Genetic sex determination of mice by simplex PCR.

BACKGROUND: Investigating fetal development in mice necessitates the determination of fetal sex. However, whilst the sex of adult and juvenile mice can be readily distinguished from anogenital distance, the sex of fetal and neonatal mice cannot be identified visually. Instead, genetic sex must be determined by PCR amplification of X chromosome genes with divergent Y chromosome gametologs. Existing simplex PCR methods are confounded by small size differences between amplicons, amplification of unexpected products, and biased amplification of the shorter amplicon. RESULTS: Primers were designed flanking an 84 bp deletion of the X-linked Rbm31x gene relative to its Y-linked gametolog Rbm31y. A single product was amplified from XX samples, with two products amplified from XY samples. Amplicons were resolved by gel electrophoresis for 20 min, with unbiased amplification of both products observed in XY samples. CONCLUSION: This method achieves rapid and unequivocal genetic sex determination of mice in low volume PCR reactions, reducing reagent usage and simultaneously eliminating shortcomings of previous methods

Characterising the dynamics of placental glycogen stores in the mouse.

INTRODUCTION: The placenta performs a range of functions to support fetal growth. In addition to facilitating nutrient transport, the placenta also stores glucose as glycogen, which is thought to maintain fetal glucose supply during late gestation. However, evidence to support such a role is currently lacking. Similarly, our understanding of the dynamics of placental glycogen metabolism in normal mouse pregnancy is limited. METHODS: We quantified the placental glycogen content of wild type C57BL/6JOlaHsd mouse placentas from mid (E12.5) to late (E18.5) gestation, alongside characterising the temporal expression pattern of genes encoding glycogenesis and glycogenolysis pathway enzymes. To assess the potential of the placenta to produce glucose, we investigated the spatiotemporal expression of glucose 6-phosphatase by qPCR and in situ hybridisation. Separate analyses were undertaken for placentas of male and female conceptuses to account for potential sexual dimorphism. RESULTS: Placental glycogen stores peak at E15.5, having increased over 5-fold from E12.5, before declining by a similar extent by E18.5. Glycogen stores were 17% higher in male placentas than in females at E15.5. Expression of glycogen branching enzyme (Gbe1) was reduced ~40% towards term. Expression of the glucose 6-phosphatase isoform G6pc3 was enriched in glycogen trophoblast cells and increased towards term. DISCUSSION: Reduced expression of Gbe1 suggests a decline in glycogen branching towards term. Expression of G6pc3 by glycogen trophoblasts is consistent with an ability to produce and release glucose from glycogen stores. However, the ultimate destination of the glucose generated from placental glycogen remains to be elucidated.Centre for Trophoblast Researc

BACs as tools for the study of genomic imprinting.

Genomic imprinting in mammals results in the expression of genes from only one parental allele. Imprinting occurs as a consequence of epigenetic marks set down either in the father's or the mother's germ line and affects a very specific category of mammalian gene. A greater understanding of this distinctive phenomenon can be gained from studies using large genomic clones, called bacterial artificial chromosomes (BACs). Here, we review the important applications of BACs to imprinting research, covering physical mapping studies and the use of BACs as transgenes in mice to study gene expression patterns, to identify imprinting centres, and to isolate the consequences of altered gene dosage. We also highlight the significant and unique advantages that rapid BAC engineering brings to genomic imprinting research

Excess Phlda2 as a mouse model of intrauterine growth restriction

A small fraction of mammalian genes exhibit parent-of-origin specific monoallelic expression. They are expressed from only one allele and this is determined by modifications established in the germline. Approximately 100 imprinted genes have been identified to date. Most imprinted genes are located in discrete clusters and are controlled by shared regulatory elements. Imprinted genes play important roles in regulating embryonic and placental development, with overt growth phenotypes resulting both from loss of expression and from over-expression of imprinted genes. The maternally expressed Phlda2 gene has been implicated in placental development. Loss of expression leads to placentomegaly as a consequence of the disproportionate expansion of the spongiotrophoblast layer. In this study, the consequences of over-expressing Phlda2 and the adjacent Slc22a18 were investigated in four independent lines of transgenic mice driving incrementally increasing doses of the two genes and on two genetic backgrounds. In all cases, transgenic placentae were significantly lighter throughout gestation, which was entirely due to a reduction in the spongiotrophoblast layer. There was also a reduction in glycogen staining and a progressive mislocalisation of cells from the spongiotrophoblast layer. These phenotypes were essentially restored by restored by normalising Phlda2 gene dosage in a single copy line. In addition, transgenic embryos were significantly lighter than wild type littermates from E16.5 onwards and were born 13% lighter. These embryos were asymmetrically growth restricted and displayed rapid post-natal catch up growth within two weeks of birth. Adult transgenic females that had undergone embryonic growth restriction also displayed increased adiposity and reduced glucose tolerance at one year of age. These data suggest that altered expression of Phlda2 and possibly Slc22a18 drive IUGR and program adult disease susceptibility. Recent human studies have found an association between elevated placental PHLDA2 and low birth weight or IUGR infants. This mouse model may thus provide a genetic tool that recapitulates a known human condition for further investigation of the fetal programming of metabolic syndrome.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Increased dosage of the imprinted Ascl2 gene restrains two key endocrine lineages of the mouse Placenta

