Sex differences in rat placental development: from pre-implantation to late gestation.

BACKGROUND: A male fetus is suggested to be more susceptible to in utero and birth complications. This may be due in part to altered morphology or function of the XY placenta. We hypothesised that sexual dimorphism begins at the blastocyst stage with sex differences in the progenitor trophectoderm (TE) and its derived trophoblast lineages, as these cells populate the majority of cell types within the placenta. We investigated sex-specific differences in cell allocation in the pre-implantation embryo and further characterised growth and gene expression of the placental compartments from the early stages of the definitive placenta through to late gestation. METHODS: Naturally mated Sprague Dawley dams were used to collect blastocysts at embryonic day (E) 5 to characterise cell allocation; total, TE, and inner cell mass (ICM), and differentiation to downstream trophoblast cell types. Placental tissues were collected at E13, E15, and E20 to characterise volumes of placental compartments, and sex-specific gene expression profiles. RESULTS: Pre-implantation embryos showed no sex differences in cell allocation (total, TE and ICM) or early trophoblast differentiation, assessed by outgrowth area, number and ploidy of trophoblasts and P-TGCs, and expression of markers of trophoblast stem cell state or differentiation. Whilst no changes in placental structures were found in the immature E13 placenta, the definitive E15 placenta from female fetuses had reduced labyrinthine volume, fetal and maternal blood space volume, as well as fetal blood space surface area, when compared to placentas from males. No differences between the sexes in labyrinth trophoblast volume or interhaemal membrane thickness were found. By E20 these sex-specific placental differences were no longer present, but female fetuses weighed less than their male counterparts. Coupled with expression profiles from E13 and E15 placental samples may suggest a developmental delay in placental differentiation. CONCLUSIONS: Although there were no overt differences in blastocyst cell number or early placental development, reduced growth of the female labyrinth in mid gestation is likely to contribute to lower fetal weight in females at E20. These data suggest sex differences in fetal growth trajectories are due at least in part, to differences in placenta growth.

Review: Nutrient sulfate supply from mother to fetus: Placental adaptive responses during human and animal gestation.

Nutrient sulfate has numerous roles in mammalian physiology and is essential for healthy fetal growth and development. The fetus has limited capacity to generate sulfate and relies on sulfate supplied from the maternal circulation via placental sulfate transporters. The placenta also has a high sulfate requirement for numerous molecular and cellular functions, including sulfate conjugation (sulfonation) to estrogen and thyroid hormone which leads to their inactivation. Accordingly, the ratio of sulfonated (inactive) to unconjugated (active) hormones modulates endocrine function in fetal, placental and maternal tissues. During pregnancy, there is a marked increase in the expression of genes involved in transport and generation of sulfate in the mouse placenta, in line with increasing fetal and placental demands for sulfate. The maternal circulation also provides a vital reservoir of sulfate for the placenta and fetus, with maternal circulating sulfate levels increasing by 2-fold from mid-gestation. However, despite evidence from animal studies showing the requirement of maternal sulfate supply for placental and fetal physiology, there are no routine clinical measurements of sulfate or consideration of dietary sulfate intake in pregnant women. This is also relevant to certain xenobiotics or pharmacological drugs which when taken by the mother use significant quantities of circulating sulfate for detoxification and clearance, and thereby have the potential to decrease sulfonation capacity in the placenta and fetus. This article will review the physiological adaptations of the placenta for maintaining sulfate homeostasis in the fetus and placenta, with a focus on pathophysiological outcomes in animal models of disturbed sulfate homeostasis.

Review: Sexual dimorphism in the formation, function and adaptation of the placenta.

Exposure of the embryo or fetus to perturbations in utero can result in intrauterine growth restriction, a primary risk factor for the development of adult disease. However, despite similar exposures, males and females often have altered disease susceptibility or progression from different stages of life. Fetal growth is largely mediated by the placenta, which, like the fetus is genetically XX or XY. The placenta and its associated trophoblast lineages originate from the trophectoderm (TE) of the early embryo. Rodent models (rat, mouse, spiny mouse), have been used extensively to examine placenta development and these have demonstrated the growth trajectory of the placenta in females is generally slower compared to males, and also shows altered adaptive responses to stressful environments. These placental adaptations are likely to depend on the type of stressor, duration, severity and the window of exposure during development. Here we describe the divergent developmental pathways between the male and female placenta contributing to altered differentiation of the TE derived trophoblast subtypes, placental growth, and formation of the placental architecture. Our focus is primarily genetic or environmental perturbations in rodent models which show altered placental responsiveness between sexes. We suggest that perturbations during early placental development may have greater impact on viability and growth of the female fetus whilst those occurring later in gestation may preferentially affect the male fetus. This may be of great relevance to human pregnancies which result from assisted reproductive technologies or complications such as pre-eclampsia and diabetes.

