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.

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.