Evolution of the Chick Embryo Chorioallantoic Membrane Proteome during Early Development

In avian species, the chorioallantoic membrane (CAM) is a vital, highly vascularized extraembryonic structure that supports embryonic respiration, calcium transport, and innate immune defense. In this study, we applied LC/MS/MS-based proteomics to CAM tissue harvested at embryonic days (ED) 6, 8, 10, and 12 to characterize its protein profile during the expression of different CAM functionalities during embryonic development and gain insight into possible sex-based distinctions. A total of 2688 proteins were identified, with 2347, 2265, 2351, and 1267 proteins detected at ED 6, 8, 10, and 12, respectively. Notably, 1191 common proteins were identified across all stages, while 124, 47, 86, and 2 proteins were uniquely expressed at ED 6, 8, 10, and 12, respectively. Functional annotation revealed correlations with abundant CAM protein constituents (as per their emPAI); for example: calcium mobilization - v-type proton ATPase subunit E1 (ATP6V1E1) and G1 (ATP6V1G1); intracellular transport-calcium-binding protein 39 (CAB39); vascular system and gaseous exchange - annexin A2 (ANXA2); lymphatics-actin, gamma 1 (ACTG1); blood elements-hemoglobin subunit alpha-1 (HBA1); immune defense-cathelicidin-1 (CATH1), cathelicidin-2 (CATH2); and protection against luminal toxic contents-thioredoxin (TXN). Notably, a sex-specific analysis identified 614, 320, 314, and 212 proteins that were uniquely expressed in female embryos, and 212, 273, 144, and 56 proteins only in male embryos at ED 6, 8, 10, and 12, respectively. The identification of sex-linked proteins during early CAM development may provide insight into their functional roles and highlight the CAM's potential as a target for the development of in-ovo sex identification technology.