DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals ­ where testis differentiation is driven by a different gene called SRY ­ is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.

Identification of the Inner Cell Mass and the Trophectoderm Responses after an In Vitro Exposure to Glucose and Insulin during the Preimplantation Period in the Rabbit Embryo.

The prevalence of metabolic diseases is increasing, leading to more women entering pregnancy with alterations in the glucose-insulin axis. The aim of this work was to investigate the effect of a hyperglycemic and/or hyperinsulinemic environment on the development of the preimplantation embryo. In rabbit embryos developed in vitro in the presence of high insulin (HI), high glucose (HG), or both (HGI), we determined the transcriptomes of the inner cell mass (ICM) and the trophectoderm (TE). HI induced 10 differentially expressed genes (DEG) in ICM and 1 in TE. HG ICM exhibited 41 DEGs involved in oxidative phosphorylation (OXPHOS) and cell number regulation. In HG ICM, proliferation was decreased (p < 0.01) and apoptosis increased (p < 0.001). HG TE displayed 132 DEG linked to mTOR signaling and regulation of cell number. In HG TE, proliferation was increased (p < 0.001) and apoptosis decreased (p < 0.001). HGI ICM presented 39 DEG involved in OXPHOS and no differences in proliferation and apoptosis. HGI TE showed 16 DEG linked to OXPHOS and cell number regulation and exhibited increased proliferation (p < 0.001). Exposure to HG and HGI during preimplantation development results in common and specific ICM and TE responses that could compromise the development of the future individual and placenta.

Predicting male fertility from the sperm methylome: application to 120 bulls with hundreds of artificial insemination records.

BACKGROUND: Conflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility. RESULTS: A unique collection of 100 sperm samples was constituted by pooling 2-5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls. CONCLUSION: This study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.

Accelerating Onset of Puberty Through Modification of Early Life Nutrition Induces Modest but Persistent Changes in Bull Sperm DNA Methylation Profiles Post-puberty.

In humans and model species, alterations of sperm DNA methylation patterns have been reported in cases of spermatogenesis defects, male infertility and exposure to toxins or nutritional challenges, suggesting that a memory of environmental or physiological changes is recorded in the sperm methylome. The objective of this study was to ascertain if early life plane of nutrition could have a latent effect on DNA methylation patterns in sperm produced post-puberty. Holstein-Friesian calves were assigned to either a high (H) or moderate (M) plane of nutrition for the first 24 weeks of age, then reassigned to the M diet until puberty, resulting in HM and MM groups. Sperm DNA methylation patterns from contrasted subgroups of bulls in the HM (ejaculates recovered at 15 months of age; n = 9) and in the MM (15 and 16 months of age; n = 7 and 9, respectively) were obtained using Reduced Representation Bisulfite Sequencing. Both 15 and 16 months were selected in the MM treatment as these bulls reached puberty approximately 1 month after the HM bulls. Hierarchical clustering demonstrated that inter-individual variability unrelated to diet or age dominated DNA methylation profiles. While the comparison between 15 and 16 months of age revealed almost no change, 580 differentially methylated CpGs (DMCs) were identified between the HM and MM groups. Differentially methylated CpGs were mostly hypermethylated in the HM group, and enriched in endogenous retrotransposons, introns, intergenic regions, and shores and shelves of CpG islands. Furthermore, genes involved in spermatogenesis, Sertoli cell function, and the hypothalamic-pituitary-gonadal axis were targeted by differential methylation when HM and MM groups were compared at 15 months of age, reflecting the earlier timing of puberty onset in the HM bulls. In contrast, the genes still differentially methylated in MM bulls at 16 months of age were enriched for ATP-binding molecular function, suggesting that changes to the sperm methylome could persist even after the HM and MM bulls reached a similar level of sexual maturity. Together, results demonstrate that enhanced plane of nutrition in pre-pubertal calves associated with advanced puberty induced modest but persistent changes in sperm DNA methylation profiles after puberty.

Bacillus subtilis Regulators MntR and Zur Participate in Redox Cycling, Antibiotic Sensitivity, and Cell Wall Plasticity.

The Bacillus subtilis MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of mntR and zur deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in ΔmntR and Δzur cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg2+ accumulation in the ΔmntR mutant. Phenotypic and morphological analyses of ΔmntR and Δzur mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure.IMPORTANCE Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

Proteomic Response of Pseudomonas aeruginosa PAO1 Adhering to Solid Surfaces.

Pseudomonas aeruginosa is a pathogenic micro-organism responsible for many hospital-acquired infections. It is able to adhere to solid surfaces and develop an immobilized community or so-called biofilm. Many studies have been focusing on the use of specific materials to prevent the formation of these biofilms, but the reactivity of the bacteria in contact to surfaces remains unknown. The aim of this study was to evaluate the impact of the abiotic surface on the physiology of adherent bacteria. Three different materials, stainless steel (SS), glass (G), and polystyrene (PS) that were relevant to industrial or medical environments were characterized at the physicochemical level in terms of their hydrophobicity and roughness. We showed that SS was moderately hydrophilic and rough, potentially containing crevices, G was hydrophilic and smooth while PS was hydrophobic and smooth. We further showed that P. aeruginosa cells were more likely able to adhere to SS and G rather than PS surfaces under our experimental conditions. The physiological response of P. aeruginosa when adhering to each of these materials was then evaluated by global proteomic analysis. The abundance of 70 proteins was shown to differ between the materials suggesting that their abundance was modified as a function of the material to which bacteria adhered. Our data lead to enabling the identification of abundance patterns that appeared to be specific to a given surface. Taken together, our data showed that P. aeruginosa is capable of sensing and responding to a surface probably via specific programmes to adapt its physiological response accordingly.

Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans.

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

The MarR-like protein PchR (YvmB) regulates expression of genes involved in pulcherriminic acid biosynthesis and in the initiation of sporulation in Bacillus subtilis.

BACKGROUND: Cyclodipeptides and their derivatives constitute a large class of peptide natural products with noteworthy biological activities. In some yeasts and bacterial species, pulcherriminic acid derived from cyclo-L-leucyl-L-leucyl is excreted and chelates free ferric ions to form the pulcherrimin. In Bacillus subtilis, the enzymes YvmC and CypX are known to be involved in pulcherriminic acid biosynthesis. However, the mechanisms controlling the transcription of the yvmC-cypX operon are still unknown. RESULTS: In this work, we demonstrated that the B. subtilis YvmB MarR-like regulator is the major transcription factor controlling yvmC-cypX expression. A comprehensive quantitative proteomic analysis revealed a wide and prominent effect of yvmB deletion on proteins involved in cellular processes depending on iron availability. In addition, expression of yvmB depends on iron availability. Further analysis with real-time in vivo transcriptional profiling allowed us to define the YvmB regulon. We identified yvmBA, yvmC-cypX and yvnB for negative regulation and yisI for positive regulation. In combination with genetic approaches, gel mobility shift assays indicated that a 14-bp palindromic motif constitutes the YvmB binding site. It was unexpected that YvmB controls expression of yisI, whose encoding protein plays a negative role in the regulation of the sporulation initiation pathway. YvmB appears as an additional regulatory element into the cell's decision to grow or sporulate. CONCLUSION: Our findings reveal a possible role of the B. subtilis YvmB regulator in the regulatory networks connected to iron metabolism and to the control of proper timing of sporulation. YvmB was renamed as PchR controlling the pulcherriminic acid biosynthetic pathway of B. subtilis.