# Placental Transcriptome Adapations to Maternal Nutrient Restriction in Sheep
## 3 Discussion
Multiple genes from a variety of steps in the cytotoxicity process were upregulated, from target cell ligands and NK cell receptors to _TNF-\(\alpha\)_, genes involved in cytotoxic granule exocytosis, and apoptotic genes. Considering the histological changes, such as decreased surface area at the maternal-fetal interface in placentomes from SGA fetuses that were seen previously [5], the implications of increased cytotoxic activity may manifest in both histoarchitectural and functional aspects of placentome development. Of note, increased NK-cell-mediated cytotoxicity has also been associated with recurrent pregnancy loss in vitamin-D-deficient women, lending weight to the negative impact it can have on placental development [24,25]. Major histocompatibility complex (MHC) class II molecule expression serves as a mediator of T-lymphocyte response [26,27]. This is of interest because, at day 70, MHC class II protein binding was a molecular function that was altered in NR SGA compared to 100% NRC placentomes. MHC class II receptor activity was also the only molecular function that was altered in NR NonSGA compared to 100% NRC placentomes and, indeed, two of the three genes involved--_HLA-DQA2_ and _HLA-DQB2_--were downregulated in the NR placentomes (Figure 2). While these two genes are considered to be nonfunctional pseudogenes in humans, they are transcribed and translated in sheep and cattle, although their exact roles are still unknown, and may be of interest to study in the context of placentome development [28]. One limitation to this study is that after mapping reads to the available sheep genome, those gene identifiers were then matched to human identifiers for further bioinformatics analyses, due to the availability of resources to analyze the human transcriptome as opposed to that of the sheep. This resulted in the loss of sheep-specific genes from the GSEA analyses that may have an important role that could be further elucidated by working through specific pathways of interest. The other area where this swap from sheep to human identifiers would need to be explored further is in the major histocompatibility complex molecules and NK cell complex molecules. While there are a number of similarities between the sheep and human genomes in these pathways, there are also documented differences that reinforce that these bioinformatics analyses are exploratory tools only, and specific mechanisms must be worked out directly [28; 29]. Alterations in amino acid transport in the late-gestation placenta, and resultant changes in fetal plasma amino acid concentrations, have been well documented in NR pregnancies resulting in SGA offspring [5; 30; 31; 32; 33]. However, one of the pathways that were enriched in this study in SGA compared to NonSGA placentomes in mid-pregnancy that is unique is beta-alanine metabolism. Beta-alanine is a non-essential amino acid that is not incorporated into proteins, but serves as an intermediary that can be used for fatty acid synthesis, pyrimidine metabolism, or carnosine production [34]. The specific enzymes that are upregulated in the SGA fetuses appear to drive the production of beta-alanine from L-aspartate and uracil, with beta-alanine itself then being utilized in fatty acid biosynthesis (Figure 8). There appears to be little knowledge about a role for beta-alanine in the placenta, but its use could very well be an adaptive measure undertaken by the NR placentome to maintain function. The GO term signaling receptor binding (GO:0005102) was identified as differential between SGA and NonSGA placentomes, and identifies genes that bind to a receptor molecule to initiate a change in cell function. At day 70, 9 of the 22 differentially expressed genes fit into this category, and especially interesting is that 4 of those--_FAM3C_, _MSTN_, _TNFRSF11B_, and _WNT9B_--were also identified in the cytokine activity molecular function (GO:0005125), which is defined as genes that interact with a receptor to control survival, growth, and differentiation.