Loss of Rearranged L-Myc Fusion (RLF) results in defects in heart development in the mouse.

Recently we reported that Rearranged L-Myc Fusion, RLF, acts as an epigenetic modifier maintaining low levels of DNA methylation at CpG island shores and enhancers across the genome. Here we focus on the phenotype of Rlf null mutant mice generated via an ENU mutagenesis screen, to identify genes required for epigenetic regulation. RLF is expressed in a range of fetal mouse tissues, including the fetal heart. Comprehensive timed-mating studies are consistent with our previously reported findings that Rlf homozygous mutant mice rarely survive to adulthood, with the majority dying shortly after birth. Histological analysis of two independent Rlf ENU mutant lines at E11.5-E14.5 showed heart defects resembling those present in humans with Left Ventricular Non-Compaction (LVNC). In situ hybridisation analysis localized expression of Rlf to the endocardium and epicardium of embryonic and postnatal hearts, and transiently to cardiomyocytes during heart looping and early chamber formation stages. RNA-seq analysis of Rlf mutant hearts highlighted defective NOTCH pathway signalling, recently describe as one cause of LVNC. This study provides the first evidence that RLF is required for normal heart development in the mouse. The heart morphological defects present at high penetrance in Rlf mutants are consistent with features of LVNC in humans, and molecular analysis identified attenuated JAGGED 1 expression and NOTCH signalling as likely contributors to these defects. Our study highlights the importance of RLF-dependent epigenetic modifications to DNA for maintaining correct gene regulatory network and intercellular signalling interactions during heart chamber and septal development. Further investigations are needed to define the biochemical role of RLF in the developing heart, and whether RLF mutations are a cause of heart defects in humans.

Alcohol exposure impairs trophoblast survival and alters subtype-specific gene expression in vitro.

Maternal alcohol consumption is common prior to pregnancy recognition and in the rat results in altered placental development and fetal growth restriction. To assess the effect of ethanol (EtOH) exposure on the differentiation of trophoblast stem (TS) cells, mouse TS lines were differentiated in vitro for 6 days in 0%, 0.2% or 1% EtOH. This reduced both trophoblast survival and expression of labyrinth and junctional zone trophoblast subtype-specific genes. This suggests that fetal growth restriction and altered placental development associated with maternal alcohol consumption in the periconceptional period could be mediated in part by direct effects on trophoblast development.

Genetic ablation of placental sinusoidal trophoblast giant cells causes fetal growth restriction and embryonic lethality.

A specialized subtype of trophoblast giant cells (TGCs) line the torturous sinusoids of the murine placental labyrinth, and can be distinguished from most other TGCs by the expression of Ctsq. We generated a transgenic mouse line expressing Cre recombinase from the Ctsq promoter. Crosses with Cre-inducible tdTomato reporter mice indicated Cre activity was restricted to the sinusoidal TGCs of the labyrinth, as well as the recently characterized channel TGCs. When crossed with Cre-inducible DTA transgenic mice, ablation of sinusoidal TGCs was achieved in double transgenic embryos, resulting in fetal growth restriction by E16.5, and embryonic lethality by term.

Placental and fetal cysteine dioxygenase gene expression in mouse gestation.

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.

Expression of aldehyde dehydrogenase family 1, member A3 in glycogen trophoblast cells of the murine placenta.

INTRODUCTION: Retinoic acid (RA) signaling is a well known regulator of trophoblast differentiation and placental development, and maternal decidual cells are recognized as the source of much of this RA. We explored possible trophoblast-derived sources of RA by examining the expression of RA synthesis enzymes in the developing mouse placenta, as well as addressed potential sites of RA action by examining the ontogeny of gene expression for other RA metabolizing and receptor genes. Furthermore, we investigated the effects of endogenous RA production on trophoblast differentiation. METHODS: Placental tissues were examined by in situ hybridization and assayed for RARE-LacZ transgene activity to locate sites of RAR signaling. Trophoblast stem cell cultures were differentiated in the presence of ALDH1 inhibitors (DEAB and citral), and expression of labyrinth (Syna, Ctsq) and junctional zone (Tpbpa, Prl7b1, Prl7a2) marker genes were analyzed by qRT-PCR. RESULTS: We show Aldh1a3 is strongly expressed in a subset of ectoplacental cone cells and in glycogen trophoblast cells of the definitive murine placenta. Most trophoblast subtypes of the placenta express RA receptor combinations that would enable them to respond to RA signaling. Furthermore, expression of junctional zone markers decrease in differentiating trophoblast cultures when endogenous ALDH1 enzymes are inhibited. DISCUSSION: Aldh1a3 is a novel marker for glycogen trophoblast cells and their precursors and may play a role in the differentiation of junctional zone cell types via production of a local source of RA.