AbstractImprinted genes are expressed primarily from one parental allele by virtue of a germ line epigenetic process. Achaete-scute complex homolog 2 (Ascl2 aka Mash2) is a maternally expressed imprinted gene that plays a key role in placental and intestinal development. Loss-of-function of Ascl2 results in an expansion of the parietal trophoblast giant cell (P-TGC) lineage, an almost complete loss of Trophoblast specific protein alpha (Tpbpa) positive cells in the ectoplacental cone and embryonic failure by E10.5. Tpbpa expression marks the progenitors of some P-TGCs, two additional trophoblast giant cell lineages (spiral artery and canal), the spongiotrophoblast and the glycogen cell lineage. Using a transgenic model, here we show that elevated expression of Ascl2 reduced the number of P-TGC cells by 40%. Elevated Ascl2 also resulted in a marked loss of the spongiotrophoblast and a substantial mislocalisation of glycogen cells into the labyrinth. In addition, Ascl2-Tg placenta contained considerably more placental glycogen than wild type. Glycogen cells are normally located within the junctional zone in close contact with spongiotrophoblast cells, before migrating through the P-TGC layer into the maternal decidua late in gestation where their stores of glycogen are released. The failure of glycogen cells to release their stores of glycogen may explain both the inappropriate accumulation of glycogen and fetal growth restriction observed late in gestation in this model. In addition, using in a genetic cross we provide evidence that Ascl2 requires the activity of a second maternally expressed imprinted gene, Pleckstrin homology-like domain, family a, member 2 (Phlda2) to limit the expansion of the spongiotrophoblast. This “belts and braces” approach demonstrates the importance of genomic imprinting in regulating the size of the placental endocrine compartment for optimal placental development and fetal growth

Placental glycogen stores and fetal growth: insights from genetic mouse models

The placenta performs a range of crucial functions that support fetal growth during pregnancy, including facilitating the supply of nutrients and gases to the fetus, removal of waste products from the fetus, and the endocrine modulation of maternal physiology. The placenta also stores glucose in the form of glycogen, the function of which remains unknown. Aberrant placental glycogen storage in humans is associated with maternal diabetes during pregnancy and pre-eclampsia, thus linking placental glycogen storage and metabolism to pathological pregnancies. To understand the role of placental glycogen in normal and complicated pregnancies, we must turn to animal models. Over 40 targeted mutations in mice demonstrate defects in placental cells that store glycogen and suggest that placental glycogen represents a source of readily mobilised glucose required during periods of high fetal demand. However, direct functional evidence is currently lacking. Here, we evaluate these genetic mouse models with placental phenotypes that implicate glycogen trophoblast cell differentiation and function to illuminate the common molecular pathways that emerge and to better understand the relationship between placental glycogen and fetal growth. We highlight current limitations to exploring key questions regarding placental glycogen storage and metabolism and define how to experimentally overcome these constraints.Centre for Trophoblast Research Next Generation Fellowshi

