Tammar wallabies can arrest embryonic development at the blastocyst stage for 11 months with no embryonic growth or cell death. However reactivation can be induced by removing the sucking pouch young (RPY). The blastocyst is surrounded by an acellular shell coat, so diapause and reactivation must be controlled by soluble factors inthe uterine secretions. We investigated the proteins in the uterine environment during diapause and reactivation.
Endometrium and uterine flushings from day 0 RPY (embryo in diapause), and reactivated stages at RPY days 4, 5, 6, 8 and 9 (expansion of blastocyst starts), day 11 and day 24 (2 days before birth) were analysed by liquid chromatography-mass spectrometry (LC-MS/MS), MALDI imaging and quantitation using iTRAQ labelling. With all 3 techniques there was a distinct difference in protein profiles in diapause compared to the reactivation stages. The increased secretory activity of the endometrium from d4 RPY is accompanied by increases in metabolic enzymes, heat shock proteins and growth factors. These were also in congruence with the results from MS imaging where significant changes were noted between d0 and d4. The d4 and d5 had distinct set of peptides which might initiate reactivation with an increase in metabolism and d6 and d8 were similar which might contain proteins which promote growth and initiate the expansion of blastocyst. iTRAQ quantitation demonstrated significant increases of about 3-6 fold increase in metabolic enzymes, heat shock proteins and structural proteins by d 4. Analysis of the dynamic proteomic changes with reactivation is underway to identify potential candidates that may be regulating diapause and reactivation. Understanding the fundamental controls of cell division that must dominate control of diapause may be applicable to understanding implantation failures and infertility, and the control of cell proliferation in cancer and other disease states.