The effects of gestational age and maternal hypoxia on the placental renin angiotensin system in the mouse.

INTRODUCTION: The renin angiotensin system (RAS) is an important mediator of placental development. However, a comprehensive expression profile for 8 key components of the placental RAS throughout murine gestation has not been performed. Furthermore, maternal hypoxia induces dysregulation of RAS expression in fetal tissues but the effects on the murine placental RAS are less well known. METHODS: Placentas were collected from male and female CD1 mouse fetuses at seven gestational ages for qPCR analysis of Agt, Ren1, Atp6ap2, Ace, Ace2, Agtr1a, Agtr2 and Mas1. mRNA localisation of Agtr1 and Mas1 and protein localisation of ACE and ACE2 was determined at E18.5. To determine the effects of maternal hypoxia on the placental RAS, mice were housed in 12% oxygen from E14.5-E18.5 and placentas examined at E18.5. RESULTS: All RAS genes were expressed in the placenta throughout pregnancy and expression varied with fetal sex and age. Agtr1 was expressed within the labyrinth while Mas1 was expressed within the intraplacental yolk sac. ACE and ACE2 were localised to both labyrinth and junctional zones. In response to maternal hypoxia the expression of Agt, Ace and Ace2 was decreased but expression of Agtr1a was increased. Ace and Agtr1a mRNA levels were affected to a greater extent in females compared to males. DISCUSSION: Collectively, the location within the placenta as well as the expression profiles identified, support a role for the placental RAS in labyrinth development. The placental RAS is disturbed by maternal hypoxia in a sexually dimorphic manner and may contribute to impairment of placental vascular development.

Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta.

In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6E⁺ cells, which we found to express Syna at a much higher level than did LY6E⁻ cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type.

Human placental sulfate transporter mRNA profiling from term pregnancies identifies abundant SLC13A4 in syncytiotrophoblasts and SLC26A2 in cytotrophoblasts.

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.

Molecular analysis of the human SLC13A4 sulfate transporter gene promoter.

The human solute linked carrier (SLC) 13A4 gene is primarily expressed in the placenta where it is proposed to mediate the transport of nutrient sulfate from mother to fetus. The molecular mechanisms involved in the regulation of SLC13A4 expression remain unknown. To investigate the regulation of SLC13A4 gene expression, we analysed the transcriptional activity of the human SLC13A4 5'-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Basal transcriptional activity was identified in the region -57 to -192 nucleotides upstream of the SLC13A4 transcription initiation site. Mutational analysis of the minimal promoter region identified Nuclear factor Y (NFY), Specificity protein 1 (SP1) and Krüppel like factor 7 (KLF7) motifs which conferred positive transcriptional activity, as well as Zinc finger protein of the cerebellum 2 (ZIC2) and helix-loop-helix protein 1 (HEN1) motifs that repressed transcription. The conserved NFY, SP1, KLF7, ZIC2 and HEN1 motifs in the SLC13A4 promoter of placental species but not in non-placental species, suggests a potential role for these putative transcriptional factor binding motifs in the physiological control of SLC13A4 mRNA expression.

Maternal corticosterone exposure in the mouse has sex-specific effects on placental growth and mRNA expression.

Maternal exposure to increased synthetic glucocorticoids (GC) during pregnancy is known to disturb fetal development and increase the risk of long-term disease. Maternal exposure to elevated levels of natural GC is likely to be common yet is relatively understudied. The placenta plays an important role in regulating fetal exposure to maternal GC but is itself vulnerable to maternal insults. This study uses a mouse model of maternal corticosterone (Cort) exposure to investigate its effects on the developing placenta. Mice were treated with Cort (33 µg/kg·h) for 60 h starting at embryonic d 12.5 (E12.5) before collection of placentas at E14.5 and E17.5. Although Cort exposure did not affect fetal size, placentas of male fetuses were larger at E17.5 in association with changes in placental Igf2. This increase in size was associated with an increase in placental thickness and an increase in placental junctional zone volume. Placentas from female fetuses were of normal size and had no changes in growth factor mRNA levels. The expression of the protective enzyme 11ß-hydroxysteroid dehydrogenase type 2 was increased at E14.5 but was decreased in males at E17.5. In contrast, the expression of Nr3c1 (which encodes the GC receptor) was increased during the Cort exposure and remained elevated at E17.5 in the placentas of male fetuses. Our study has shown that maternal Cort exposure infers a sex-specific alteration to normal placental growth and growth factor expression, thus further adding to our understanding of the mechanisms of male dominance of programmed disease.

Crim1 has an essential role in glycogen trophoblast cell and sinusoidal-trophoblast giant cell development in the placenta.

