This study yielded two important results regarding the interaction between maternal stress and dietary DHA enrichment in embryonic day 12.5 conceptuses. First, maternal stress during the first week of gestation showed a marked influence on the composition of the litter and gene expression patterns in the placenta, with offspring sex largely determining the magnitude of disruption. Second, a maternal diet enriched with preformed DHA during periods of high stress shows partial rescue of stress-dependent dysregulation of gene expression in the placenta.
The observation that the placenta responds to maternal diet is not surprising given its specialized metabolic niche that is particularly sensitive to maternal resource availability. Maternal malnutrition during pregnancy, presumably through constrained exchange of maternal nutrients across the placenta, exerts long-term changes in morbidity and mortality, growth trajectory, and increased disease risk in adulthood(Waterland and Garza, 1999). Maternal diet also appears to exhibit contrasting programing on the placental transcriptome based on offspring sex, consistent with maternal diet influencing resource exchange through the placenta in a sex-specific manner(Mao et al., 2010).
In the present study, maternal diet was associated with some aspects of growth and development. Mothers consuming the DHA-enriched diet exhibited lower fetal loss relative to offspring of CTL-diet mothers, suggesting that the availability of DHA in maternal diet may be critical to fetal development at this development window. Although there were no sex differences in placenta weight within the same maternal diet, DHA-enrichment increased male placenta weight relative to that of males in the CTL-diet group. Previous reports have shown that placenta weight is associated with offspring size, which may be attributed to differential resource demand for growth-related nutrients and downstream consequences on newborn size(Godfrey et al., 1996). Indeed, DHA-enrichment increased embryo weight at E12.5 relative to offspring exposed to CTL diet, and this effect was independent number of offspring given no diet-related differences in litter size. These results must be interpreted with caution, however, as high rates of infanticide have been reported for the C57Bl/6J strain used in this study, and, as a result, increased offspring size at E12.5 may not reflect litter size or survival rates following birth(Brown et al., 1999).
Similar to maternal nutrition status, maternal stress during pregnancy has direct consequences on offspring growth and development in humans, and later phenotypic outcomes in adulthood across multiple species, including increased risk for neurodevelopmental and neuropsychiatric disorders in humans(Bale, 2015). Consistent with previous results, we observed that maternal stress trended toward reduction in male placental weight, with no effect of prenatal stress on female placenta weight(Mairesse et al., 2007). To assess the potential factors involved in the sex differences in placenta and embryo size, we measured the expression of placental genes previously reported to be sensitive to sex-specific disruption following EPS. Similar to previous reports, EPS resulted in male-specific upregulation of placental HIF3α and PPARA, with no effect on females(Mueller and Bale, 2008). However, there was no sex or EPS-related difference in expression of OGT, IGFBP1 or GLUT4(Howerton et al., 2013; Mueller and Bale, 2008). The discrepancy between previous and current study is likely related to the differences in genetic background of mouse strains (C57Bl/6J vs. mixed background of C57Bl/6J*129), which exhibit differential sensitivity to stress(Chan et al., 2017). DHA-enrichment exhibited no effect on placental expression in either EPS-exposed or control females, further suggesting male-specific vulnerability to early prenatal stress may be buffered by maternal diet.
Based on the observation that maternal diet and maternal stress result in similar sex-specific changes to placenta and embryo size, we examined whether maternal diet and maternal stress converge upon similar transcriptional pathways. DHA-enrichment reversed the EPS induced male-specific upregulation of placental HIF3α and PPARA back to comparable levels of males that were not exposed to early prenatal stress. Low oxygen conditions activate a cascade of physiological response that includes the upregulation of large class of hypoxia inducible factor (Forristal et al., 2010; Lee et al., 2004; Semenza, 2000). HIF proteins control a broad family of genes, including VEGFA, a canonical regulator of angiogenesis that is highly sensitive to hypoxic conditions(Forristal et al., 2010; Lee et al., 2004; Semenza, 2000). EPS increased expression of HIF3α but did not result in a parallel EPS-dependent upregulation of VEGFA. The inability of HIF3α to increase expression of VEGFA may be related to the unique structural properties of HIF3α. In contrast to family protein members HIF1 and HIF2, HIF3α lacks a C-terminal activation domain required for co-activator binding, and, as a result, is unable to recruit co-transcriptional regulators and basal transcriptional machinery to gene targets, including VEGFA(Forristal et al., 2010; Lee et al., 2004; Semenza, 2000).
An alternative interpretation to the finding that EPS induces male-specific reprogramming of candidate genes in the placenta would be that this represents a sex-specific adaptation to increase resources transfer during periods of stress, and, as a result, DHA-enrichment is hampering this adaptation(Burton and Fowden, 2012; Díaz et al., 2014). Such an alternative hypothesis would predict either no difference in placenta and embryo weight for EPS and non-stress animals exposed to the CTL diet or a negative effect of DHA diet on placenta and embryo weight. The present results show the opposite trend: DHA-enrichment increases placenta and embryo weight while concomitantly decreasing expression of genes that are normally expressed in low oxygen or constrained nutrient conditions.
Nevertheless, the present results can be readily understood within maternal life history trade-offs(Stearns, 1989). During pregnancy, mothers require resources to meet both maternal and offspring requirements. However, environmental cues, such as malnutrition and stress, may decrease the optimal resource transfer from mothers to offspring in a way that maximizes maternal reproductive success at the detriment to offspring. Sex-specific reductions in male, but not female, placenta and embryo in EPS exposed animals are consistent with reduction of maternal investment in male relative to female offspring. From an evolutionary perspective, the shift in maternal resource allocation may be related to sex differences in reproductive payoffs for mothers; specifically, smaller, less fit males are less likely to reproduce as adults than their sisters (Trivers, 1974). Indeed, rescue of this sex-specific vulnerability with dietary DHA-enrichment may suggests that EPS-dependent deficiency of nutrient and oxygen transfer may trigger this shift in maternal investment. The goal of future studies should focus on whether DHA-enrichment buffers from prenatal-stress dependent deficits in adulthood and on identifying sex- and diet-dependent broad programmatic pathways following exposure to prenatal stress by leveraging whole transcriptome profiling approaches. Moreover, determining the possibility that DHA-enrichment may buffer from stress that extend across pregnancy and later developmental windows is a key avenue for future research.