Prolonged dialysis during ex vivo lung perfusion promotes inflammatory responses.

Ex-vivo lung perfusion (EVLP) has extended the number of transplantable lungs by reconditioning marginal organs. However, EVLP is performed at 37°C without homeostatic regulation leading to metabolic wastes' accumulation in the perfusate and, as a corrective measure, the costly perfusate is repeatedly replaced during the standard of care procedure. As an interesting alternative, a hemodialyzer could be placed on the EVLP circuit, which was previously shown to rebalance the perfusate composition and to maintain lung function and viability without appearing to impact the global gene expression in the lung. Here, we assessed the biological effects of a hemodialyzer during EVLP by performing biochemical and refined functional genomic analyses over a 12h procedure in a pig model. We found that dialysis stabilized electrolytic and metabolic parameters of the perfusate but enhanced the gene expression and protein accumulation of several inflammatory cytokines and promoted a genomic profile predicting higher endothelial activation already at 6h and higher immune cytokine signaling at 12h. Therefore, epuration of EVLP with a dialyzer, while correcting features of the perfusate composition and maintaining the respiratory function, promotes inflammatory responses in the tissue. This finding suggests that modifying the metabolite composition of the perfusate by dialysis during EVLP can have detrimental effects on the tissue response and that this strategy should not be transferred as such to the clinic.

Reinforcement of repressive marks in the chicken primordial germ cell epigenetic signature: divergence from basal state resetting in mammals.

BACKGROUND: In mammals, primordial germ cells (PGCs), the embryonic precursors of the germline, arise from embryonic or extra-embryonic cells upon induction by the surrounding tissues during gastrulation, according to mechanisms which are elucidated in mice but remain controversial in primates. They undergo genome-wide epigenetic reprogramming, consisting of extensive DNA demethylation and histone post-translational modification (PTM) changes, toward a basal, euchromatinized state. In contrast, chicken PGCs are specified by preformation before gastrulation based on maternally-inherited factors. They can be isolated from the bloodstream during their migration to the genital ridges. Our prior research highlighted differences in the global epigenetic profile of cultured chicken PGCs compared with chicken somatic cells and mammalian PGCs. This study investigates the acquisition and evolution of this profile during development. RESULTS: Quantitative analysis of global DNA methylation and histone PTMs, including their distribution, during key stages of chicken early development revealed divergent PGC epigenetic changes compared with mammals. Unlike mammalian PGCs, chicken PGCs do not undergo genome-wide DNA demethylation or exhibit a decrease in histone H3 lysine 9 dimethylation. However, chicken PGCs show 5­hydroxymethylcytosine loss, macroH2A redistribution, and chromatin decompaction, mirroring mammalian processes. Chicken PGCs initiate their epigenetic signature during migration, progressively accumulating high global levels of H3K9me3, with preferential enrichment in inactive genome regions. Despite apparent global chromatin decompaction, abundant heterochromatin marks, including repressive histone PTMs, HP1 variants, and DNA methylation, persists in chicken PGCs, contrasting with mammalian PGCs. CONCLUSIONS: Chicken PGCs' epigenetic signature does not align with the basal chromatin state observed in mammals, suggesting a departure from extensive epigenetic reprogramming. Despite disparities in early PGC development, the persistence of several epigenetic features shared with mammals implies their involvement in chromatin-regulated germ cell properties, with the distinctive elevation of chicken-specific H3K9me3 potentially participating in these processes.

Long-term exposure from perinatal life to food-grade TiO2 alters intestinal homeostasis and predisposes to food allergy in young mice.

BACKGROUND: Food allergy (FA) is an inappropriate immunological response to food proteins resulting from an impaired induction of oral tolerance. Various early environmental factors can affect the establishment of intestinal homeostasis, predisposing to FA in early life. In this context, we aimed to assess the effect of chronic perinatal exposure to food-grade titanium dioxide (fg-TiO2 ), a common food additive. METHODS: Dams were fed a control versus fg-TiO2 -enriched diet from preconception to weaning, and their progeny received the same diet at weaning. A comprehensive analysis of baseline intestinal and systemic homeostasis was performed in offspring 1 week after weaning by assessing gut barrier maturation and microbiota composition, and local and systemic immune system and metabolome. The effect of fg-TiO2 on the susceptibility of progeny to develop oral tolerance versus FA to cow's milk proteins (CMP) was performed starting at the same baseline time-point, using established models. Sensitization to CMP was investigated by measuring ß-lactoglobulin and casein-specific IgG1 and IgE antibodies, and elicitation of the allergic reaction by measuring mouse mast cell protease (mMCP1) in plasma collected after an oral food challenge. RESULTS: Perinatal exposure to fg-TiO2 at realistic human doses led to an increased propensity to develop FA and an impaired induction of oral tolerance only in young males, which could be related to global baseline alterations in intestinal barrier, gut microbiota composition, local and systemic immunity, and metabolism. CONCLUSIONS: Long-term perinatal exposure to fg-TiO2 alters intestinal homeostasis establishment and predisposes to food allergy, with a clear gender effect.

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.

Alteration of the embryonic microenvironment and sex-specific responses of the preimplantation embryo related to a maternal high-fat diet in the rabbit model.

The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Strain-specific changes in nucleus accumbens transcriptome and motivation for palatable food reward in mice exposed to maternal separation.