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1(KST264), we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1(KST264/KST264) mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1(KST264) allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1(KST264/KST264) placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Sex specific changes in placental growth and MAPK following short term maternal dexamethasone exposure in the mouse.

OBJECTIVES: Maternal glucocorticoid (GC) exposure during pregnancy can alter fetal development and program the onset of disease in adult offspring. The placenta helps protect the fetus from excess GC exposure but is itself susceptible to maternal insults and may be involved in sex dependant regulation of fetal programming. This study aimed to investigate the effects of maternal GC exposure on the developing placenta. STUDY DESIGN AND MAIN OUTCOME MEASURES: Pregnant mice were treated with dexamethasone (DEX-1 µg/kg/h) or saline (SAL) for 60 h via minipump beginning at E12.5. Placentas were collected at E14.5 and E17.5 and the expression of growth factors and placental transporters examined by real-time PCR and/or Western blot. Histological analysis was performed to assess for morphological changes. RESULTS: At E14.5, DEX exposed male and female fetuses had a lower weight compared to SAL animals but placental weight was lower in females only. Hsd11b2 and Vegfa gene expression was increased and MAPK1 protein expression decreased in the placentas of females only. At E17.5 placental and fetal body weights were similar and differences in MAPK were no longer present although HSD11B2 protein was elevated in placentas of DEX females. Levels of glucose or amino acid transporters were unaffected. CONCLUSIONS: Results suggest sex specific responses to maternal GCs within the placenta. Decreased levels of MAPK protein in placentas of female fetuses suggest alterations in the MAPK pathway may contribute to the lower placental weights in this sex. This may contribute towards sex specific fetal programming of adult disease.

Review: Sex specific programming: a critical role for the renal renin-angiotensin system.

The "Developmental Origins of Health and Disease" hypothesis has caused resurgence of interest in understanding the factors regulating fetal development. A multitude of prenatal perturbations may contribute to the onset of diseases in adulthood including cardiovascular and renal diseases. Using animal models such as maternal glucocorticoid exposure, maternal calorie or protein restriction and uteroplacental insufficiency, studies have identified alterations in kidney development as being a common feature. The formation of a low nephron endowment may result in impaired renal function and in turn may contribute to disease. An interesting feature in many animal models of developmental programming is the disparity between males and females in the timing of onset and severity of disease outcomes. The same prenatal insult does not always affect males and females in the same way or to the same degree. Recently, our studies have focused on changes induced in the kidney of both the fetus and the offspring, following a perturbation during pregnancy. We have shown that changes in the renin-angiotensin system (RAS) occur in the kidney. The changes are often sex specific which may in part explain the observed sex differences in disease outcomes and severity. This review explores the evidence suggesting a critical role for the RAS in sex specific developmental programming of disease with particular reference to the immediate and long term changes in the local RAS within the kidney.

Regulation of trophoblast invasion - a workshop report.

Trophoblast invasion during placental development helps to establish efficient physiological exchange between maternal and fetal circulatory systems. Trophoblast stem cells differentiate into multiple subtypes, including some that are highly invasive. Signalling to the trophoblast from decidua, uterine natural killer cells and vascular smooth muscle can regulate extravillous trophoblast differentiation. Important questions remain about how these cellular interactions promote trophoblast invasion and the signalling pathways that are involved. New and established biological models are being used to experimentally examine these interactions and the underlying molecular mechanisms.

Genes, development and evolution of the placenta.

Through studies of transgenic and mutant mice, it is possible to describe molecular pathways that control the development of all major trophoblast cell subtypes and structures of the placenta. For example, the proliferation of trophoblast stem cells is dependent on FGF signalling and downstream transcription factors Cdx2, Eomes and Err2. Several bHLH transcription factors regulate the progression from trophoblast stem cells to spongiotrophoblast and to trophoblast giant cells (Id1/2, Mash2, Hand1, Stra13). Intercellular actions critical for maintaining stable precursor cell populations are dependent on the gap junction protein Cx31 and the growth factor Nodal. Differentiation towards syncytiotrophoblast as well as the initiation of chorioallantoic (villous) morphogenesis is regulated by the Gcm1 transcription factor, and subsequent labyrinth development is dependent on Wnt, HGF and FGF signalling. These insights suggest that most of the genes that evolved to regulate placental development are either identical to ones used in other organ systems (e.g., FGF and epithelial branching morphogenesis), were co-opted to take on new functions (e.g., AP-2gamma, Dlx3, Hand1), or arose via gene duplication to take on a specialized placental function (e.g., Gcm1, Mash2). Many of the human orthologues of these critical genes show restricted expression patterns that are consistent with a conserved function. Such information is aiding the comparison of the human and mouse placenta. In addition, the prospect of a conserved function clearly suggests potential mechanisms for explaining complications of human placental development.

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.