Fetal endocrine axes mRNA expression levels are related to sex and intrauterine position.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes are two major pathways that connect the neural and endocrine systems in vertebrates. Factors such as prenatal stress and maternal exposure to exogenous steroids have been shown to affect these pathways during fetal development. Another less studied factor is the transfer of hormones across fetuses in multifetal pregnancies. This form of transfer has been shown to influence the morphology, anatomy, physiology, and behavior of the offspring in litter-bearing mammals, an influence termed the intrauterine position (IUP) effect. In this study, we sought to delineate how the IUP effects HPA and HPG brain receptors, peptides, and enzymes (hereafter components) in utero and how these influences may differ between males and females. METHODS: We utilized the unconventional model of culled free-ranging nutria (Myocastor coypus), with its large natural variation. We collected brain tissues from nutria fetuses and quantified the expression of key HPA and HPG components in three brain regions: prefrontal cortex, hypothalamus, and striatum. RESULTS: We found an interaction between sex and IUP in the mineralocorticoid receptor (MR), gonadotropin-releasing hormone receptor (GNRHR), androgen receptor (AR), and estrogen receptor alpha (ESR1). IUP was significant in both gonadotropin-releasing hormone (GnRH) and its receptor GNRHR, but in different ways. In the hypothalamus, fetuses adjacent to same-sex neighbors had higher expression of GnRH than fetuses neighboring the opposite sex. Conversely, in the cortex, GNRHR exhibited the inverse pattern, and fetuses that were neighboring the opposite sex had higher expression levels than those neighboring the same sex. Regardless of IUP, in most components that showed significant sex differences, female fetuses had higher mRNA expression levels than male fetuses. We also found that HPA and HPG components were highly related in the early stages of gestation, and that there was an interaction between sex and developmental stage. In the early stages of pregnancy, female component expression levels were more correlated than males', but in the last trimester of pregnancy, male components were more related to each other than female's. CONCLUSIONS: This study suggests that there are sexually different mechanisms to regulate the HPA and HPG axes during fetal development. Higher mRNA expression levels of endocrine axes components may be a mechanism to help females cope with prolonged androgen exposure over a long gestational period. Additionally, these findings suggest different coordination requirements of male and female endocrine axes during stages of fetal development.

A co-ordinated transcriptional programme in the maternal liver supplies long chain polyunsaturated fatty acids to the conceptus using phospholipids.

The long and very long chain polyunsaturated fatty acids (LC-PUFAs) are preferentially transported by the mother to the fetus. Failure to supply LC-PUFAs is strongly linked with stillbirth, fetal growth restriction, and impaired neurodevelopmental outcomes. However, dietary supplementation during pregnancy is unable to simply reverse these outcomes, suggesting imperfectly understood interactions between dietary fatty acid intake and the molecular mechanisms of maternal supply. Here we employ a comprehensive approach combining untargeted and targeted lipidomics with transcriptional profiling of maternal and fetal tissues in mouse pregnancy. Comparison of wild-type mice with genetic models of impaired lipid metabolism allows us to describe maternal hepatic adaptations required to provide LC-PUFAs to the developing fetus. A late pregnancy-specific, selective activation of the Liver X Receptor signalling pathway dramatically increases maternal supply of LC-PUFAs within circulating phospholipids. Crucially, genetic ablation of this pathway in the mother reduces LC-PUFA accumulation by the fetus, specifically of docosahexaenoic acid (DHA), a critical nutrient for brain development.

Global Transcriptomic and Characteristics Comparisons between Mouse Fetal Liver and Bone Marrow Definitive Erythropoiesis.

