Major transcriptomic, epigenetic and metabolic changes underlie the pluripotency continuum in rabbit preimplantation embryos.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.

A Cross-Sectional Quantitative Metabolomics Study Evidencing the Metabolic Signature in Six Organs during a 14-Week High-Fat High-Sucrose and Standard Diet in Mice.

Obesity is a risk factor for many diseases, such as type 2 diabetes and cardiovascular diseases. In line with the need for precision medicine, the search for biomarkers reporting the progression of obesity- and diet-associated disorders is urgent. We used NMR to determine the metabolomics profile of key organs (lung, liver, heart, skeletal muscle, kidney, and brain) and serum from male C57Bl/6J mice (5 weeks old) fed for 6, 10, and 14 weeks on a high-fat and high-sucrose diet (HFHSD) vs. a standard diet (STD). We determined metabolite concentrations in the organs at each time point, which allowed us to discriminate age- and diet-related effects as well as the interactions between both, highlighting the need to evaluate the influence of age as a confounding factor on metabolic signatures. Notably, the analysis revealed the influence of time on metabolite concentrations in the STD condition, probably reflecting the juvenile-to-adult transition. Variations impacted the liver and lung metabolites, revealing the strong influence of the HFHS diet on normal metabolism maturation during youth.

Inhibition of intestinal FXR activity as a possible mechanism for the beneficial effects of a probiotic mix supplementation on lipid metabolism alterations and weight gain in mice fed a high fat diet.

Supplementation with probiotics has emerged as a promising therapeutic tool to manage metabolic diseases. We investigated the effects of a mix of Bifidobacterium animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 on high-fat (HF) diet -induced metabolic disease in mice. Supplementation with the probiotic mix in HF diet-fed mice (HF-Pr2) reduced weight and fat mass gains, decreased hepatic lipid accumulation, and lowered plasma triglyceride peak during an oral lipid tolerance test. At the molecular level, the probiotic mix protected against HF-induced rise in mRNA levels of genes related to lipid uptake, metabolism, and storage in the liver and white adipose tissues, and strongly decreased mRNA levels of genes related to inflammation in the white adipose tissue and to oxidative stress in the liver. Regarding intestinal homeostasis, the probiotic mix did not prevent HF-induced gut permeability but slightly modified microbiota composition without correcting the dysbiosis induced by the HF diet. Probiotic supplementation also modified the cecal bile acid (BA) profile, leading to an increase in the Farnesoid-X-Receptor (FXR) antagonist/agonist ratio between BA species. In agreement, HF-Pr2 mice exhibited a strong inhibition of FXR signaling pathway in the ileum, which was associated with lipid metabolism protection. This is consistent with recent reports proposing that inhibition of intestinal FXR activity could be a potent mechanism to overcome metabolic disorders. Altogether, our results demonstrate that the probiotic mix evaluated, when administered preventively to HF diet-fed mice could limit obesity and associated lipid metabolism disorders, likely through the inhibition of FXR signaling in the intestinal tract.

Glycine Supplementation in Obesity Worsens Glucose Intolerance through Enhanced Liver Gluconeogenesis.

Interactions between mitochondria and the endoplasmic reticulum, known as MAMs, are altered in the liver in obesity, which contributes to disruption of the insulin signaling pathway. In addition, the plasma level of glycine is decreased in obesity, and the decrease is strongly correlated with the severity of insulin resistance. Certain nutrients have been shown to regulate MAMs; therefore, we tested whether glycine supplementation could reduce insulin resistance in the liver by promoting MAM integrity. Glycine (5 mM) supported MAM integrity and insulin response in primary rat hepatocytes cultured under control and lipotoxic (palmitate 500 µM) conditions for 18 h. In contrast, in C57 BL/6 JOlaHsd mice (male, 6 weeks old) fed a high-fat, high-sucrose diet (HFHS) for 16 weeks, glycine supplementation (300 mg/kg) in drinking water during the last 6 weeks (HFHS-Gly) did not reverse the deleterious impact of HFHS-feeding on liver MAM integrity. In addition, glycine supplementation worsened fasting glycemia and glycemic response to intraperitoneal pyruvate injection compared to HFHS. The adverse impact of glycine supplementation on hepatic gluconeogenesis was further supported by the higher oxaloacetate/acetyl-CoA ratio in the liver in HFHS-Gly compared to HFHS. Although glycine improves MAM integrity and insulin signaling in the hepatocyte in vitro, no beneficial effect was found on the overall metabolic profile of HFHS-Gly-fed mice.

