Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues.

African clawed frog Xenopus sp. is used extensively for developmental biology and toxicology research. Amid concerns of environmental pollutants disrupting endocrine systems and causing altered reproductive development in wildlife, eco-toxicology research has led to a focus on linking molecular initiating events to population-level effects. As such, efforts to better understand reproductive development at the molecular level in these model species are warranted. To that end, transcriptomes were characterized in differentiating Xenopus tropicalis gonad tissues at Nieuwkoop and Faber (NF) stage 58 (pro-metamorphosis), NF66 (completion of metamorphosis), one week post-metamorphosis (1WPM), and two weeks post-metamorphosis (2WPM). Differential expression analysis between tissue types at each developmental stage revealed a substantial divergence of ovary and testis transcriptomes starting between NF58 and NF66; transcriptomes continued to diverge through 2WPM. Generally, testis-enriched transcripts were expressed at relatively constant levels, while ovary-enriched transcripts were up-regulated within this developmental period. Functional analyses of differentially expressed transcripts allowed linkages to be made between their putative human orthologues and specific cellular processes associated with differentiating gonad tissues. In ovary tissue, genetic programs direct germ cells through meiosis to the diplotene stage when maternal mRNAs are transcribed and trafficked to oocytes for translation following fertilization. In the testis, gene expression is consistent with connective tissue development, tubule formation, and germ cell support (Leydig and Sertoli cells). This dataset exhibited remarkable consistency with transcript profiles previously described in gonad tissues across species, and emphasizes the universal importance of certain transcripts for germ cell development and preparation of these tissues for reproduction. Four developmental time-points were considered for global expression analysis: NF58 (pro-metamorphosis), NF66 (completion of metamorphosis), one week post-metamorphosis (1WPM), and two weeks post-metamorphosis (2WPM). Gonad tissues were time-matched and stage-matched, so collection occurred on a single day for each developmental time-point and all animals used were from the same spawn. For the 1WPM and 2WPM time-points, all animals reached NF66 on the same day and were separated from the rest of the spawn until sample collection one and two weeks later respectively. On the day of tissue collection, animals were developmentally staged (Nieuwkoop and Faber, 1994) and euthanized by submersion in a lethal concentration of tricane methanesulfonate ([MS-222], Argent Chemical Laboratories, Redmond, WA, USA) buffered with sodium bicarbonate. Gonad tissues were then excised independently of any other tissues (i.e. “pure” gonad tissue) and transferred to a separate vial for each individual, homogenized in lysis buffer from an RNeasy micro kit (Qiagen Inc., Valencia, CA, USA), and immediately frozen on dry ice until samples could be moved to -80oC storage. Tail or toe tissue was also collected from each individual for genetic sex determination using methods previously described by Olmstead et al. (2010). Once genetic sex was determined for each individual, testes and ovaries were randomly assigned to pools of four individuals (eight gonads per pool) to make a total of five pools of testis tissue and five pools of ovary tissue at each developmental time-point, with the exception of NF58 which had four pools of testis tissue and four pools of ovary tissue (38 total sample pools).

非洲爪蟾（Xenopus sp.）被广泛应用于发育生物学与毒理学研究领域。鉴于环境污染物会干扰野生动物内分泌系统并引发生殖发育异常的担忧，生态毒理学研究的重点逐渐转向将分子起始事件与种群层面效应建立关联。因此，深入解析这类模式物种生殖发育的分子机制具有重要意义。为此，本研究对不同发育阶段的热带爪蟾（Xenopus tropicalis）性腺组织的转录组（transcriptome）进行了表征，涵盖的发育节点包括Nieuwkoop-Faber（NF）58期（前变态期）、NF66期（变态完成期）、变态后1周（1WPM）以及变态后2周（2WPM）。对各发育阶段的组织进行差异表达分析后发现，卵巢与睾丸的转录组差异在NF58至NF66阶段间已显著显现，并在直至2WPM的过程中持续扩大。整体而言，富集于睾丸的转录本表达水平相对稳定，而富集于卵巢的转录本在该发育阶段中呈现上调趋势。对差异表达转录本的功能分析显示，其推定的人类同源基因可与性腺分化相关的特定细胞过程建立关联。在卵巢组织中，遗传程序引导生殖细胞经减数分裂进入双线期，此时母源mRNA会被转录并转运至卵母细胞，以供受精后翻译使用。在睾丸组织中，基因表达模式与结缔组织发育、小管形成以及生殖细胞支持（莱迪希细胞与支持细胞）的过程相符。本数据集与此前跨物种性腺组织转录谱的研究结果具有高度一致性，凸显了部分转录本在生殖细胞发育及性腺生殖功能准备过程中的普遍重要性。本研究选取4个发育时间点开展全局表达分析：NF58期（前变态期）、NF66期（变态完成期）、变态后1周（1WPM）以及变态后2周（2WPM）。所有性腺组织均经过时间与发育阶段匹配，即每个发育时间点的样本均在同一天采集，且所有实验动物均来自同一批产卵个体。针对1WPM与2WPM两个时间点，所有实验动物均在同一天达到NF66期，并从同批次产卵个体中分离出来，分别于1周和2周后进行样本采集。样本采集当日，先根据Nieuwkoop与Faber（1994）的标准对动物进行发育阶段鉴定，随后将其浸没于经碳酸氢钠缓冲的致死浓度三甲磺酸三卡因（tricaine methanesulfonate，[MS-222]，Argent Chemical Laboratories，美国华盛顿州雷德蒙德市）中实施安乐死。随后独立剥离性腺组织（即获得"纯净"性腺组织），将每只个体的组织转移至独立离心管中，用RNeasy微量试剂盒（Qiagen Inc.，美国加利福尼亚州瓦伦西亚市）的裂解液进行匀浆，立即置于干冰上冷冻，直至样品转移至-80℃冰箱保存。同时采集每只个体的尾部或趾部组织，用于按照Olmstead等人（2010）报道的方法进行遗传性别鉴定。完成每只个体的遗传性别鉴定后，将睾丸与卵巢分别随机分组，每4只个体的性腺（即每样本池包含8个性腺）混合为一个样本。每个发育时间点共设置5个睾丸样本池与5个卵巢样本池，仅NF58期除外：该阶段仅设置4个睾丸样本池与4个卵巢样本池，总样本池共计38个。