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

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

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

Placental expression of imprinted genes varies with sampling site and mode of delivery

Imprinted genes, which are monoallelically expressed by virtue of an epigenetic process initiated in the germline, are known to play key roles in regulating fetal growth and placental development. Numerous studies are investigating the expression of these imprinted genes in the human placenta in relation to common complications of pregnancy such as fetal growth restriction and preeclampsia. This study aimed to determine whether placental sampling protocols or other factors such as fetal sex, gestational age and mode of delivery may influence the expression of imprinted genes predicted to regulate placental signalling. Methods. Term placentas were collected from Caucasian women delivering at University Hospital of Wales or Royal Gwent Hospital within two hours of delivery. Expression of the imprinted genes PHLDA2, CDKN1C, PEG3 and PEG10 was assayed by quantitative real time PCR. Intraplacental gene expression was analysed (N = 5). Placental gene expression was compared between male (N = 11) and female (N = 11) infants, early term (N = 8) and late term (N = 10) deliveries and between labouring (N = 13) and non-labouring (N = 21) participants. Results. The paternally expressed imprinted genes PEG3 and PEG10 were resilient to differences in sampling site, fetal sex, term gestational age and mode of delivery. The maternally expressed imprinted gene CDKN1C was elevated over 2-fold (p < 0.001) in placenta from labouring deliveries compared with elective caesarean sections. In addition, the maternally expressed imprinted gene PHLDA2 was elevated by 1.8 fold (p = 0.01) in samples taken at the distal edge of the placenta compared to the cord insertion site. Conclusion. These findings support the reinterpretation of existing data sets on these genes in relation to complications of pregnancy and further reinforce the importance of optimising and unifying placental collection protocols for future studies

Blastocyst transfer in mice alters the placental transcriptome and growth.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.This work was supported by grants from the Centre for Trophoblast Research (CTR) (to EDW), Lister Institute for Preventative Medicine (to EDW), and Canadian Institutes for Health Research (to JCC). KM was funded by a Newnham College (Cambridge) studentship and A.G. Leventis scholarship. SJT was funded by a Next Generation Fellowship (CTR). GETB was funded by a Wellcome Trust PhD studentship in Developmental Mechanisms. RSH and MP were funded by the CTR

Imprinted genes in mouse placental development and the regulation of fetal energy stores

Imprinted genes, which are preferentially expressed from one or other parental chromosome as a consequence of epigenetic events in the germline, are known to functionally converge on biological processes that enable in utero development in mammals. Over 100 imprinted genes have been identified in the mouse, the majority of which are both expressed and imprinted in the placenta. The purpose of this review is to provide a summary of the current knowledge regarding imprinted gene function in the mouse placenta. Few imprinted genes have been assessed with respect to their dosage-related action in the placenta. Nonetheless, current data indicate that imprinted genes converge on two key functions of the placenta, nutrient transport and placental signalling. Murine studies may provide a greater understanding of certain human pathologies, including low birth weight and the programming of metabolic diseases in the adult, and complications of pregnancy, such as pre-eclampsia and gestational diabetes, resulting from fetuses carrying abnormal imprints

The significance of elevated placental PHLDA2 in human growth restricted pregnancies

In utero growth restriction is the failure of a fetus to achieve its genetic growth potential during gestation. Elevated expression of the maternally expressed imprinted gene PHLDA2, has been reported in the human placenta of growth restricted pregnancies. A mouse modelling this alteration has been generated and also displays fetal growth restriction suggesting that increased expression of PHLDA2 is not an effect but rather a cause of growth restriction in human pregnancies. Here we review the current data linking PHLDA2 to growth restriction and the data from human and animal model studies suggesting that placental PHLDA2 expression may be responsive to environmental stimuli such as maternal lifestyle. Further investigation is warranted in larger studies of human placentas with the aim of determining whether placental PHLDA2 expression could be used as a diagnostic tool to identify or sub-classify growth restricted infants and to inform more effective interventions and treatment for IUGR in the future

Impact of genetic background on placental glycogen storage in mice.

129 and C57BL/6 are two of the most widely used laboratory mouse strains. While it is well known that genetic modifiers between the two strains can directly influence embryonic and adult phenotypes, less is known regarding morphological differences in placental development. Here we identify differences in the junctional zone, glycogenstorage and the maternal-fetal interface between these two strains and provide examples where these differences impact the phenotypic characterisation of placental mutations