Excess Phlda2 as a mouse model of intrauterine growth restriction

A small fraction of mammalian genes exhibit parent-of-origin specific monoallelic expression. They are expressed from only one allele and this is determined by modifications established in the germline. Approximately 100 imprinted genes have been identified to date. Most imprinted genes are located in discrete clusters and are controlled by shared regulatory elements. Imprinted genes play important roles in regulating embryonic and placental development, with overt growth phenotypes resulting both from loss of expression and from over-expression of imprinted genes. The maternally expressed Phlda2 gene has been implicated in placental development. Loss of expression leads to placentomegaly as a consequence of the disproportionate expansion of the spongiotrophoblast layer. In this study, the consequences of over-expressing Phlda2 and the adjacent Slc22a18 were investigated in four independent lines of transgenic mice driving incrementally increasing doses of the two genes and on two genetic backgrounds. In all cases, transgenic placentae were significantly lighter throughout gestation, which was entirely due to a reduction in the spongiotrophoblast layer. There was also a reduction in glycogen staining and a progressive mislocalisation of cells from the spongiotrophoblast layer. These phenotypes were essentially restored by restored by normalising Phlda2 gene dosage in a single copy line. In addition, transgenic embryos were significantly lighter than wild type littermates from E16.5 onwards and were born 13% lighter. These embryos were asymmetrically growth restricted and displayed rapid post-natal catch up growth within two weeks of birth. Adult transgenic females that had undergone embryonic growth restriction also displayed increased adiposity and reduced glucose tolerance at one year of age. These data suggest that altered expression of Phlda2 and possibly Slc22a18 drive IUGR and program adult disease susceptibility. Recent human studies have found an association between elevated placental PHLDA2 and low birth weight or IUGR infants. This mouse model may thus provide a genetic tool that recapitulates a known human condition for further investigation of the fetal programming of metabolic syndrome

Placental PHLDA2 expression is increased in cases of fetal growth restriction following reduced fetal movements

Background Maternal perception of reduced fetal movements (RFM) is associated with increased risk of fetal growth restriction (FGR) and stillbirth, mediated by placental insufficiency. The maternally expressed imprinted gene PHLDA2 controls fetal growth, placental development and placental lactogen production in a mouse model. A number of studies have also demonstrated abnormally elevated placental PHLDA2 expression in human growth restricted pregnancies. This study examined whether PHLDA2 was aberrantly expressed in placentas of RFM pregnancies resulting in delivery of an FGR infant and explored a possible relationship between PHLDA2 expression and placental lactogen release from the human placenta. Methods Villous trophoblast samples were obtained from a cohort of women reporting RFM (N = 109) and PHLDA2 gene expression analysed. hPL levels were assayed in the maternal serum (N = 74). Results Placental PHLDA2 expression was significantly 2.3 fold higher in RFM pregnancies resulting in delivery of an infant with FGR (p < 0.01), with highest levels of PHLDA2 expression in the most severe cases. Placental PHLDA2 expression was associated with maternal serum hPL levels (r = −0.30, p = 0.008, n = 74) although this failed to reach statistical significance in multiple linear regression analysis controlling for birth weight (p = 0.07). Conclusions These results further highlight a role for placental PHLDA2 in poor perinatal outcomes, specifically FGR associated with RFM. Furthermore, this study suggests a potential relationship between placental PHLDA2 expression and hPL production by the placenta, an association that requires further investigation in a larger cohort

The imprinted Phlda2 gene modulates a major endocrine compartment of the placenta to regulate placental demands for maternal resources.

Imprinted genes, which are expressed from a single parental allele in response to epigenetic marks first established in the germline, function in a myriad of processes to regulate mammalian development. Recent work suggests that imprinted genes may regulate the signalling function of the placenta by modulating the size of the endocrine compartment. Here we provide in vivo evidence that this hypothesis is well founded. Elevated expression of the imprinted Pleckstrin homology-like domain, family a, member 2 (Phlda2) gene drives a reduction of the spongiotrophoblast endocrine compartment, diminished placental glycogen and asymmetric foetal growth restriction. Using both loss-of-function and gain-in-expression mouse models, here we further show that Phlda2 exclusively modulates the spongiotrophoblast compartment of the placenta without significantly altering the composition of the trophoblast giant cell endocrine lineages that share a common progenitor with this lineage. Additionally, we show that Phlda2 loss-of-function placentae contain nearly three times more placental glycogen than non-transgenic placentae. Remarkably, relative to a fully wild type scenario, wild type placentae also accumulate excessive glycogen. While loss-of-function of Phlda2 increased both placental weight and placental glycogen, the weight of both mutant and non-transgenic fetuses was lower than that found in a fully wild type scenario indicating that excessive glycogen accumulation comes at the cost of foetal growth. This work firstly highlights a novel signalling function for the spongiotrophoblast in stimulating the global accumulation of placental glycogen. Furthermore, this work suggests that Phlda2 manipulates the placenta's demands for maternal resources, a process that must be tightly regulated by epigenetic marks to ensure optimal foetal growth