Methods: The mouse was used in this study as the endocrine function of the placenta is performed by a discrete zone, the junctional zone (Jz). Primary cell cultures from the whole placenta (WP) and isolated Jz from dams on day 14 and 16 of gestation were established (term=20 days). Cell viability necrosis and apoptosis were measured over the 5 day culture using XTT and LDH release assays and Tunel, p53 and Bax expression. The expression of Jz cell marker genes was assessed by qPCR and proteins released into the conditioned media on day 2 of culture were identified by mass spectrometry and bioinformatic analyses. Data are from n=5-6.
Results: Cell culture characteristics were similar for those prepared from WP and Jz at both pregnancy days. Cell viability was reduced by 20% at 24h of culturing, with a corresponding increase in necrosis (but not apoptosis). At 48h-72h, cell viability increased by 200-500%, and necrosis levels reduced by 100%. At 96h-120h of culture, cell viability reduced in line with increased apoptosis. All three Jz cell types were present in all cultures at 24h to 96h. Conditioned media of day 16 WP cultures contained a total of 932 proteins in at least 4 out of 5 samples, of which 167 are also expressed reported expressed by human placenta. Gene ontology suggested these proteins are involved in activities such as metabolic regulation, immune modulation, signalling and growth. Mass spectrometry analysis revealed ~800 proteins secreted from the placenta of the mouse which share homologue to human proteins. These proteins were further analysed using bioinformatic tools for there involvement in know pregnancy conditions such as gestational diabetes and pre-eclampsia.
Conclusion: A map of the mouse placental secretome has been created using primary cell cultures. This map will be the first step in identifying new biomarkers for pregnancy comlications in humans and understanding these comlications mechanism and their impact on maternal physiology and feta development.
