RETRACTED: Analysis of the Molecular Mechanism of Energy Metabolism in the Sex Differentiation of Chickens Based on Transcriptome Sequencing

Abstract

The determination of sex in mammals is established and controlled by various complex mechanisms. In contrast, sex control in poultry remains an unresolved issue. In this study, RNA-sequencing was conducted for male gonads and ovarian tissues in chicken embryos of up to 18.5 days to identify metabolic factors influencing male and female sex differentiation, as well as gonadal development. Our results reveal that PKM2, a critical glycolysis-related protein, plays a significant role in chicken sex differentiation via PPARG, a crucial hormone gene. We propose that our discoveries bolster the notion that glycolysis and oxidative phosphorylation function as antecedent contributors to sexual phenotypic development and preservation.

Keywords: energy metabolism; primordial germ cell; sex differentiation; transcriptome sequencing.

Figure 1

Sampling, transcriptome sequencing, and validation of male and female gonads: (A) White dashed lines denote the locations of the male (upper panel) and female (lower panel) gonads. (B) PAS staining of chicken male and female gonads, with a scale bar indicating 50 µm. (C) Schematic overview of the sampling process, RNA-seq sequencing, and subsequent data analysis. (D) Principal Component Analysis (PCA) of transcriptomic profiles from male and female gonads. (E) Volcano plot illustrating differentially expressed genes in male and female gonads. (F) Expression levels of sex-related genes in male and female gonads (n = 3), with significance indicated by ** p < 0.01 (Student’s t-test).

Figure 2

Characteristics of metabolism−related genes: (A) Pie chart illustrating that 26.55% of differentially expressed genes are associated with metabolic processes. (B) Heat map displaying the expression levels of glycolysis-related genes at 18.5 days of development.

Figure 3

Energy metabolism and sex determination in Chickens: (A) Expression levels of key glycolytic and oxidative phosphorylation genes in both female and male gonads. (B) RT−qPCR analysis of selected genes (PKM, LDH, α−KGDH, and SDH), with expression levels normalized to ACTB. Statistical significance is denoted as * p < 0.05, ** p < 0.01 (Student’s t−test), and “ns” indicates no significant difference (p > 0.05). (C) PAS staining reveals that 2DG treatment enhances the structural integrity of testicular spermatophores compared to the control group.

Figure 4

Energy Metabolism Maintains Gonadal Development Through Hormone Synthesis: (A) PPI data reveal that glycolysis-related genes may alter GAPDH through Pkm2, which interacts with PPARG to affect sex-regulated SOX9. (B) Box plot shows that testosterone levels were lower in the 2DG group than in the rotenone group. (C) Box plot shows that estradiol levels were lower in the 2DG group than in the rotenone group. (D) Expression of the hormone-related gene PPARG and sex regulation-related genes SOX9 and GATA4 in male gonads after adding 2DG and rotenone. (E) Expression of the hormone-related gene PPARG and sex regulation-related genes SOX9 and GATA4 in female gonads after adding 2DG and rotenone. Data are shown as mean ± SEM (n = 3 independent experiments), with statistical significance indicated by * p < 0.05, ** p < 0.01, assessed by one-way ANOVA. “ns” indicates no significant difference (p > 0.05).