Metformin treatment for 8 days impacts multiple intestinal parameters in high-fat high-sucrose fed mice.

Although the mechanism of action of the antidiabetic drug metformin is still a matter of discussions, it is well accepted that the gut plays an important role. To gain more insights into the mechanisms occurring in the different regions of the intestine, adult male mice were fed a high-fat-high sucrose (HFS) diet for 8 days and treated with metformin by gavage (300 mg/day/kg body weight) during the HFS diet. Metformin counteracted HFS diet-induced overexpression of a network of genes involved in the transport of glucose and fatty acids in the different regions of the small intestine. It also induced beneficial modification of secondary bile acid profile in the caecum, with a reduction of deoxycholic acid and lithocholic acid levels and increased abundance of ursodeoxycholic acid and tauroursodeoxycholic acid, potentially leading to FRX inhibition. In parallel, metformin treatment was associated with specific changes of the microbiota composition in the lumen of the different regions of the intestine. Metformin induced a marked increase in the abundance of Akkermansia muciniphila in the lumen all along the gut and counteracted the effects of HFS diet on the abundances of some bacterial groups generally associated with metabolic disturbances (f-Lachnospiraceae, f-Petostreptococcaceae, g-Clostidium). Therefore, the present work clearly emphasises the role of all the regions of the intestinal tract in the beneficial action of the antidiabetic drug metformin in a prediabetic mouse model.

Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells.

Progression of germ cells through meiosis is regulated by phosphorylation events. We previously showed the key role of cyclin dependent kinases in meiotic divisions of rat spermatocytes co-cultured with Sertoli cells (SC). In the present study, we used the same culture system to address the role of mitogen-activated protein kinases (MAPKs) in meiotic progression. Phosphorylated ERK1/2 were detected in vivo and in freshly isolated SC and in pachytene spermatocytes (PS) as early as 3 h after seeding on SC. The yield of the two meiotic divisions and the percentage of highly MPM-2-labeled pachytene and secondary spermatocytes (SII) were decreased in co-cultures treated with U0126, an inhibitor of the ERK-activating kinases, MEK1/2. Pre-incubation of PS with U0126 resulted in a reduced number of in vitro formed round spermatids without modifying the number of SII or the MPM-2 labeling of PS or SII. Conversely, pre-treatment of SC with U0126 led to a decrease in the percentage of highly MPM-2-labeled PS associated with a decreased number of SII and round spermatids. These results show that meiotic progression of spermatocytes is dependent on SC-activated MAPKs. In addition, high MPM-2 labeling was not acquired by PS cultured alone in Sertoli cell conditioned media, indicating a specific need for cell-cell contact between germ cells and SC.

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis.

Glial cell-line-derived neurotropic factor (GDNF) and its receptors glial cell-line-derived neurotropic factor alpha (GFR1alpha) and rearranged during transformation (RET) have been localized in the rat testis during postnatal development. The three mRNAs, and GDNF and GFR1alpha proteins were detected in testis extracts from 1- to 90-day-old rats by reverse transcriptase PCR and Western blotting respectively. The three mRNAs were present in Sertoli cells from 20- and 55-day-old rats, pachytene spermatocytes (PS), and round spermatids (RS). The GDNF and GFR1alpha proteins were detected in PS, RS, and Sertoli cells. GDNF and GFR1alpha were also detected using flow cytometry in spermatogonia and preleptotene spermatocytes, and in secondary spermatocytes. The localization of GDNF and GFR1alpha in germ and Sertoli cells was confirmed by immunocytochemistry. The hypothesis that GDNF may control DNA synthesis of Sertoli cells and/or spermatogonia in the immature rat was addressed using cultures of seminiferous tubules from 7- to 8-day-old rats. Addition of GDNF for 48 h resulted in a twofold decrease in the percentage of spermatogonia able to duplicate DNA, whereas Sertoli cells were not affected. These results are consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia. Moreover, the wide temporal and spatial expression of GDNF and its receptors in the rat testis suggest that it might act at several stages of spermatogenesis.

Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates.

The imprinted genes of primate embryonic stem cells (ESCs) often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs), SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency.

A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency.

Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes.

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.