Molecular Mechanism of HINTW (HINT Gene in W-Chromosome)-Mediated UBE2I (Ubiquitin Conjugating Enzyme E2 I) Interaction in Female Differentiation of Chicken Embryos.

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.