The Placental Ferroxidase Zyklopen Is Not Essential for Iron Transport to the Fetus in Mice.

BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.

Molecular analysis of the human placental cysteine dioxygenase type 1 gene.

Sulfate is essential for healthy fetal growth and development. Cysteine dioxygenase type 1 (CDO1) plays an important role in the catabolism of cysteine to sulfate. Cdo1 knockout mice exhibit severe and lethal fetal phenotypes but the involvement of CDO1 gene variants in human development is unknown. We searched the NCBI and Ensembl gene databases and identified four alternatively spliced CDO1 coding mRNA transcripts, as well as 148 validated CDO1 gene variants, including 138 missense, 6 nonsense, 1 frameshift, 1 in-frame deletion, and 2 splice site variants. In silico analyses predicted 68 of the missense variants to be deleterious to CDO1 protein structure and function. We examined the relative abundance of the four CDO1 coding mRNA transcripts in human term placentas using qRT-PCR. CDO1 mRNA variant 2 was the most abundant transcript, with intermediate levels of variant 4 and lower levels of variants 1 and 3. Using in situ hybridization, we localised CDO1 mRNA expression to the syncytiotrophoblast layer of human term placenta. To investigate the regulation of CDO1 gene expression, we analysed the transcriptional activity of the human CDO1 5'-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Transcriptional activities were identified in the regions -5 to -269 and - 269 to -1200 nucleotides upstream of the CDO1 transcription initiation site. Mutational analyses of a single nucleotide polymorphism -289C > G that is common in the general population (allele frequency = 0.37) and a putative transcription factor binding motif (CCAAT enhancer binding protein beta) did not alter transcriptional activity of the CDO1 5'-flanking region. Collectively, this study provides an overview and analysis of human CDO1 for future investigations of this gene in human health.

Complex patterns of cell growth in the placenta in normal pregnancy and as adaptations to maternal diet restriction.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

Secondary Placental Defects in Cxadr Mutant Mice.

The Coxsackie virus and adenovirus receptor (CXADR) is an adhesion molecule known for its role in virus-cell interactions, epithelial integrity, and organogenesis. Loss of Cxadr causes numerous embryonic defects in mice, notably abnormal development of the cardiovascular system, and embryonic lethality. While CXADR expression has been reported in the placenta, the precise cellular localization and function within this tissue are unknown. Since impairments in placental development and function can cause secondary cardiovascular abnormalities, a phenomenon referred to as the placenta-heart axis, it is possible placental phenotypes in Cxadr mutant embryos may underlie the reported cardiovascular defects and embryonic lethality. In the current study, we determine the cellular localization of placental Cxadr expression and whether there are placental abnormalities in the absence of Cxadr. In the placenta, CXADR is expressed specifically by trophoblast labyrinth progenitors as well as cells of the visceral yolk sac (YS). In the absence of Cxadr, we observed altered expression of angiogenic factors coupled with poor expansion of trophoblast and fetal endothelial cell subpopulations, plus diminished placental transport. Unexpectedly, preserving endogenous trophoblast Cxadr expression revealed the placental defects to be secondary to primary embryonic and/or YS phenotypes. Moreover, further tissue-restricted deletions of Cxadr suggest that the secondary placental defects are likely influenced by embryonic lineages such as the fetal endothelium or those within the extraembryonic YS vascular plexus.

C5a receptor signaling prevents folate deficiency-induced neural tube defects in mice.

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid-deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic 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.

Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta.

Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf+ TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from Tpbpa+ ectoplacental cone precursors.

Expression of Cystatin C in the rat endometrium during the peri-implantation period.

Endometrial receptivity for embryo implantation in the rat is a transient state occurring on day 5 of pregnancy or pseudopregnancy and is controlled by estrogen and progesterone. To identify genes potentially involved in receptivity, a uterine cDNA library was screened. An interesting pattern for Cystatin C (Cst3) expression was discovered with a peak in abundance just prior to embryo implantation (day 4 of pregnancy) followed by a significant drop the following day when implantation is initiated. Histology localized Cst3 mRNA and CST3 protein to the glandular epithelium on day 4 of pregnancy suggesting that it is secreted into the uterine lumen at this time. In ovariectomized rats endometrial Cst3 mRNA levels decreased within 3h of treatment with estradiol; this effect was inhibited by the anti-estrogen, ICI 182, 780. The data suggest that the endometrial expression of the cysteine protease inhibitor, Cst3, is modulated by estrogen during the peri-implantation period.

Determinants of trophoblast lineage and cell subtype specification in the mouse placenta.

Cells of the trophoblast lineage make up the epithelial compartment of the placenta, and their rapid development is essential for the establishment and maintenance of pregnancy. A diverse array of specialized trophoblast subtypes form throughout gestation and are responsible for mediating implantation, as well as promotion of blood to the implantation site, changes in maternal physiology, and nutrient and gas exchange between the fetal and maternal blood supplies. Within the last decade, targeted mutations in mice and the study of trophoblast stem cells in vitro have contributed greatly to our understanding of trophoblast lineage development. Here, we review recent insights into the molecular pathways regulating trophoblast lineage segregation, stem cell maintenance, and subtype differentiation.

Rat endometrial Vdup1 expression: changes related to sensitization for the decidual cell reaction and hormonal control.

During implantation in rodents, attachment and invasion of embryonic trophoblast is accompanied by decidualization of the adjacent endometrial stroma. Decidualization can be initiated only when the endometrium is receptive, and this occurs for a short period in pregnancy. The molecular mechanisms underlying this phenomenon remain unclear. In the current study, using differential display and northern blot analysis, we found that steady-state levels of mRNA for vitamin D3 upregulated protein 1 (Vdup1) were significantly higher in 'refractory' and 'delayed' endometrium compared with 'receptive' endometrium or endometrium undergoing artificially induced decidualization. Conversely, thioredoxin (Txn), a ubiquitously expressed cellular redox regulator known to promote growth and proliferation, was found to have elevated transcript levels within the decidualizing endometrium. VDUP1 has previously been shown to bind TXN and inhibit its action. In an inverse, but cooperative, relationship, these molecules have been implicated in regulating cell growth and proliferation in a number of tissues and during transformation to cancer. The Vdup1 mRNA is localized to the uterine stroma in the nonreceptive endometrium, the site of increased Txn mRNA levels during decidualization. In addition, Vdup1 mRNA levels are inversely regulated by progesterone and estrogen; prolonged progesterone exposure stimulates an increase in Vdup1 mRNA levels whereas estrogen decreases Vdup1 transcript levels. Together, these results suggest a novel mechanism by which suppression of the decidual response in the nonreceptive endometrium may occur.

Uterine sensitization-associated gene-1: a novel gene induced within the rat endometrium at the time of uterine receptivity/sensitization for the decidual cell reaction.

For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.