The placenta is essential for the growth and development of all Eutherian mammals. Its primary function is to serve as a surface for the exchange of nutrients and waste products between the developing fetus and its mother. It also acts as a barrier to fetopaternal antigens, and produces hormones for the establishment and maintenance of the pregnancy (18
). In the mouse, the labyrinth is the site of placental nutrient exchange. The mature fetal labyrinth contains mononuclear and syncytial trophoblast cells, as well as mesenchymal (stromal) and endothelial cells of mesodermal origin. Nutrient exchange occurs across three cell layers of syncytiotrophoblasts and cytotrophoblasts that separate endothelial lined fetal vessels from sinuses of maternal blood (18
Following fusion of the fetal allantois with the placental chorion at ~E8.5 (21
), there is rapid proliferation and differentiation of both trophoblast and fetal-derived cells of the placental labyrinth, the latter originating in allantoic mesoderm. This is also a critical time for the fetus, with substantial growth as organogenesis proceeds. Large amounts of cholesterol are necessary for the production of membranes for this fetal and placental growth. Unlike the adult where cholesterol is in steady state, there is net accrual in the fetus [reviewed in (23
)]. Cholesterol is also necessary for the synthesis of steroid hormones, bile acids and oxysterols. The latter are involved in lipid homeostasis through their regulation of orphan nuclear receptors, such as LXRs (24
). Lipid rafts, which are involved in signaling at the plasma membrane, are enriched in cholesterol. Finally, a cholesterol molecule is covalently bound to hedgehog proteins during their processing. Secreted hedgehog proteins act as dose-dependent morphogens and play important roles in numerous processes throughout embryogenesis (26
There are two sources of fetal cholesterol, endogenous synthesis and transport from the mother (13
). In the mouse, significant cholesterol transport from the mother to the fetus occurs throughout gestation. On the basis of a recent study (14
), prior to E12.5, most fetal cholesterol is maternal in origin. Thereafter, endogenous synthesis contributes up to ~50% of the total fetal cholesterol in peripheral tissues. However, by midgestation in the mouse, most cholesterol found in the developing brain is synthesized in situ
due to the presence of a functional blood-brain-barrier (14
We reasoned that heterozygosity for Nsdhl in a pregnant dam might contribute to the phenotype in affected embryos: affected males die before E12.5 at a time when most cholesterol is maternal in origin. To assess possible contributions of the maternal genotype to the embryonic phenotype, we first examined plasma cholesterol levels in non-pregnant and gravid WT and Bpa1H/+ females fed our institutional cholesterol-free breeder chow. The significant decrease in serum cholesterol levels that we noted in pregnant dams beginning at ~E8.5 (Fig. ) corresponds with the phase of rapid growth following chorioallantoic fusion and likely reflects increased demands for cholesterol in both the fetus and placenta. We believe that the lag in recovery of cholesterol levels in Bpa1H/+ dams reflects less cholesterol stores in the mutant females and/or a lower capacity for de novo synthesis, although further experiments will be necessary to prove this hypothesis.
The generation of mice expressing a human NSDHL transgene enabled us to ask whether the maternal and extraembryonic environments make significant contributions to the overall fetal placental pathology by examining female Bpa1H/+ embryos. Surprisingly, although the genotype of the embryo and the mother contributed to the overall placental size and area, the major contributing factor was the presence or absence of Nsdhl expression in the fetal membranes (Table ). The maternal effect could come from the maternal decidua of the placenta, a source of cholesterol-derived steroid hormones, or lipids, such as cholesterol, transported from the pregnant dam. The apparent delay in recovery of maternal plasma cholesterol levels in Bpa/+ dams (Fig. ), possibly related to a lower capacity for de novo synthesis, could accentuate effects of the physiologic decrease found at midgestation. Thus, the increased demands of the growing fetus and placenta for maternal cholesterol and the reduced capacity of a Bpa1H dam to produce cholesterol could create a bottleneck between ~E9.5 and E11.5, where cholesterol is limiting for growth.
The murine placenta becomes functional after ~E10.5 (18
). On the basis of the much higher expression of Nsdhl
in the yolk sac than the fetal placenta at this time in gestation (Fig. ), we believe that the former is likely to be the important determinant in the extraembryonic tissues. In support of this hypothesis, we previously demonstrated that yolk sacs from E9.5 to E10.5 mutant Nsdhl
embryos are pale, with possible defects in vascular remodeling (5
). However, it is not clear how a primary yolk sac defect would result in a smaller placenta. It is possible that the yolk sac defects result in overall slower growth with suboptimal transport of nutrients to the fetus. Alternatively, we have demonstrated the migration of small numbers of cells from yolk sac visceral endoderm into placental mesoderm following chorioallantoic fusion (6
). It is possible that this migration, and signaling by one or more pathways between the two extraembryonic tissues, is compromised. Unfortunately, we cannot separate effects of trophoblast-derived placental lineages from those of yolk sac endoderm using the genetic approaches employed here. It would require a conditional Nsdhl
allele with inactivation in specific extraembryonic tissues using cre recombinase or similar technology.
Although these studies were performed using a mutant mouse model, they could have implications for human pregnancy and placental function. Rare, inherited human disorders of cholesterol biosynthesis, such as Smith–Lemli–Opitz syndrome (SLOS), demonstrate that cholesterol deficiency is highly teratogenic to the developing human fetus [reviewed in (28
)]. The presence of detectable cholesterol levels in SLOS fetuses carrying two null alleles further demonstrates that there is maternal cholesterol transport during human pregnancy, although the extent and duration are not known. Further, the maternal, but not the paternal, ApoE genotype influences the severity of SLOS in the offspring, suggesting an effect of maternal cholesterol levels (30
It should be noted that there are many similarities, but also some important differences, between human and rodent placentas (18
). Many genes and proteins, including transcription factors and those involved in cellular signaling pathways, originally described in one system have subsequently been found to play a similar role in the other (31
). Both share similar trophoblast cell lineages, such as giant cells and syncytiotrophoblasts, although some nomenclature and morphologic or molecular details differ. However, the human placenta is fully functional earlier in gestation, at 8–10 weeks post-conception, whereas the murine placenta becomes fully functional only in midgestation after E10.5 (18
). This difference in timing is due in large part to the presence of the yolk sac and active choriovitelline circulation that persists throughout gestation in the rodent embryo. The human yolk sac functions only during the first trimester. Further, the rodent yolk sac is inverted, with endodermal cells facing out, enabling substantial uptake from maternal tissues and plasma. Finally, although some skewing of X-inactivation can occur in human female placentas, extreme skewing (>85%) is rare (33
). Expression of NSDHL
and other cholesterol biosynthetic enzymes in human fetal membranes has not been well studied.
Interest in understanding lipid metabolism in the human fetus and placenta has increased recently with the recognition that maternal hypercholesterolemia during pregnancy is a predictor of cardiovascular disease in offspring, an example of ‘fetal programming’ (11
). Effects of maternal hypocholesterolemia on the fetus are not well studied, although Edison et al
) have recently demonstrated that lower maternal plasma cholesterol is associated with lower birth weight, and there was a trend with microcephaly. Further, Steffen et al
) noted suggestive associations for SNPs at several loci involved in cholesterol metabolism with prematurity and birth weight. It is likely that risks of low maternal cholesterol in pregnancy will become better defined as more studies are performed.