Furthermore, we recorded the development of secondary sexual characteristics and the body growth of sex-reversed chickens, and analyzed the mechanisms underlying their infertility, which might have resulted from the low expression of several gametogenesis-related genes, such as SPEF2, DNAI1, and TACR3. Overall, our findings enable a better understanding of gonadal development in sex-reversed chickens and facilitate the construction of a gene regulatory network for chicken sex determination and differentiation. This evidence concerns the gene SPEF2 and Infertility.