The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system

In reptiles,such as the red-eared slider turtle(Trachemys scripta elegans),gonadal sex determination is highly dependent on the environmental temperature during embryonic stages.This complex process,which leads to differentiation into either testes or ovaries,is governed by the finely tuned expression of upstream genes,notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2.Recent studies have identified epigenetic regulation as a crucial factor in testis development,with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T.s.elegans.However,whether KDM6B alone can induce testicular differentiation remains unclear.In this study,we found that overexpression of Kdm6b in T.s.elegans embryos induced the male development pathway,accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C,a temperature typically resulting in female development.Notably,this sex reversal could be entirely rescued by Dmrt1 knockdown.These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway,underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

红耳龟Amh基因在温度依赖型性别决定中的功能

为研究Amh基因在TSD(Temperature-dependent sex determination,温度依赖型性别决定)中的功能,文章以红耳龟Trachemys scripta为TSD动物模型,分析了Amh在胚胎性腺中的精细表达特征和细胞定位;通过基因功能缺失和获得研究手段,验证了Amh在TSD中的具体功能。表达分析结果显示,Amh基因在性腺分化启动前的第15期便已呈现产雄温度(Male-producing temperature,MPT)性腺高表达;AMH蛋白主要定位在MPT性腺sertoli前体细胞上,而在各个发育时期的FPT(Female-producing temperature,产雌温度)性腺中仅检测到极其微弱的Amh mRNA和蛋白表达信号。RNA干扰实验显示,敲低Amh后的MPT性腺出现了雄性向雌性完全性逆转,雄性分化因子Sox9明显下调,雌性分化因子Foxl2和Cyp19a1显著上调;相反地,异位表达Amh后的FPT性腺则转向睾丸方向分化,Foxl2和Cyp19a1表达被抑制,Sox9表达上升。上述研究表明,Amh是启动红耳龟早期睾丸分化必需且充分的关键因子,处于TSD雄性分化分子通路上游。

Sex ratio variation and sex determination in the mallee dragon Ctenophorus fordi

Recent evidence suggests that many Australian agamids show temperature-dependent sex determination(TSD)with variation in sex determining mechanisms among closely related taxa.However,as shown in other vertebrates,sex ratios can also be influenced by genetic or phenotypic differences among females in their propensity to produce sons or daughters,and these influences might confound any thermal effects of incubation per se.To address these issues,we investigated the determinants of sex ratios in the mallee dragon Ctenophorus fordi,together with a detailed analysis of karyotypes.There was no detectable variation in sex ratios arising from variation among females,clutches or incubation temperatures,which might indicate genetic sex determination for this species.However,there was no evidence of cytologically distinct sex chromosomes using standard banding techniques.The sex ratio pattern in C.fordi strongly contrasts with the results for the congener Ctenophorus pictus,where sex ratios show variation among females.Thus,Australian agamids offer promising opportunities to address fundamental issues in sex ratio biology.

遗传和温度因素对鱼类性别分化相关基因表达及性别比例的影响

鱼类的性别决定机制较为复杂,是遗传和环境共同作用的结果,具有很大的可塑性。随着分子生物技术的不断更新,对鱼类性别决定及分化相关基因的鉴定和研究有了新的进展,环境因子如温度、光照、p H、低氧、水压等均能影响大多数鱼类的性别决定和分化过程。本研究中,阐述了鱼类遗传型性别决定(GSD)和温度依赖型性别决定(TSD)方式,并对TSD与遗传型性别加温度影响(GSD+TE)性别决定机制下,性别决定相关基因在雌雄异型中的表达及结合温度诱导性逆转进行概述,分析了温度对鱼类性别比例的影响,并提出今后应加强对性别决定基因的鉴定、解析和基因功能研究,为科学制定鱼类繁殖计划提供指导。