Erythropoiesis occurs first in the yolk sac as a transit "primitive" form, then is gradually replaced by the "definitive" form in the fetal liver (FL) during fetal development and in the bone marrow (BM) postnatally. While it is well known that differences exist between primitive and definitive erythropoiesis, the similarities and differences between FL and BM definitive erythropoiesis have not been studied. Here we performed comprehensive comparisons of erythroid progenitors and precursors at all maturational stages sorted from E16.5 FL and adult BM. We found that FL cells at all maturational stages were larger than their BM counterparts. We further found that FL BFU-E cells divided at a faster rate and underwent more cell divisions than BM BFU-E. Transcriptome comparison revealed that genes with increased expression in FL BFU-Es were enriched in cell division. Interestingly, the expression levels of glucocorticoid receptor Nr3c1, Myc and Myc downstream target Ccna2 were significantly higher in FL BFU-Es, indicating the role of the Nr3c1-Myc-Ccna2 axis in the enhanced proliferation/cell division of FL BFU-E cells. At the CFU-E stage, the expression of genes associated with hemoglobin biosynthesis were much higher in FL CFU-Es, indicating more hemoglobin production. During terminal erythropoiesis, overall temporal patterns in gene expression were conserved between the FL and BM. While biological processes related to translation, the tricarboxylic acid cycle and hypoxia response were upregulated in FL erythroblasts, those related to antiviral signal pathway were upregulated in BM erythroblasts. Our findings uncovered previously unrecognized differences between FL and BM definitive erythropoiesis and provide novel insights into erythropoiesis.

Small non-coding RNA transcriptomic profiling in adult and fetal human brain.

Small non-coding RNAs (sncRNAs) make up ~1% of the transcriptome; nevertheless, they play significant roles in regulating cellular processes. Given the complexity of the central nervous system, sncRNAs likely hold particular importance in the human brain. In this study, we provide sncRNA transcriptomic profiles in a range of adult and prenatal brain regions, with a focus on piRNAs, due to their underexplored expression in somatic cells and tissue-specific nature. Using the WIND workflow, which combines two detection methods, we found 1333 (731 miRNAs, 249 piRNAs, 285 snoRNAs, and 68 other sncRNAs) and 1445 unique sncRNAs (770 miRNAs, 307 piRNAs, 289 snoRNAs, and 79 other sncRNAs) in developing and adult brains, respectively. Significant variations were found upon comparison of fetal and adult brain groups, with 82 miRNAs, 17 piRNAs, and 70 snoRNAs enriched in fetal brains and 22 miRNAs, 11 piRNAs in adult brains. This dataset represents a valuable resource for exploring the sncRNA roles in brain function, their involvement in neurological diseases, and the molecular mechanisms behind brain region interactions.

Transcriptome analysis reveals hepatic disordered lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses due to maternal undernutrition during late pregnancy.

