MicroRNAs-140-5p/140-3p modulate Leydig cell numbers in the developing mouse testis.

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.

Folate-depletion alters mouse trophoblast stem cell regulation in vitro.

Maternal folate deficiency increases risk of congenital malformations, yet its effect on placenta development is unclear. Here, we investigated how folate-depleted culture medium affects the developmental potential of mouse trophoblast stem cells (TSCs). When cultured in stem cell conditions, TSC viability was unaffected by folate depletion, but ectopic differentiation of trophoblast cell subtypes occurred. When cultured in conditions that promote differentiation, folate-depleted TSCs were driven towards a syncytiotrophoblast cell fate potentially at the expense of other lineages. Additionally, trophoblast giant cell nuclei were small implicating folate in the regulation of endoreduplication. Therefore, dietary folate intake likely promotes trophoblast development.

Placental, renal, and ileal sulfate transporter gene expression in mouse 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 ≈2-fold 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 (≈8-fold) 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.

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations.

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.

Biomechanical conditioning of the motor unit transitory force decrease following a reduction in stimulation rate.

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.

Dynamic expression of TET1, TET2, and TET3 dioxygenases in mouse and human placentas throughout gestation.

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.

Multigenerational analysis of sex-specific phenotypic differences at midgestation caused by abnormal folate metabolism.

The exposure to adverse environmental conditions (e.g. poor nutrition) may lead to increased disease risk in an individual and their descendants. In some cases, the results may be sexually dimorphic. A range of phenotypes has been associated with deficiency in or defective metabolism of the vitamin folate. However, the molecular mechanism linking folate metabolism to development is still not well defined nor is it clear whether phenotypes are sex-specific. The enzyme methionine synthase reductase (MTRR) is required for the progression of folate metabolism and the utilization of methyl groups from the folate cycle. Previously, we showed that the hypomorphic Mtrrgt mutation in mice results in metabolic disruption, epigenetic instability, and a wide spectrum of developmental phenotypes (e.g. growth defects, congenital malformations) at midgestation that appear in subsequent wild-type generations. This transgenerational effect only occurs through the maternal lineage. Here, we explore whether the phenotypes that result from either intrinsic or ancestral Mtrr deficiency are sexually dimorphic. We found that no sexual dimorphism is apparent in either situation when the phenotypes were broadly or specifically defined. However, when we focused on the group of phenotypically normal conceptuses derived from maternal grandparental Mtrr deficiency, we observed an apparent increase in placental efficiency in each subsequent generation leading to F4 generation female embryos that weigh more than controls. These data suggest that ancestral abnormal folate metabolism may lead to male grandprogeny that are less able to adapt or female grandprogeny that are programmed to become more sensitive to folate availability in subsequent generations.

Fat eggs shape offspring health.

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.

The effect of hyperpolarization-activated cyclic nucleotide-gated ion channel inhibitors on the vagal control of guinea pig airway smooth muscle tone.

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.

Anterograde neuronal circuit tracing using a genetically modified herpes simplex virus expressing EGFP.

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.