龟鳖类温度依赖型性别决定机制的研究进展

近几十年来,发现很多爬行动物具有温度依赖型性别决定机制(TSD),即性别分化取决于胚胎发育过程中温度敏感期(TSP)的环境温度高低。龟鳖类存在两种TSD模式,即低温产雄性、高温产雌性的TSDⅠa,低温、高温均产雌性而中间温度产雄性的TSDⅡ。TSD机制在生理水平的作用机制主要受到性腺类固醇激素的控制,温度通过影响芳香化酶和5α-还原酶的活性控制雌、雄激素转化,进而决定了个体的性别分化。在分子水平的研究发现:Sf1、Mis、Sox9、Dax1、Wt1和Dmrt1等基因的表达受到温度的影响,参与了龟鳖类性别分化。介绍了关于TSD的进化意义提出的假说,有待进一步验证。

Sox9在温度依赖型性别决定中的功能初探

在一些爬行动物中,个体的性别完全取决于胚胎发育过程中的环境温度,称之为温度依赖型性别决定(temperaturedependent sex determination,TSD).TSD的分子机制长期是个谜,特别是调控早期性腺分化的分子基础仍不清楚.本文通过表达分析和基因敲低手段研究了Sox9基因在红耳龟雄性性腺分化中的生物学功能,为TSD动物的性别决定和性腺发育的分子机制的研究奠定了基础.qRT-PCR显示,从性腺分化前的17期起,Sox9呈现产雄温度(male-producing temperature,MPT)性腺特异性高表达,而在产雌温度(female-producing temperature,FPT)性腺中表达水平极低.免疫组化进一步证实了SOX9蛋白的MPT特异性表达趋势,其定位于Sertoli前体细胞核中.温度置换实验显示,与MPT性腺相比,MPT→FPT性腺中(16期置换)的Sox9表达量从17期起就显著降低,表明Sox9能快速响应温度变化.同时MPT性腺经过雌激素处理后,Sox9表达量亦快速下调.功能缺失研究显示,经过Sox9-RNAi处理后,90.9%(20/22)的MPT性腺结构明显雌性化,皮质区高度发育,髓质区退化,揭示Sox9的敲低能导致雄性向雌性性逆转.上述研究表明,Sox9是红耳龟早期睾丸分化的关键调控因子,参与TSD的雄性分化通路.

温度依赖型性别决定在爬行动物中的研究进展

爬行动物性别决定方式主要有遗传依赖型性别决定(genetic sex determination,GSD)和环境依赖型性别决定(environmental sex determination,ESD),而ESD又以温度依赖型性别决定(temperature sex determination,TSD)为主。研究爬行动物TSD有助于人们弄清楚环境条件对物种表型的影响,从而更好地利用环境条件和遗传基础的共同机制来人为的改善或者诱导具TSD型物种的进化方向,以实现自然和人类的最大利益。该篇综述从母系活动、气候变化(全球气候变暖)、类固醇以及TSD机制四个方面总结了近年来关于爬行动物TSD的最新研究。

鱼类性别调控机制的研究进展

有性生殖生物的性别发育主要分为性别决定和性别分化两个阶段。鱼类的性别决定机制复杂,即使是关系密切的物种也有不同的性别决定系统,一般归结于两种模式:遗传决定模式(GSD)和环境决定模式(ESD)。此外,环境决定模式还有两种主要类型:温度依赖型性别决定类型(TSD)和行为性别决定类型(BSD)。笔者对上述两种决定模式及类型进行了综述,同时涉及鱼类性逆转现象。