Although lipid metabolism in fetal livers under intrauterine growth restriction (IUGR) conditions has been widely studied, the implications of maternal undernutrition on fetal hepatic lipid metabolism, lipotoxic injury, and abnormal development remain largely unknown. Therefore, this study investigated the effects of maternal undernutrition on disordered hepatic lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses using transcriptome analysis. Seventeen singleton ewes were randomly divided into three groups on day 90 of pregnancy: a control group (CG; 0.63 MJ metabolic energy/body weight (ME/BW)0.75/day, n = 5), maternal undernutrition group 1 (MU1; 0.33 MJ ME/BW0.75/day, n = 6), and maternal undernutrition group 2 (MU2; 0.20 MJ ME/BW0.75/day, n = 6). The fetuses were euthanized and recovered on day 130 of pregnancy. The levels of free fatty acids (FFA) in maternal blood (P < 0.01), fetal blood (P < 0.01), and fetal livers (P < 0.05) were increased in the MU1 and MU2 groups, but fetal hepatic triglyceride (TG) levels in the MU2 group (P < 0.01) and ß-hydroxybutyrate levels in the MU1 and MU2 groups (P < 0.01) were decreased compared to the CG. Severe inflammatory cell infiltration and increased non-alcoholic fatty liver disease activity scores were observed in MU1 and MU2 fetuses (P < 0.01). Progressive deposition of fetal hepatic reticular fibers and collagen fibers in the fetal livers of the MU1 and MU2 groups and significant hepatic fibrosis were observed in the MU2 fetuses (P < 0.05). Gene set enrichment analysis showed that genes involved in lipid accumulation and FFA beta oxidation were downregulated in both MU groups compared to those in the controls. The fetal liver mRNA expression of the ß-oxidation regulator, acetyl-CoA acetyltransferase 1, and the TCA regulator, isocitrate dehydrogenase were reduced in MU1 (P < 0.05) and MU2 (P < 0.01) fetuses, and downregulated mRNA expression of long chain fatty acid CoA ligase 1 (P < 0.05) and glycerol-3-phosphate acyltransferase (P < 0.01) was observed in MU2 fetuses. Differentially expressed genes (DEGs) in MU1 versus CG (360 DEGs) and MU2 versus CG (746 DEGs) were identified using RNA sequencing. Bioinformatics analyses of the 231 intersecting DEGs between MU1 versus CG and MU2 versus CG indicated that neutrophil extracellular traps (NETs) were induced and played a central role in fetal hepatic injury in IUGR sheep. Increased maternal blood myeloperoxidase (MPO) levels (P < 0.01), NE (Elane)-positive areas in fetal liver sections (P < 0.05), and fetal liver MPO protein expression (P < 0.01) were found in the MU1 and MU2 groups; however, MPO levels were reduced in the fetal membrane (P < 0.01) and fetal blood (P < 0.05) in the MU1 group, and in the maternal-fetal placenta and fetal blood in the MU2 group (P < 0.01). Analysis of gene expression trends in the intersecting DEGs between MU1 versus CG (129 DEGs) and MU2 versus CG (515 DEGs) further revealed that 30 hub genes were essential regulators of the G2/M cell cycle, all of which were associated with hepatocellular carcinoma. G0/G1 phase cells of the fetal liver were reduced in the MU1 (P < 0.05) and MU2 (P < 0.01) groups, whereas G2/M phase cells were elevated in the MU1 and MU2 groups (P < 0.01). The representatives of upregulated hub genes and fetal liver protein expression of maternal embryonic leucine zipper kinase and protein regulator of cytokinesis 1 were progressively enhanced in the MU1 and MU2 groups (P < 0.01), and topoisomerase II alpha protein expression in the MU2 group (P < 0.05), as expected. These results indicate that FFA overload, severe lipotoxic injury, and NETs were induced, and disease-promoting regulators of the G2/M cell cycle were upregulated in the fetal liver of IUGR sheep. These findings provide new insights into the pathogenesis of impaired hepatic lipid metabolism and abnormal development and the molecular origin of post-natal liver disease in IUGR due to maternal undernutrition. This information can support the development of new therapeutic strategies.

Resveratrol as modulator of PSA-NCAM expression in the hippocampus of diazinon-injured rat fetuses.

Polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed in the developing central nervous system (CNS) and plays an important role in neurogenesis. Organophosphorus (OP) toxins, including diazinon (DZN), cause oxidative stress (OS) and damage the CNS. Resveratrol (RV), with its antioxidant effect, leads to the reduction of OS. Therefore, this research was conducted with the aim of the effect of RVon the expression of PSA-NCAM in the hippocampus (HPC) of rat fetuses treated with DZN. In this study, 24 female Wistar rats were divided into 4 groups (n = 6): Control, DZN (40 mg/kg), RV(10 mg/kg), and DZN + RV(40 mg/kg + 10 mg/kg) after confirming they were pregnant. On the 21st day of pregnancy, the mother mice were anesthetized with ketamine and xylazine, and the fetuses were removed; after anesthesia, their brains were removed for immunohistochemistry and western blot (WB) technique. The results of the study showed that in the group receiving DZN, the level of PSA-NCAM protein expression decreased significantly compared to the control group, and the group receiving RV with its antioxidant property increased the expression of PSA-NCAM protein compared to the DZN group. All in all, the exposure of pregnant mice to DZN causes disorders in the CNS, especially the level of PSA-NCAM protein expression in the HPC of fetuses, and the use of RV as an antioxidant by pregnant mothers neutralizes the effects of DZN in the HPC of their fetuses.

HES1 is required for mouse fetal hematopoiesis.

BACKGROUND: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. METHODS: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. RESULTS: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. CONCLUSION: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

One-carbon metabolite supplementation increases vitamin B12, folate, and methionine cycle metabolites in beef heifers and fetuses in an energy dependent manner at day 63 of gestation.

