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Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrr gt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect., Lister Institute of Preventative Medicine Newton International Fellowship, Royal Society AG Leventis Fund

Background The biomechanical background of the transitory force decrease following a sudden reduction in the stimulation frequency under selected experimental conditions was studied on fast resistant motor units (MUs) of rat medial gastrocnemius in order to better understand the mechanisms of changes in force transmission. Methods Firstly, MUs were stimulated with three-phase trains of stimuli (low–high–low frequency pattern) to identify patterns when the strongest force decrease (3–36.5%) following the middle high frequency stimulation was observed. Then, in the second part of experiments, the MUs which presented the largest force decrease in the last low-frequency phase were alternatively tested under one of five conditions to analyse the influence of biomechanical factors of the force decrease: (1) determine the influence of muscle stretch on amplitude of the force decrease, (2) determine the numbers of interpulse intervals necessary to evoke the studied phenomenon, (3) study the influence of coactivation of other MUs on the studied force decrease, (4) test the presence of the transitory force decrease at progressive changes in stimulation frequency, (5) and perform mathematical analysis of changes in twitch-shape responses to individual stimuli within a tetanus phase with the studied force decrease. Results Results indicated that (1) the force decrease was highest when the muscle passive stretch was optimal for the MU twitch (100 mN); (2) the middle high-frequency burst of stimuli composed of at least several pulses was able to evoke the force decrease; (3) the force decrease was eliminated by a coactivation of 10% or more MUs in the examined muscle; (4) the transitory force decrease occured also at the progressive decrease in stimulation frequency; and (5) a mathematical decomposition of contractions with the transitory force decrease into twitch-shape responses to individual stimuli revealed that the force decrease in question results from the decrease of twitch forces and a shortening in contraction time whereas further force restitution is related to the prolongation of relaxation. Conclusions High sensitivity to biomechanical conditioning indicates that the transitory force decrease is dependent on disturbances in the force transmission predominantly by collagen surrounding active muscle fibres.

Introduction Throughout pregnancy, the placenta dynamically changes as trophoblast progenitors differentiate into mature trophoblast cell subtypes. This process is in part controlled by epigenetic regulation of DNA methylation leading to the inactivation of ‘progenitor cell’ genes and the activation of ‘differentiation’ genes. TET methylcytosine dioxygenases convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) during DNA demethylation events. Here, we determine the spatiotemporal expression of TET1, TET2, and TET3 in specific trophoblast cell populations of mouse and human placentas throughout gestation, and consider their role in trophoblast cell differentiation and function. Methods In situ hybridization analysis was conducted to localize Tet1, Tet2, and Tet3 mRNA at key stages of mouse placental development. The distribution of 5-mC and 5-hmC in these samples was also evaluated. In comparison, expression patterns of TET1, TET2, and TET3 protein in human placentas were determined in first trimester and term pregnancies. Results In mouse, Tet1-3 mRNA was widely expressed in trophoblast cell populations from embryonic (E) day 8.5 to E12.5 including in progenitor and differentiated cells. However, expression became restricted to specific trophoblast giant cell subtypes by late gestation (E14.5 to E18.5). This coincided with cellular changes in 5-mC and 5-hmC levels. In human, cell columns, extravillous trophoblast and syncytiotrophoblast expressed TET1-3 whereas only TET3 was expressed in villus cytotrophoblast cells in first trimester and term placentas. Discussion Altogether, our data suggest that TET enzymes may play a dynamic role in the regulation of transcriptional activity of trophoblast progenitors and differentiated cell subtypes in mouse and human placentas.

Background and Purpose Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. Experimental Approach The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs+ were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. Key Results HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs+, but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. Conclusions and Implications HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

Sulfate is an important nutrient for fetal growth and development. The fetus has no mechanism for producing its own sulfate and is therefore totally reliant on sulfate from the maternal circulation via placental sulfate transport. To build a model of directional sulfate transport in the placenta, we investigated the relative abundance of the 10 known sulfate transporter mRNAs in human placenta from uncomplicated term pregnancies. SLC13A4 and SLC26A2 were the most abundant sulfate transporter mRNAs, which localized to syncytiotrophoblast and cytotrophoblast cells, respectively. These findings indicate important physiological roles for SLC13A4 and SLC26A2 in human placental sulfate transport.

How maternal diet influences offspring metabolism is unclear, as it is difficult to distinguish between the effects of the in utero environment and epigenetic factors contributed by the oocyte. In a mouse model of high-fat diet, a new study teases apart these mechanisms by using in vitro fertilization and shows that susceptibility of offspring to metabolic disorder can likely be attributed to epigenetic inheritance via the oocyte.

