Abstract
BACKGROUND/OBJECTIVE: It has been established that HINTW plays a pivotal role in the female differentiation of chickens; nevertheless, the underlying molecular mechanism remains to be fully elucidated. METHOD: To investigate the role of HINTW in avian sex determination, a prokaryotic expression vector containing its key structural domain was constructed, and its in vitro expression was achieved. Pull-down assays were performed to capture interacting proteins from male and female gonadal tissues, followed by a silver staining analysis to compare interaction profiles between ovaries and testes. Mass spectrometry was utilized to identify differentially bound proteins. Additionally, functional characterization and co-immunoprecipitation (Co-IP) assays were conducted to validate the interaction between HINTW and its candidate binding partner. RESULT: A total of 1590 differentially bound proteins were identified between ovarian and testicular tissues. Functional analysis and Co-IP assays confirmed a specific interaction between HINTW and UBE2I in the ovary, suggesting that HINTW may facilitate female differentiation in chicken embryos through its interaction with UBE2I. CONCLUSIONS: This study provides novel insights into the molecular mechanisms underlying HINTW-mediated female differentiation in chickens and contributes to a deeper understanding of avian sex determination.