One-carbon metabolites (OCM) are metabolites and cofactors which include folate, vitamin B12, methionine, and choline that support methylation reactions. The objectives of this study were to investigate the effects of moderate changes in maternal body weight gain in combination with OCM supplementation during the first 63 d of gestation in beef cattle on (1) B12 and folate concentrations in maternal serum (2) folate cycle intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF) and (3) metabolites involved in one-carbon metabolism and related metabolic pathways in maternal and fetal liver. Heifers were either intake restricted (RES) and fed to lose 0.23 kg/d, or fed to gain 0.60 kg/d (CON). Supplemented (+ OCM) heifers were given B12 and folate injections weekly and fed rumen-protected methionine and choline daily, while non-supplemented (-OCM) heifers were given weekly saline injections. These two treatments were combined in a 2 × 2 factorial arrangement resulting in 4 treatments: CON-OCM, CON + OCM, RES-OCM, and RES + OCM. Samples of maternal serum, maternal and fetal liver, ALF, and AMF were collected at slaughter on day 63 of gestation. Restricted maternal nutrition most notably increased (./ ≤ 0.05) the concentration of vitamin B12 in maternal serum, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate in maternal liver, and cystathionine in the fetal liver; conversely, maternal restriction decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentration in fetal liver. Supplementing OCM increased (P ≤ 0.05) the concentrations of maternal serum B12, folate, and folate intermediates, ALF and AMF 5-methyltetrahydrofolate concentration, and altered (P ≤ 0.02) other maternal liver intermediates including S-adenosylmethionine, dimethylglycine, cystathionine Glutathione reduced, glutathione oxidized, taurine, serine, sarcosine, and pyridoxine. These data demonstrate that OCM supplementation was effective at increasing maternal OCM status. Furthermore, these data are similar to previously published literature where restricted maternal nutrition also affected maternal OCM status. Altering OCM status in both the dam and fetus could impact fetal developmental outcomes and production efficiencies. Lastly, these data demonstrate that fetal metabolite abundance is highly regulated, although the changes required to maintain homeostasis may program altered metabolism postnatally.

Maternal stresses that occur during pregnancy, such as restricted nutrition, can impact the developmental outcomes of the offspring in a process known as developmental programming. This programming can occur through epigenetics, which involves changes in fetal gene expression and can occur through the addition of methyl groups to DNA. These changes regulate gene transcription in the offspring and can alter offspring health, efficiency, and life-long outcomes. One-carbon metabolites (OCM), which are nutrients like the amino acid methionine and the vitamins B12, folate, and choline, act as intermediates or cofactors for the donation of methyl groups to DNA. This study investigated the effects of differing maternal rates of gain along with OCM supplementation during early gestation on OCM and related metabolite concentrations in the dam and fetus. We found that supplementing OCM to beef heifers increased maternal OCM and related metabolite concentrations and fetal fluid OCM concentrations. We also found that low maternal gain increased maternal serum and liver OCM concentrations. We can conclude from these findings that both maternal rate of gain and OCM supplementation can impact maternal OCM concentrations at day 63 of gestation and further research is needed to see if those maternal impacts will affect the developing fetus or calf later in its life.

Developmentally dynamic changes in DNA methylation in the human pancreas.

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.

Expression of transforming growth factor ß signalling molecules and their correlations with genes in loci linked to polycystic ovary syndrome in human foetal and adult tissues.

Context Altered signalling of androgens, anti-Müllerian hormone or transforming growth factor beta (TGFß) during foetal development have been implicated in the predisposition to polycystic ovary syndrome (PCOS) in later life, aside from its genetic predisposition. In foetal ovarian fibroblasts, TGFß1 has been shown to regulate androgen signalling and seven genes located in loci associated with PCOS. Since PCOS exhibits a myriad of symptoms, it likely involves many different organs. Aims To identify the relationships between TGFß signalling molecules and PCOS candidate genes in different tissues associated with PCOS. Methods Using RNA sequencing data, we examined the expression patterns of TGFß signalling molecules in the human ovary, testis, heart, liver, kidney, brain tissue, and cerebellum from 4 to 20weeks of gestation and postnatally. We also examined the correlations between gene expression of TGFß signalling molecules and PCOS candidate genes. Key results TGFß signalling molecules were dynamically expressed in most tissues prenatally and/or postnatally. FBN3 , a PCOS candidate gene involved in TGFß signalling, was expressed during foetal development in all tissues. The PCOS candidate genes HMGA2, YAP1 , and RAD50 correlated significantly (P TGFBR1 in six out of the seven tissues examined. Conclusions This study suggests that possible crosstalk occurs between genes in loci associated with PCOS and TGFß signalling molecules in multiple tissues, particularly during foetal development. Implications Thus, alteration in TGFß signalling during foetal development could affect many tissues contributing to the multiple phenotypes of PCOS in later life.