Introduction: In humans, adversity in childhood exerts enduring effects on brain and increases the vulnerability to psychiatric diseases. It also leads to a higher risk of eating disorders and obesity. Maternal separation (MS) in mice has been used as a proxy of stress during infancy. We hypothesized that MS in mice affects motivation to obtain palatable food in adulthood and changes gene expression in reward system. Methods: Male and female pups from C57Bl/6J and C3H/HeN mice strains were subjected to a daily MS protocol from postnatal day (PND) 2 to PND14. At adulthood, their motivation for palatable food reward was assessed in operant cages. Results: Compared to control mice, male and female C3H/HeN mice exposed to MS increased their instrumental response for palatable food, especially when the effort required to obtain the reward was high. Importantly, this effect is shown in animals fed ad libitum. Transcriptional analysis revealed 375 genes differentially expressed in the nucleus accumbens of male MS C3H/HeN mice compared to the control group, some of these being associated with the regulation of the reward system (e.g., Gnas, Pnoc). Interestingly, C57Bl/6J mice exposed to MS did not show alterations in their motivation to obtain a palatable reward, nor significant changes in gene expression in the nucleus accumbens. Conclusion: MS produces long-lasting changes in motivation for palatable food in C3H/HeN mice, but has no impact in C57Bl/6J mice. These behavioral alterations are accompanied by drastic changes in gene expression in the nucleus accumbens, a key structure in the regulation of motivational processes.

Evolution of T cell receptor beta loci in salmonids.

T-cell mediated immunity relies on a vast array of antigen specific T cell receptors (TR). Characterizing the structure of TR loci is essential to study the diversity and composition of T cell responses in vertebrate species. The lack of good-quality genome assemblies, and the difficulty to perform a reliably mapping of multiple highly similar TR sequences, have hindered the study of these loci in non-model organisms. High-quality genome assemblies are now available for the two main genera of Salmonids, Salmo and Oncorhynchus. We present here a full description and annotation of the TRB loci located on chromosomes 19 and 25 of rainbow trout (Oncorhynchus mykiss). To get insight about variations of the structure and composition of TRB locus across salmonids, we compared rainbow trout TRB loci with other salmonid species and confirmed that the basic structure of salmonid TRB locus is a double set of two TRBV-D-J-C loci in opposite orientation on two different chromosomes. Our data shed light on the evolution of TRB loci in Salmonids after their whole genome duplication (WGD). We established a coherent nomenclature of salmonid TRB loci based on comprehensive annotation. Our work provides a fundamental basis for monitoring salmonid T cell responses by TRB repertoire sequencing.

Cell type- and time-dependent biological responses in ex vivo perfused lung grafts.

In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4+ and CD8+ T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments.

AKT activity orchestrates marginal zone B cell development in mice and humans.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D+CD27+ B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD+CD27- and memory IgD-CD27+ B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans.

Bisphenol A and bisphenol S both disrupt ovine granulosa cell steroidogenesis but through different molecular pathways.

BACKGROUND: Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERß). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis. RESULTS: Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively. CONCLUSION: In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.

Toll-like receptors control the accumulation of neutrophils in lymph nodes that expand CD4+ T cells during experimental autoimmune encephalomyelitis.

Toll-like receptors (TLR) control the activation of dendritic cells that prime CD4+ T cells in draining lymph nodes, where these T cells then undergo massive clonal expansion. The mechanisms controlling this clonal T cell expansion are poorly defined. Using the CD4+ T cell-mediated disease experimental autoimmune encephalomyelitis (EAE), we show here that this process is markedly suppressed when TLR9 signaling is increased, without noticeably affecting the transcriptome of primed T cells, indicating a purely quantitative effect on CD4+ T cell expansion. Addressing the underpinning mechanisms revealed that CD4+ T cell expansion was preceded and depended on the accumulation of neutrophils in lymph nodes a few days after immunization. Underlying the importance of this immune regulation pathway, blocking neutrophil accumulation in lymph nodes by treating mice with a TLR9 agonist inhibited EAE progression in mice with defects in regulatory T cells or regulatory B cells, which otherwise developed a severe chronic disease. Collectively, this study demonstrates the key role of neutrophils in the quantitative regulation of antigen-specific CD4+ T cell expansion in lymph nodes, and the counter-regulatory role of TLR signaling in this process.

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.

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.

Corrigendum: An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

[This corrects the article DOI: 10.3389/fcimb.2021.761945.].

Prepubertal nutritional modulation in the bull and its impact on sperm DNA methylation.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Molecular and Cellular Analysis of the Repair of Zebrafish Optic Tectum Meninges Following Laser Injury.

We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrß-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain-blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.

Maternal age affects equine day 8 embryo gene expression both in trophoblast and inner cell mass.

BACKGROUND: Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. RESULTS: Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected. CONCLUSIONS: This is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

BPA disrupts meiosis I in oogonia by acting on pathways including cell cycle regulation, meiosis initiation and spindle assembly.

The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.

Clonotypic IgH Response against Systemic Viral infection in Pronephros and Spleen of a Teleost Fish.

Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHµ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5' RACE-cDNA sequencing approach to characterize modifications of the IgHµ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.

Antigen receptor-engineered Tregs inhibit CNS autoimmunity in cell therapy using nonredundant immune mechanisms in mice.

CD4+ FOXP3+ Tregs are currently explored to develop cell therapies against immune-mediated disorders, with an increasing focus on antigen receptor-engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG-3 and CTLA-4 in LNs, while IL-10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL-10, CTLA-4, and LAG-3 were nonredundantly required for the protective function of antigen receptor-engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg-derived IL-10 and CTLA-4 were both required to suppress acute autoreactive CD4+ T-cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor-engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks.