Micro-bulk RNA-seq of trilaminar omphalopleure of Monodelphis domestica to investigate placental cytokine signaling

The brief placental attachment of the gray short-tailed opossum Monodelphis domestica spans only the final two days of the 14-day pregnancy. During this time the fetus grows rapidly and a pronounced spike in inflammatory signaling occurs. It was recently discovered that these cytokines are produced primarily by fetal cells, the syncytial knots which develop at the end stage of placentation after day 13 of gestation. To investigate this further, we conducted laser microdissection on placental membranes (omphalopleure) at 13.5 days post copulation to compare to published transcriptomes at 12.5 days post copulation. Analysis revealed indeed a pronounced expression of inflammatory cytokines in the 13.5 samples. We hypothesized that placental cytokines act as solicitation signals benefitting the fetus by effects on maternal nutrient transfer. To test this, we administered anti-inflammatory drugs to pregnant opossums during the attachment phase targeted at three arms of this response: IL1A via the receptor inhibitor anakinra, IL6 via the receptor inhibitor tocilizumab, and prostaglandin E2 via the PTGS2 synthetase inhibitor meloxicam. Inhibition of IL-1 and IL-6 signaling resulted in significant increases in fetal biomass, suggesting that they impose a cost to biomass acquisition by offspring rather than acting in direct solicitation. However, in the case of anakinra, saline-treated control individuals showed a greater mean surviving litter size compared to IL-1-inhibited animals. Together, our results suggest that marsupial end-pregnancy inflammation has acquired a novel function modulating both fetal growth and survival during pregnancy. Overall design: Opossums were mated (no drug treatment or other intervention) and pregnant uteri were collected at 13.5 days post copulation. Placental tissues, which normally appose maternal uterine lining, were isolated by laser microdissection and subjected to RNA sequencing.