Fetal Brain-Derived Exosomal miRNAs from Maternal Blood: Potential Diagnostic Biomarkers for Fetal Alcohol Spectrum Disorders (FASDs).

Fetal alcohol spectrum disorders (FASDs) are leading causes of neurodevelopmental disability but cannot be diagnosed early in utero. Because several microRNAs (miRNAs) are implicated in other neurological and neurodevelopmental disorders, the effects of EtOH exposure on the expression of these miRNAs and their target genes and pathways were assessed. In women who drank alcohol (EtOH) during pregnancy and non-drinking controls, matched individually for fetal sex and gestational age, the levels of miRNAs in fetal brain-derived exosomes (FB-Es) isolated from the mothers' serum correlated well with the contents of the corresponding fetal brain tissues obtained after voluntary pregnancy termination. In six EtOH-exposed cases and six matched controls, the levels of fetal brain and maternal serum miRNAs were quantified on the array by qRT-PCR. In FB-Es from 10 EtOH-exposed cases and 10 controls, selected miRNAs were quantified by ddPCR. Protein levels were quantified by ELISA. There were significant EtOH-associated reductions in the expression of several miRNAs, including miR-9 and its downstream neuronal targets BDNF, REST, Synapsin, and Sonic hedgehog. In 20 paired cases, reductions in FB-E miR-9 levels correlated strongly with reductions in fetal eye diameter, a prominent feature of FASDs. Thus, FB-E miR-9 levels might serve as a biomarker to predict FASDs in at-risk fetuses.

Genetic insights into fetal kidney development: Variants in HNF1A and PKHD1 genes.

The orchestration of fetal kidney development involves the precise control of numerous genes, including HNF1A, HNF1B and PKHD1. Understanding the genetic factors influencing fetal kidney development is essential for unraveling the complexities of renal disorders. This study aimed to search for disease-causing variants in HNF1A, HNF1B, PKHD1 genes, among fetus and babies or via parental samples, using sanger sequencing, NGS technologie and MLPA. The study revealed an absence of gene deletions and disease-causing variants in the HNF1B gene. However, five previously SNPs in the HNF1A gene were identified in four patients (patients 1, 2, 3, and 4). These include c.51C > G (Exon1, p. Leu17=), c.79A > C (Exon1, p. Ile27Leu), c.1375C > T (Exon7, p. Leu459=), c.1460G > A (Exon7, p. Ser487Asn), and c.1501 + 7G > A (Intron7). Additionally, in addition to previously SNPs identified, a de novo heterozygous missense mutation (p.E508K) was detected in patient 4. Furthermore, a heterozygous mutation in exon 16 (p. Arg494*; c.1480C > T) was identified in both parents of patient 5, allowing predictions of fetal homozygosity. Bioinformatic analyses predicted the effects of the c.1522G > A mutation (p.E508K) on splicing processes, pre-mRNA structures, and protein instability and conformation. Similarly, the c.1480C > T mutation (p. Arg494*) was predicted to introduce a premature codon stop, leads to the production of a shorter protein with altered or impaired function. Identification of variants in the HNF1A and in PKHD1 genes provides valuable insights into the genetic landscape of renal abnormalities in affected patients. These findings underscore the heterogeneity of genetic variants contributing to renal disorders and emphasize the importance of genetic screening.

Pulmonary maternal immune activation does not cross the placenta but leads to fetal metabolic adaptation.

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.

Comprehensive analysis of the expression profiles of mRNA, lncRNA, circRNA, and miRNA in primary hair follicles of coarse sheep fetal skin.

