function. This study may contribute to elucidating the mechanism of thermogenetic adaptation in cold and heat environments among mammals.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

AIM: The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. METHODS: We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Furthermore, we examined the frequencies of known BA-related single-nucleotide polymorphisms in the BA and control groups. RESULTS: SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a Toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value = 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated single-nucleotide polymorphisms, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness, but not to BA, and the p.Ala421Thr of THOC2 represents a relatively common single-nucleotide polymorphism in Asia. CONCLUSIONS: The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, whereas the contribution of other monogenic variants to the disease predisposition is limited.

LPS Administration during Fertilization Affects Epigenetic Inheritance during Embryonic Development.

Intrauterine inflammation can cause infertility by disrupting reproductive function. The pathogenesis underlying this process may primarily involve endotoxins from lipopolysaccharides (LPS), which are produced by Gram-negative bacteria. However, the long-term effects of endotoxins in mammalian pregnancy following LPS exposure during fertilization have not been clarified. In this study, we performed experiments to analyze the influence of LPS on early embryonic development and fetal development in mice. Mice uteruses were examined for the expression of genes related to the inflammatory response. The expression of Il-1ß and Il-6 increased following the administration of 200 and 1000 µg/kg LPS. Exposure to LPS using in vitro fertilization (IVF) significantly decreased the embryonic developmental rate. A concentration of 100 µg/kg LPS significantly increased the placental weight and fetal crown -rump length (CRL), whereas a concentration of 200 µg/kg LPS significantly decreased the placenta weight and fetal weight in vivo. These findings indicate that maternal LPS during fertilization affects fetal development until the late stage of pregnancy. Thus, maternal endotoxins may affect epigenetic inheritance during embryonic development from the early to late stages of pregnancy.

Comparison of Placental HSD17B1 Expression and Its Regulation in Various Mammalian Species.

During mammalian gestation, large amounts of progesterone are produced by the placenta and circulate for the maintenance of pregnancy. In contrast, primary plasma estrogens are different between species. To account for this difference, we compared the expression of ovarian and placental steroidogenic genes in various mammalian species (mouse, guinea pig, porcine, ovine, bovine, and human). Consistent with the ability to synthesize progesterone, CYP11A1/Cyp11a1, and bi-functional HSD3B/Hsd3b genes were expressed in all species. CYP17A1/Cyp17a1 was expressed in the placenta of all species, excluding humans. CYP19A/Cyp19a1 was expressed in all placental estrogen-producing species, whereas estradiol-producing HSD17B1 was only strongly expressed in the human placenta. The promoter region of HSD17B1 in various species possesses a well-conserved SP1 site that was activated in human placental cell line JEG-3 cells. However, DNA methylation analyses in the ovine placenta showed that the SP1-site in the promoter region of HSD17B1 was completely methylated. These results indicate that epigenetic regulation of HSD17B1 expression is important for species-specific placental sex steroid production. Because human HSD17B1 showed strong activity for the conversion of androstenedione into testosterone, similar to HSD17B1/Hsd17b1 in other species, we also discuss the biological significance of human placental HSD17B1 based on the symptoms of aromatase-deficient patients.

Progesterone stimulates cortisol production in the maturing bovine cumulus-oocyte complex.

In the bovine cumulus oophorus, 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1)-mediated cortisol production dramatically increases during the periovulatory period. This event is closely associated with increased progesterone (P4) production, implying a functional connection between these C21 steroids. In this study, we investigated the mutual regulation of P4 and cortisol production in the bovine cumulus oophorus. Bovine cumulus-oocyte complexes (COCs) were aspirated from follicles 2-5 mm in diameter and subjected to in vitro maturation (IVM) for 24 h in an M199 supplemented with fetal calf serum (FCS) and follicle-stimulating hormone (FSH). COCs were treated with trilostane (0, 0.1, 1, 10 mM), an inhibitor of P4 synthesis, RU486 (0, 0.1, 1, 10 mM), a receptor antagonist for the progesterone receptor (PR) and glucocorticoid receptor (GR), and various concentrations of a synthetic progestogen nomegestrol acetate (NA; 0, 0.001, 0.01, 0.1, 1, 10 mM) to examine effect of P4. The effects of cortisol (0, 0.1, 1, 10 mM) were also examined in the presence or absence of trilostane. Trilostane and RU486 suppressed cumulus expansion, cortisol production, and HSD11B1 but not hexose-6-phosphate dehydrogenase (H6PDH) expression. Concomitant treatment with NA reversed the effects of trilostane. Unlike NA, cortisol did not alter the antagonistic effects of trilostane on cumulus expansion and HSD11B1 expression. Cortisol did not affect P4 production or steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1), and HSD11B1 expression. Collectively, these results indicate that locally produced P4 is crucial in regulating the local glucocorticoid environment through PRtg in the maturing bovine cumulus oophorus. Cortisol, however, does not appear to regulate P4 or its production.

