Our previous observations proposed Pelophylax nigromaculatus as a model species for studying the masculinizing effects of androgenic EDCs in amphibians. To better develop this model species, we studied the process of the gonadal differentiation/development and the sensitive stage to androgens. We found that the earliest sexual dimorphism in gonads at morphological and histological levels occurred at stages 38–40 and stage 36 respectively. Further examination of molecular markers for testicular and ovarian differentiation during development revealed that the cyp17 and cyp19 expressions were sexually dimorphic from stage 32 and stage 36 respectively. Further, we investigated the sex-reversal induced by 100 ng/L 5α-dihydrotestosterone (DHT) when exposures were initiated at stages 24, 26 and 28. We found that when exposed from stage 24, DHT resulted masculinization of all tadpoles with no typical ovaries, whereas exposures from stage 26 or 28 dramatically reduced the effect of DHT. Our findings show that gonads of P. nigromaculatus are bipotential at stage 24, in the process of differentiation at stage 26 and determined to become either testis or ovary at stage 28. Altogether, exposure of P. nigromaculatus should begin at stage 24 in order to sensitively detect masculinizing effects of EDCs. Present study provides useful information about the gonadal differentiation and development in P. nigromaculatus for effectively evaluating masculinizing effects of EDCs on gonads.

我们之前的观察结果提出了黑腹侧耳（Pelophylax nigromaculatus）作为研究两栖动物中雄性EDC的男性化作用的模型物种。为了更好地发展这种模式物种，我们研究了性腺的分化/发育过程以及对雄激素的敏感阶段。我们发现，在性腺和形态学水平上，性腺的最早性二态性分别发生在第38–40和第36阶段。进一步检查发育过程中睾丸和卵巢分化的分子标志物表明，cyp17和cyp19的表达分别在第32期和第36期呈性别双态。此外，我们调查了在第24、26和28阶段开始暴露时100 ng / L5α-二氢睾丸激素（DHT）引起的性逆转。我们发现，从24阶段暴露时，DHT导致所有t的男性化，没有典型的t。卵巢 而来自第26或28阶段的暴露会大大降低DHT的作用。我们的发现表明，P. nigromaculatus在第24阶段分化时处于第24阶段的双电位状态，并在第28阶段被确定为变成睾丸或卵巢。总而言之，P。nigromaculatus的暴露应在第24阶段开始，以便敏感地检测EDC的男性化作用。 。本研究提供了有关性腺分化和发育中的有用信息P. nigromaculatus有效地评估对性腺的内分泌干扰物的男性化作用。