Early Gonadal Differentiation in the Viviparous Red Stingray: Histological Characterization of Sexual Dimorphism

The mechanisms underlying sexual differentiation in viviparous chondrichthyans remain poorly understood, particularly regarding the impact of the maternal environment on early gonadal differentiation. In this study, we examined gonadal and reproductive duct differentiation in red stingray (Hemitrygon akajei) embryos before and after clasper formation through detailed histological analyses. Histological observations revealed that early embryos (stage 3, before clasper formation) exhibited undifferentiated gonads with scattered primordial germ cells, whereas later embryos (stage 4, during clasper development) exhibited clear sexual dimorphism. Female gonads contained numerous germ cells localized in the dorsal region, whereas male gonads contained fewer scattered germ cells. Both the Müllerian and Wolffian ducts were present in both sexes, with the Wolffian ducts emerging from the Müllerian ducts in a segmental pattern. Stage 4 female embryos exhibited a selective enlargement of the right Müllerian duct, establishing reproductive tract asymmetry. The sex ratio of red stingray embryos was 1:1. These findings provide the first detailed histological characterization of gonadal differentiation and reproductive duct development in a viviparous chondrichthyan and establish an essential foundation for future studies on sexual development in these evolutionarily significant vertebrates.