Artiodactyl livestock species have a uniform vomeronasal system with a vomeronasal type 1 receptor (V1R) pathway.

Artiodactyl livestock animals have a vomeronasal system that detects pheromones. Vomeronasal receptors comprise type 1 (V1R) coupled with G protein α-i2 (Gαi2) and type 2 (V2R) coupled with G protein α-o (Gαo). Laboratory rodents have two segregated V1R and V2R pathways that reach separately to the accessory olfactory bulb (AOB). In contrast, the AOBs of goats and sheep are entirely positive for Gαi2, indicating that they have only the V1R pathway. However, we detected a few V2R genes in the genome of cattle, goats, sheep and pigs by genome assembly. Thus, we immunohistochemically analyzed the AOBs of cattle and pigs to confirm which type of the vomeronasal system is present in artiodactyl livestock species. The glomerular layer of the AOB in cattle and pigs was entirely positive for anti-Gαi2 and weakly positive for anti-Gαo, as in the V1R uniform type of vomeronasal system in other mammal species. These findings indicated that artiodactyl livestock species have a uniform type of vomeronasal system composing the V1R pathway. Therefore, caution is advised when extrapolating knowledge of laboratory rodents with two vomeronasal pathways to livestock animals that have one.

When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3.

17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

BACKGROUND & AIMS: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells. METHODS: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition. RESULTS: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion. CONCLUSIONS: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

[Urinary Retention Caused by Necrotic Tissues in the Bladder of a Female Recipient of a Living-Donor Kidney Transplant : A Case Report].

We report a patient who developed urinary retention due to the presence of necrotic tissues in the bladder 5 months after kidney transplantation. The patient was a 47-year-old female who had been diagnosed with immunoglobulin A nephropathy. She requested to receive a living-donor kidney transplant from her husband, and was referred to our hospital. Given that the patient had anuria preoperatively, her bladder capacity was presumed to have decreased following the transplantation. There were no events regarding vascular anastomosis during the surgery. However, since ureteroneocystostomy was difficult to perform due to the thinning of the bladder wall, the recipient's own ureter was anastomosed to the ureter of the transplanted kidney. Since the patient had difficulty voiding soon after the indwelling urinary catheter was removed, clean intermittent self-catheterization was initiated. Abdominal computed tomography revealed perforation of the bladder and extravesical urinary leakage on postoperative day 15. An indwelling urinary catheter was reinserted as conservative treatment. We removed the indwelling urinary catheter on postoperative day 25. The patient was discharged on postoperative day 30. Five months after transplantation, the patient suddenly developed urinary retention. Cystoscopy revealed some tissue hanging from the anterior wall of the bladder. The tissue was removed, and her voiding function improved. On pathology, the tissue was found to be non-specific necrotic tissue. This finding suggested that the necrotic tissue had caused urinary retention 5 months after transplantation. The symptoms of urinary retention markedly improved after the treatment.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

Method for collecting mouse milk without exogenous oxytocin stimulation.

It has been reported that breast-feeding more than 6 months strongly decreases the risk of allergy, diabetes, obesity, and hypertension in humans. In order to understand the mechanisms responsible for this benefit, it is important to evaluate precisely the composition of maternal milk, especially in response to environmental cues. Mouse models offer a unique opportunity to study the impact of maternal milk composition on the development and health of offspring. Oxytocin injection of the dam is usually used to stimulate milk ejection; however, exogenous oxytocin might have deleterious effects under some experimental conditions by modifying milk content as well as the physiology and behavior of the dam. Taking advantage of the natural stimulation of the mammary gland that occurs after the reunion of a dam that has been separated from her pups, we developed a new procedure to collect mouse milk without the injection of oxytocin. This method is easy to use, low-cost ,and non-invasive. Moreover, it provides a sufficient amount of milk for use in a wide range of biological analyses.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina.

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina.

In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders.

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.

Perinatal protein malnutrition affects mitochondrial function in adult and results in a resistance to high fat diet-induced obesity.

Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed "nutritional programming". We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases.

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids.

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.

Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation.

CHOP encodes a ubiquitous transcription factor that is one of the most important components in the network of stress-inducible transcription. In particular, this factor is known to mediate cell death in response to stress. The focus of this work is to study its pivotal role in the control of cell viability according to the duration of a stress like amino acid starvation. We show that during the first 6h of starvation, CHOP upregulates a number of autophagy genes but is not involved in the first steps of the autophagic process. By contrast, when the amino acid starvation is prolonged (16-48h), we demonstrated that CHOP has a dual role in both inducing apoptosis and limiting autophagy through the transcriptional control of specific target genes. Overall, this study reveals a novel regulatory role for CHOP in the crosstalk between autophagy and apoptosis in response to stress.