BACKGROUND: The Qinghai Tibetan sheep, a local breed renowned for its long hair, has experienced significant deterioration in wool characteristics due to the absence of systematic breeding practices. Therefore, it is imperative to investigate the molecular mechanisms underlying follicle development in order to genetically enhance wool-related traits and safeguard the sustainable utilization of valuable germplasm resources. However, our understanding of the regulatory roles played by coding and non-coding RNAs in hair follicle development remains largely elusive. RESULTS: A total of 20,874 mRNAs, 25,831 circRNAs, 4087 lncRNAs, and 794 miRNAs were annotated. Among them, we identified 58 DE lncRNAs, 325 DE circRNAs, 924 DE mRNAs, and 228 DE miRNAs during the development of medullary primary hair follicle development. GO and KEGG functional enrichment analyses revealed that the JAK-STAT, TGF-ß, Hedgehog, PPAR, cGMP-PKG signaling pathway play crucial roles in regulating fibroblast and epithelial development during skin and hair follicle induction. Furthermore, the interactive network analysis additionally identified several crucial mRNA, circRNA, and lncRNA molecules associated with the process of primary hair follicle development. Ultimately, by investigating DEmir's role in the ceRNA regulatory network mechanism, we identified 113 circRNA-miRNA pairs and 14 miRNA-mRNA pairs, including IGF2BP1-miR-23-x-novel-circ-01998-MSTRG.7111.3, DPT-miR-370-y-novel-circ-005802-MSTRG.14857.1 and TSPEAR-oar-miR-370-3p-novel-circ-005802- MSTRG.10527.1. CONCLUSIONS: Our study offers novel insights into the distinct expression patterns of various transcription types during hair follicle morphogenesis, establishing a solid foundation for unraveling the molecular mechanisms that drive hair development and providing a scientific basis for selectively breeding desirable wool-related traits in this specific breed.

Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1.

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, and impairments in social behavior. Patients with the clinically heterogeneous genetic disorder Neurofibromatosis Type 1 (NF1) may present with similar deficits, but it is currently unclear whether environmental factors such as maternal diet influence the development of these phenotypes, and if so, the mechanism by which such an effect would occur. To enable evaluation of how maternal obesogenic diet exposure affects systemic factors relevant to neurodevelopment in NF1, we have developed a method to simultaneously collect non-hemolyzed serum and whole or regionally micro-dissected brains from fetal offspring of murine dams fed a control diet versus a high-fat, high-sucrose diet. Brains were processed for cryosectioning or flash frozen to use for subsequent RNA or protein isolation; the quality of the collected tissue was verified by immunostaining. The quality of the serum was verified by analyzing macronutrient profiles. Using this technique, we have identified that maternal obesogenic diet increases fetal serum cholesterol similarly between WT and Nf1-heterozygous pups.

Intrauterine position effects in a mouse model of maternal immune activation.

Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools in preclinical research of immune-mediated neurodevelopmental disorders and mental illnesses. Using a viral-like MIA model that is based on prenatal poly(I:C) exposure in mice, we have recently identified the existence of subgroups of MIA-exposed offspring that show dissociable behavioral, transcriptional, brain network and inflammatory profiles even under conditions of genetic homogeneity and identical MIA. Here, we tested the hypothesis that the intrauterine positions of fetuses, which are known to shape individual variability in litter-bearing mammals through variations in fetal hormone exposure, may contribute to the variable outcomes of MIA in mice. MIA was induced by maternal administration of poly(I:C) on gestation day 12 in C57BL/6N mice. Determining intrauterine positions using delivery by Cesarean section (C-section), we found that MIA-exposed offspring developing between female fetuses only (0M-MIA offspring) displayed significant deficits in sociability and sensorimotor gating at adult age, whereas MIA-exposed offspring developing between one or two males in utero (1/2M-MIA offspring) did not show the same deficits. These intrauterine position effects similarly emerged in male and female offspring. Furthermore, while MIA elevated fetal brain levels of pro- and anti-inflammatory cytokines independently of the precise intrauterine position and sex of adjacent fetuses during the acute phase, fetal brain levels of TNF-α remained elevated in 0M-MIA but not 1/2M-MIA offspring until the post-acute phase in late gestation. As expected, 1/2M offspring generally showed higher testosterone levels in the fetal brain during late gestation as compared to 0M offspring, confirming the transfer of testosterone from male fetuses to adjacent male or female fetuses. Taken together, our findings identify a novel source of within-litter variability contributing to heterogeneous outcomes of short- and long-term effects in a mouse model of MIA. In broader context, our findings highlight that individual differences in fetal exposure to hormonal and inflammatory signals may be a perinatal factor that shapes risk and resilience to MIA.

HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.