雌激素受体Esr1诱导红耳龟早期卵巢分化

Estrogen receptor Esr1 induces early ovarian differentiation in Trachemys scripta

摘要:

为探究3种雌激素受体(estrogen receptor) Esr1、Esr2和Gper1在红耳龟早期胚胎性腺分化中的作用，本研究在分析受体基因表达特征的基础上，通过向性腺分化启动前的产雄温度(male-producing temperature, MPT)龟胚分别注射Esr1、Esr2和Gper1激动剂PPT、WAY 200070和G-1，从性腺形态结构、生殖细胞分布模式、性别分化关键基因和蛋白表达分布方面对处理后的胚胎性腺进行了性逆转分析。表达分析结果显示，esr1在性别分化关键时期性腺中的表达量显著高于esr2和gper1 (表达极低)，且呈现产雌温度(female-producing temperature, FPT)性腺高表达。功能验证实验显示，PPT处理后的MPT性腺形态结构明显雌性化，生殖细胞呈现雌性分布模式；雄性分化关键基因dmrt1、amh和sox9 mRNA表达明显下降，雌性分化关键基因foxl2和cyp19a1 mRNA表达则显著上升；Amh和Sox9蛋白表达的荧光信号几乎消失，Foxl2和Arom蛋白被激活出现大量表达，表明性腺由雄性逆转为雌性(性逆转率：70.27%)。而WAY 200070和G-1处理后的MPT性腺仍分化为睾丸，雌雄基因和蛋白的表达及分布与雄性性腺类似。结果表明在红耳龟中，单独激活Esr1能够充分启动早期性腺的雌性分化过程，提示雌激素可能通过其受体1 (Esr1)诱导早期卵巢分化。本研究为进一步解析雌激素在龟性别决定和分化中的调控机制提供了参考。

Abstract:

This study aims to explore the roles of three estrogen receptors (Esr1, Esr2, and Gper1) in early differentiation of embryonic gonads of Trachemys scripta. The expression characteristics of the receptor genes were studied first. The Esr1, Esr2, and Gper1 agonists PPT, WAY 200070, and G-1 were respectively injected into the embryos at the male-producing temperature (MPT) before initiation of gonadal differentiation. The sex reversal of the treated embryonic gonads was analyzed in terms of morphological structure of gonads, distribution pattern of germ cells, and expression of key genes and proteins involved in sex differentiation. The expression level of esr1 during the critical stage of sex differentiation was higher than those of esr2 and gper1(very low expression) and was particularly high in the gonads at the female-producing temperature (FPT). After treatment with PPT, the MPT gonads presented obviously feminized morphology and structure, with the germ cells exhibiting a female distribution pattern. Furthermore, the mRNA expression levels of the key genes (dmrt1, amh, and sox9) for male differentiation were down-regulated significantly, while those of the key genes (foxl2 and cyp19a1) for female differentiation were up-regulated observably. The fluorescent signals of Amh and Sox9 expression almost disappeared, while Foxl2 and Arom were activated to express abundantly, which fully demonstrated the sex reversal of the gonads from male to female (sex reversal rate: 70.27%). However, the MPT gonads treated with WAY 200070 and G-1 still differentiated into testes, and the expression patterns of the key genes and proteins were similar to those in male gonads. The above results demonstrate that activation of Esr1 alone can fully initiate the early female differentiation process of gonads, suggesting that estrogen may induce early ovarian differentiation via Esr1 in Trachemys scripta. The findings provide a basis for further revealing the mechanisms of estrogen regulation in sex determination and differentiation of turtles.