Insights into the anatomical organization of complex neural circuits provide important information about function, and thus tools that facilitate neuroanatomical studies have proved invaluable in neuroscience. Advances in molecular cloning have allowed the production of novel recombinant neuroinvasive viruses for use in transynaptic neural tracing studies. However, the vast majority of these viruses have motility in the retrograde direction only, therefore limiting their use to studies of synaptic input circuitry. Here we describe the construction of an EGFP reporting herpes simplex virus, strain H129, which preferentially moves along synaptically connected neurons in the anterograde direction. In vitro and in vivo characterization studies confirm that the HSV-1 H129-EGFP retains comparable replication and neuroinvasiveness as the wildtype H129 virus. As a proof of principle we confirm anterograde movement of the H129-EGFP along polysynaptic pathways by inoculating the upper airways and tracking time-dependent EGFP expression in previously described ascending sensory pathways. These data confirm a genomic locus for recombining HSV-1 H129 such that normal viral function and replication is maintained. Novel viral recombinants such as HSV-1 H129-EGFP will be useful tools for delineating the central organization of peripheral sensory pathways as well as the synaptic outputs from central neuronal populations.

Nutrient sulfate is important for fetal development. The fetus has a limited capacity to generate sulfate and relies on maternal sulfate supplied via the placenta. The gestational age when fetal sulfate generation begins is unknown but would require cysteine dioxygenase (CDO1) which mediates a major step of sulfate production from cysteine. We investigated the ontogeny of Cdo1 mRNA expression in mouse fetal and placental tissues, which showed increasing levels from embryonic day 10.5 and was localised to the decidua and several fetal tissues including nasal cavities and brain. These findings suggest a role for Cdo1 in sulfate generation from mid-gestation.

Sulfate is important for mammalian growth and development. During pregnancy, maternal circulating sulfate levels increase by 2-fold, enhancing sulfate availability to the fetus. We used quantitative real-time PCR to determine sulfate transporter mRNA levels during mouse gestation in three tissues: kidney and ileum, to identify transporters involved in sulfate absorption and maintaining high maternal circulating sulfate level; and placenta, to build a model of directional sulfate transport from mother to fetus. In the kidney, Slc13a1 and Slc26a1 were the most abundant sulfate transporter mRNAs, which increased by ’2fold at E4.5 or E6.5, whereas lower levels of Slc26a2, Slc26a6, and Slc26a7 mRNA increased by ’3- to 6-fold from E4.5. Ileal sulfate transporter mRNA levels were not increased in gestation, but slight decreases (by ’30–40%) were found for Slc26a3 and Slc26a6. In placentae, Slc13a4 and Slc26a2 mRNAs were most abundant, with levels increasing from E10.5 and peaking (’8fold) from E14.5 to E18.5, whereas Slc26a1 increased by ’3-fold at E18.5. The spatial expression of placental mRNAs was determined by in situ hybridization showing Slc13a4 and Slc26a6 in yolk sac, Slc26a1 in spongiotrophoblasts, and Slc13a4, Slc26a2, Slc26a3, and Slc26a7 in the labyrinthine layer. Within the labyrinth, cell-specific staining revealed Slc13a4 expression in syncytiotrophoblast-II (SynT-II) and Slc26a2 in SynT-I. Together, these data show kidney Slc13a1 and Slc26a1 and placental Slc13a4 and Slc26a2 to be the most abundant sulfate transporter mRNAs in mouse gestation, which likely play important physiological roles in maintaining high maternal serum sulfate levels during pregnancy and mediating sulfate supply to the fetus. placental transport, pregnancy, sulfate transporter