The sex ratio of wild Chinese alligators Alligator sinensis

The Chinese alligator Alligator sinensis is one of the most endangered crocodilian species, and typically exhibits temperature-dependent sex determination. It is extremely important to clarify the sex structure of Chinese alligators to implement recovery projects successfully. However, the sex ratio of wild Chinese alligators remains unknown. In this study, we collected 28 years of sex ratio data from Chinese alligators residing in the natural and artificial habitats of Changxing Nature Reserve, China, and examined the differences in the sex ratio dynamics between these two populations. We observed that the sex ratio of wild Chinese alligators is 1 male to 4.507 females, which was significantly lower compared to that of the captive population （1 to 2.040; P 〈 0.001）, and is significantly different to previously documented sex ratios for this species （all P 〈 0.01）. Furthermore, we documented an annually stable （P = 1.000） female-biased sex ratio for wild alligators at hatching [1 male to 4.747 females; 0.174 （0.167~）.182）], in contrast to a dramatically fluctuating sex ratio （P 〈 0.001） in captivity [1 male to 1.674 females; 0.374 （0.246-0.593）], showing a potential mechanism for adjusting the sex structure. Finally, we found that the hatchling sex ratios were similar to that of the population sex ratio （P = 0.748）, with little correlation to air temperature values in the 60-70 day incubation period during the breeding season （July and August; both P 〉 0.05）. Overall, this study indicates that the stabilized female-biased sex ratio of Changxing Chinese alligators might result from selection pressure caused by local mate competition and major inbreeding .

Dmrt1基因在红耳龟温度依赖型性别决定中的功能研究

除了基因型性别决定(GSD)外,温度依赖型性别决定(TSD)是另一种重要的动物性别决定方式,但其分子机制一直是个谜团.本文以TSD动物红耳龟(Trachemys scripta)Dmrt1基因为研究对象,分析了Dmrt1蛋白在不同发育时期胚胎性腺中的表达情况和细胞定位;通过慢病毒-sh RNA干扰系统对Dmrt1在TSD中的作用进行了功能验证.免疫荧光染色结果显示,Dmrt1蛋白在性腺分化之前的第15期产雄温度(male-producing temperature,MPT)性腺中开始表达,第19期后急剧增加,定位于Sertoli前体细胞的细胞核上,在整个产雌温度(female-producing temperature,FPT)性腺发育过程中几乎检测不到信号.RNA干扰实验表明,MPT胚胎经过Dmrt1-sh RNA处理后,95.34%发育成雌性或具有卵睾丸的间性(对照组MPT胚胎100%发育成雄性个体,对照组FPT胚胎100%发育成雌性个体).Dmrt1基因敲低后,MPT胚胎性腺外形和组织结构明显雌性化,生殖细胞从髓质区向皮质区转移,雄性特异性蛋白Sox9表达显著降低(甚至消失),而雌性特异性蛋白芳香化酶则开始大量表达,呈现典型的雄性向雌性逆转现象.上述研究结果表明,Dmrt1基因在TSD中是必需的,是启动未分化性腺向雄性分化的关键因子,位于TSD分子信号通路上游.本文首次对TSD进行了关键基因的功能分析,为TSD分子机制研究开辟了一条新途径.

红耳龟性别分化相关基因的筛选与分析

温度依赖型性别决定(temperature-dependent sex determination, TSD)是一种奇特的表型可塑性现象,但其背后的分子机制尚不清楚。红耳龟(Trachemysscripta)是一种典型TSD动物,个体性别完全由胚胎发育时所处的环境温度决定。为提供更多能够解释TSD机制的基础数据,选取红耳龟第16期产雄温度(male-producing temperature, MPT)和产雌温度(female-producing temperature, FPT)性腺,采用IlluminaHiseq 2500高通量平台进行转录组测序,筛选出雌雄差异表达基因474个,其中,MPT高表达基因283个,FPT高表达基因191个。通过与NR、Swiss-Prot、GO、COG、KOG、KEGG数据库进行比对,获得60个GO功能注释分类和139条KEGG代谢通路。根据差异表达基因的功能注释分析发现:参与红耳龟性别决定和分化的候选基因,包括Dmrt1、Sox9、Foxl2等高度保守的性别分化基因,Cyp11b1、Hsd11b2等类固醇激素代谢相关基因,以及Hspb6、Dnaja4、Serpinh1等热敏性蛋白编码基因。最后,通过荧光定量PCR和免疫荧光染色对部分候选基因进行表达验证,与转录组结果相一致。该研究挖掘了一些新的温度响应因子和激素代谢相关因子,为破译红耳龟TSD分子机制提供了基础。