Article

Anim Biosci. 2025; 38(6): 1081-1104.

Published online February 27, 2025.

DOI: https://doi.org/10.5713/ab.24.0679

Key events in the process of sex determination and differentiation in early chicken embryos

Xiaoqian Lv1,2 , Changhua Sun1,2,3 , Xin Liu1,2 , Guanzheng Liu1,2 , Wei Gong1,2 , Hongwu Qian1,2 , Zeyu Li1,2 , Jun Wu1,2 , Xilin Zhu1,2 , Jiuzhou Song4 , Yingjie Niu1,2 , Hongyan Sun1,2 , Wei Han5 , Guo hong Chen1,2,3 , Kai Jin1,2 , Bichun Li1,2,3,6 , and Qisheng Zuo1,2
1Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China
2Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China
3Department of Food Technology, College of Biochemical Engineering, Yangzhou Polytechnic College, Yangzhou, China
4Department of Animal & Avian Sciences, University of Maryland, College Park, MD, USA
5Jiangsu Institute of Poultry Sciences/Poultry institute, Chinese Academy of Agricultural Sciences, Yangzhou, China
6College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China
Corresponding Author: Email: 006664@yzu.edu.cn
Received October 1, 2024;    Accepted December 30, 2024.
Abstract
Objective
Current understanding of sex determination and differentiation mechanisms during early chicken embryonic development remains incomplete. To address this, we applied RNA sequencing to identify male-female expression differences at critical developmental stages (E0 blastocysts, E3.5-E6.5 genital ridges, E18.5 gonads), focusing on glycolysis, histone acetylation, and DNA methylation. This approach aims to unravel key regulatory mechanisms and advance developmental biology insights.
Methods
We analyzed molecular mechanisms of chicken sex determination at key stages (E0 blastocysts, E3.5-E6.5 genital ridges, E18.5 gonads) using RNA sequencing. Glycolysis, histone acetylation, and DNA methylation levels were assessed in embryonic stem cells and chicken embryonic fibroblasts. E18.5 gonads were treated with glycolytic activators (SB431542 and PD0325901 [2i]), a DNA demethylation activator (Vitamin C [Vc]), or an inhibitors of histone acetylation modification (valproic acid [VPA]). Sex-related gene expression, hormone levels, and gonad morphology were evaluated to determine treatment effects.
Results
Key findings revealed that sex differences emerged as early as the blastocyst stage, intensified with embryonic development and were marked by a surge in sexually dimorphic gene expression. Gene Ontology and Kyoto encyclopedia of genes and genomes analyses highlighted the pivotal roles of energy metabolism and epigenetic modification process during this critical period. 2i, VC, or VPA interventions targeting E18.5 embryo gonads, induced a remarkable transformation of ovarian tissue into a testis-like structure, characterized by cortical thinning, medulla densification, downregulation of female-specific genes (FOXL2, WNT4), upregulation of male-specific genes (SOX9, AMH), and increased testosterone secretion. This phenotypic and molecular shift underscores the ability of metabolic and epigenetic modulators to reprogram ovarian development towards a male-like pattern, preserving male sexual characteristics.
Conclusion
Our study establishes energy metabolism and epigenetic regulation as central drivers of avian sex determination. These findings advance understanding of vertebrate developmental biology and provide a framework for dissecting regulatory networks in avian sexual development.
Keywords : Chicken; DNA Methylation; Glycolysis; Histone Acetylation; Sex Determination