s / Placenta 36 (2015) A1eA60 A5 NI2.2. THE PLACENTAL SULFATE TRANSPORTER, SLC13A4, IS CRITICAL FOR SKELETAL DEVELOPMENT IN MICE. Joanna Rakoczy , Paul Dawson , David Simmons . 1 School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia; Mater Research Institute, Brisbane, Queensland, Australia Objectives: Sulfate is an important nutrient for various cellular and metabolic processes necessary for fetal development. The fetus, having a minimal capacity to produce its own sulfate, is dependent on the mother’s circulating sulfate supply, which is transported to the fetus via placental sulfate transporters. Previously, we discovered that the sulfate transporter Slc13a4 is expressed in both the human and mouse placenta, where it is restricted to the syncytiotrophoblast layer or second layer of syncytiotrophoblast respectively. Since Slc13a4 is situated in the transporting syncytia of both mouse and human placenta, and is not widely expressed in the fetus itself, we chose to investigate its role in placental sulfate transport and fetal development. Methods: We created a mouse line containing an Slc13a4 “knockout first” allele (ES cells obtained from KOMP) to analyze the effects of Slc13a4 deletion. We then converted the knockout first allele into a conditional allele using FLPeR mice to allow for tissue-specific gene deletion. Results: Slc13a4+/crosses yielded no Slc13a4-/mice, indicating that global loss of Slc13a4 is embryonic lethal. While Slc13a4-/placentae appeared normal, by E14.5 Slc13a4-/fetuses exhibited a multitude of developmental phenotypes. One major phenotype was the complete undermineralisation of the skeleton at E16.5. Further analysis revealed that Slc13a4 fetuses have a lack of vascular invasion into the long bones and osteoblast maturation is arrested. Importantly, all these phenotypes were rescued when we used an Slc13a4 conditional knockout mouse approach (crossing with Sox2-Cre), to retain Slc13a4 expression in the placenta, but achieve deletion in the fetus, indicating that the phenotypes observed in the global knockout can be attributed to a loss of placental Slc13a4 expression. Conclusion: Our novel mouse model is providing valuable insight into the critical role of placental sulfate transporters in fetal development. NI2.3. MATERNAL CORTICOSTERONE EXPOSURE IN THE MOUSE CAUSES SEX SPECIFIC ALTERATIONS IN PLACENTAL OGT AND O-LINKED GLYCOSYLATION. James Cuffe, Sarah Steane, Kathryn McMahon, Karen Moritz, Marie Pantaleon. The University of Queensland, Brisbane, Australia Objectives: Maternal stress is known to program adult disease in a sexually dimorphic manner. It is known that placentas of male and female fetuses respond differently to the same maternal challenge rendering male offspring vulnerable to diseases in adulthood. We have previously demonstrated that short term Corticosterone (Cort) exposure (60h) in the mouse induces sex specific placental adaptations and cardiovascular outcomes in offspring. Cellular ability to respond to multiple forms of stress is associated with increased capacity to uptake and metabolise glucose for use in hexosamine signalling (HS) and O-linked glycosylation (O-GlcNAcylation). These pathways are essential for cell survival in response to stress. Interestingly, O-linked-B-acetylglucosamine transferase (OGT) is an X linked gene known to be a placental biomarker of maternal stress. Methods: Using our Cort exposure model, we examined placental levels of key enzymes involved in HS and O-GlcNAcylation. Results: Firstly, we demonstrated that placentas of female fetuses had higher mRNA expression of Ogt than males. Moreover, maternal Cort exposure increased mRNA and protein levels of the stress responsive gene HSP90 in males and females indicating a stress response in both sexes. However Ogt as well as Gftpt1 (rate-limiting regulator of HS) were upregulated to a much greater extent in placentas of female fetuses compared to males. We additionally demonstrated that Cort increased expression of proteins known to be regulated by O-GlcNAcylation. Cort increased the protein expression of the stress responsive pro-survival factor AKT2 in placentas of female but not male fetuses. Conclusion: Our data demonstrates sex-specific alterations in placental HS and OGT levels as well as downstream regulators of placental development in response to Cort. These findings may underlie the sex specific placental adaptations that act to protect the developing female fetus and in turn minimises the incidence of programmed disease outcomes in female offspring. NI2.4. EARLY ALCOHOL EXPOSURE ALTERS PLACENTAL TROPHOBLAST DIFFERENTIATION. Jacinta Kalisch-Smith, Marie Pantaleon, David Simmons, Karen Moritz. The University of Queensland, Brisbane, QLD, Australia Objectives: Maternal periconceptional alcohol (PC-EtOH) exposure causes foetal growth restriction and sex-specific changes to rat placental morphology in late gestation. This may derive from perturbations to preimplantation embryo development and its subsequent capacity to form a placenta. This study aimed to examine cell allocation in the pre-implantation embryo and trophectodermal derivatives after PC-EtOH exposure. Methods: Sprague Dawley dams were administered 12.5% v/v EtOH or control diet from 4 days prior (E-4) to 4 days after conception (E4) in liquid form. Cell number and allocation in the pre-implantation embryo was assessed at E5 and embryos were cultured in vitro for 6 days and trophoblast outgrowth area assessed. To determine whether EtOH can directly affect the differentiation of placental trophoblast stem (TS) cells, one male (RS26) and one female (EGFP) mouse line were cultured in vitro for 6 days (N1⁄43 per treatment) in 0%, 0.2% or 1% EtOH and assayed for lineage-restricted trophoblast subtype markers. RNA was extracted for qPCR and gene expression specific to the labyrinth (Ctsq, Syna) and junctional zones (Tpbpa, Prl7a2, Prl7b1, Prl2c1, Prl3d1) analysed. Results: There were no differences in pre-implantation total cell number, allocation, or outgrowth area (18-51 embryos per treatment) after in vivo PC-EtOH exposure. In vitro EtOH exposure caused dose dependent reductions in the expression of Syna (syncytiotrophoblast layer 1 [SynT-I], P

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.

Loss of the sulfate transporter Slc13a4 in placenta causes severe fetal abnormalities and death in mice