Significance: Here, we show that DMRT1 dosage is the key sex determination factor in birds and is essential for testis development. Furthermore, we provide additional evidence that birds, in contrast to mammals, have acquired cell-autonomous sex identity (CASI) and that the sex hormone environment does not significantly influence avian secondary sexual characteristics. This finding highlights an evolutionary divide between mammals and nonmammalian vertebrates. In mammals, the sex chromosomes determine the type of gonad formed, and sex hormones largely define the secondary sexual phenotype. In birds, the sexual phenotype is directly determined by the sex chromosome content of individual cells in different tissues. Our findings help advance our understanding of the evolution of sex determination systems and the nature of sex identity.

Eggshell gloss is an important characteristic for the manifestation of eggshell appearance. However, no study has yet identified potential candidate genes for eggshell gloss between high-gloss (HG) and low-gloss (LG) chickens. The aim of this study was to perform a preliminary investigation into the formation mechanism of eggshell gloss and to identify potential genes. The eggshell gloss of 300-day-old Rhode Island Red hens was measured from three aspects. Uterine tissues of the selected HG and LG (n = 5) hens were collected for RNA-seq. Blood samples were also collected for whole-genome resequencing (WGRS). RNA-seq analysis showed that 150 differentially expressed genes (DEGs) were identified in the uterine tissues of HG and LG hens. These DEGs were mainly enriched in the calcium signaling pathway and the neuroactive ligand–receptor interaction pathway. Importantly, these two pathways were also significantly enriched in the WGRS analysis results. Further joint analysis of WGRS and RNA-seq data revealed that 5-hydroxytryptamine receptor 1F (HTR1F), zinc finger protein 536 (ZNF536), NEDD8 ubiquitin-like modifier (NEDD8), nerve growth factor (NGF) and calmodulin 1 (CALM1) are potential candidate genes for eggshell gloss. In summary, our research provides a reference for the study of eggshell gloss and lays a foundation for improving egg glossiness in layer breeding.

The sex chromosomes of birds are designated Z and W. The male is homogamous (ZZ), and the female is heterogamous (ZW). The chicken W chromosome is a degenerate version of the Z chromosome and harbors only 28 protein-coding genes. We studied the expression pattern of the W chromosome gene MIER3 (showing differential expression during gonadogenesis) in chicken embryonic gonads and its potential role in gonadal development. The W copy of MIER3 (MIER3–W) shows a gonad-biased expression in chicken embryonic tissues which was different from its Z copy. The overall expression of MIER3–W and MIER3–Z mRNA and protein is correlated with the gonadal phenotype being higher in female gonads than in male gonads or female-to-male sex-reversed gonads. Chicken MIER3 protein is highly expressed in the nucleus, with relatively lower expression in the cytoplasm. Overexpression of MIER3–W in male gonad cells suggested its effect on the GnRH signaling pathway, cell proliferation, and cell apoptosis. MIER3 expression is associated with the gonadal phenotype. MIER3 may promote female gonadal development by regulating EGR1 and αGSU genes. These findings enrich our knowledge of chicken W chromosome genes and support a more systematic and in-depth understanding of gonadal development in chickens.

Background: While the mechanism of asymmetric gonadal development is generally understood, the mechanism of asymmetric oviduct development remains unclear. Methods: Right and left oviducts were collected from chick embryos at three developmental stages (Embryonic day 7.5, E9.5, and E11.5) for RNA-seq analysis (RNA-seq). Whole-genome resequencing (WGRS) was performed on hens with bilateral reproductive systems (a rare natural occurrence) and unilateral controls. These data were co-analyzed with public RNA-seq data of female embryonic gonads at different developmental stages (E4.5, E5.5, and E6.5) to screen for candidate genes affecting oviduct degeneration/development. Results: RNA-seq analyses showed that a total of 27, 10, and 38 DEGs were identified between the left and right oviducts at E7.5, E9.5, and E11.5, respectively. WGRS analyses revealed 1045 differentially mutated genes (DMGs) between bilateral (D) and unilateral (S) groups. Preliminary validation highlighted BMP7, PAK3, SLC6A11, PITX2, and SMC1B as candidate genes influencing oviduct asymmetry. Conclusions: This study provides insights into the genetic basis of asymmetric oviduct development and lays the groundwork for breeding hens with bilateral reproductive systems.

Unlike mammals, where systemic sex hormones determine somatic sexual identity, chickens primarily employ a cell-autonomous mechanism, known as the cell autonomous sexual identity (CASI) hypothesis. However, tissue sensitivity to sex hormones varies significantly. This study aimed to elucidate the specific role of the estrogen/estrogen receptor α (ERα) system in this context. We assessed protein levels of key sex hormone receptors, including ERα, estrogen receptor β (ERβ), and androgen receptor (AR), in the tissues of 6-week-old and adult chickens. Results showed that ERα protein was markedly abundant in hormone-sensitive tissues (comb and wattles) but low in insensitive ones (muscle and liver); this pattern was not observed for ERβ or AR. Functionally, in the absence of estrogen, ERα overexpression in ERα-deficient primary muscle cells affected immunity but not sexual differentiation-related pathways. In contrast, analysis on the transcriptomes of ERα-rich wattle tissues indicated that the genes differentially expressed between estrogen-treated versus untreated groups were enriched in the pathways critical for development, neuroactive ligand-receptor interaction, and glycerolipid metabolism to establish sexual phenotype. In conclusion, estrogen regulates somatic sex identity via ERα in a tissue-specific manner, with regulatory intensity dictated by local ERα abundance. This provides a crucial supplement to the CASI hypothesis.

The mechanism of sex determination in chickens, especially the molecular mechanism of female ovarian development, has not yet been fully elucidated. Previous studies have shown that RSPO1, which is associated with ovarian development in mammals, might have a conserved role in chickens. In this study, we systematically investigated the spatiotemporal expression pattern of RSPO1 in various tissues, especially gonads, of male and female chicken embryos using qPCR and Western blotting, and we explored its correlation with the expression of key genes in the estrogen pathway using drug treatment or gene overexpression in vivo and in vitro. Our results reveal that RSPO1 was widely expressed in all examined tissues of chicken embryos, showing a female bias in gonadal tissues at both the mRNA and protein levels. Surprisingly, RSPO1 was not differentially expressed between male and female gonadal cells with fadrozole-induced estrogen pathway blockades, and furthermore, estradiol-induced estrogen stimulation altered the expression of RSPO1. In addition, overexpression of RSPO1 in gonadal cells induced the mRNA expression of its downstream target genes, Wnt family member 4 (WNT4) and Catenin beta 1 (CTNNB1), and that of estrogen receptor α (ERα), an estrogen pathway gene. In summary, this study provided new evidence for elucidating the role of RSPO1 in ovarian development in poultry.