Tropical Futures Institute, James Cook University Singapore, Singapore.
Centre for Sustainable Tropical Fisheries and Aquaculture, College of Science and Engineering, James Cook University, Townsville, Queensland, Australia.
PLoS One. 2018 Sep 18;13(9):e0204182. doi: 10.1371/journal.pone.0204182. eCollection 2018.
Epigenetics is involved in sex differentiation of gonochoristic and hermaphroditic fish species, whereby two genes dmrt1 (pro-male) and cyp19a1 (pro-female) are known to play major roles. Barramundi, Lates calcarifer, is an important tropical aquaculture species that undergo natural and permanent male to female sex change, a process for which the exact underlying molecular mechanisms are still unknown. To elucidate whether DNA methylation is involved in sex control of barramundi, a next-generation bisulfite amplicon sequencing approach was used to target 146 CpG sites within proximal promoters and first exons of seven sex-related genes (dmrt1, cyp19a1, amh, foxl2, nr5a2, sox8 and sox9) of 24 testis and 18 ovaries of captive and wild adult barramundi. Moreover, comparative expression profiles of the key dmrt1 and cyp19a1 genes were further investigated using RT-qPCR and Sanger sequencing approaches, whereas expression levels of remaining targeted genes were based on available literature for the species. Results showed that cyp19a1 and amh were more methylated in males, whereas dmrt1 and nr5a2 were more methylated in females (P < 0.001), with no gender differences found for foxl2, sox8 or sox9 genes (P > 0.05). Sex-biased promoter DNA methylation was inversely related to gene expression only for dmrt1 and nr5a2, and directly related to amh expression, whereas no differences in cyp19a1 expression were found between testes and ovaries. Notably, unique sex-specific alternative splicing of dmrt1 and cyp19a1 were discovered, whereby males lacked the full-length aromatase coding cyp19a1 mRNA due to partial or total exon splicing, and females lacked the dmrt1 exon containing the DM-domain sequence. This study advances the current knowledge aiming to elucidate the genetic mechanisms within male and female gonads of this large protandrous hermaphrodite by providing the first evidence of epigenetics and alternative splicing simultaneously affecting key genes (cyp19a1 and dmrt1) central to sex differentiation pathways.
表观遗传学参与了雌雄同体和雌雄异体鱼类的性别分化，其中两个基因 dmrt1（雄性前体）和 cyp19a1（雌性前体）被认为起着主要作用。金目鲈，Lates calcarifer，是一种重要的热带水产养殖物种，它经历自然和永久性的雄性到雌性的性别转变，其确切的潜在分子机制尚不清楚。为了阐明 DNA 甲基化是否参与金目鲈的性别控制，我们使用下一代亚硫酸氢盐扩增子测序方法，针对 24 个圈养和野生成体金目鲈的 24 个睾丸和 18 个卵巢中的 7 个性别相关基因（dmrt1、cyp19a1、amh、foxl2、nr5a2、sox8 和 sox9）的近端启动子和第一外显子中的 146 个 CpG 位点进行了靶向研究。此外，还使用 RT-qPCR 和 Sanger 测序方法进一步研究了关键基因 dmrt1 和 cyp19a1 的表达谱，而针对该物种的其余靶向基因的表达水平则基于现有文献。结果表明，cyp19a1 和 amh 在雄性中甲基化程度更高，而 dmrt1 和 nr5a2 在雌性中甲基化程度更高（P<0.001），foxl2、sox8 或 sox9 基因在性别间无差异（P>0.05）。性别偏向的启动子 DNA 甲基化仅与 dmrt1 和 nr5a2 的基因表达呈负相关，与 amh 表达呈直接相关，而 cyp19a1 在睾丸和卵巢之间的表达没有差异。值得注意的是，我们发现了 dmrt1 和 cyp19a1 的独特性别特异性选择性剪接，即由于部分或完全外显子剪接，雄性缺乏全长芳香酶编码 cyp19a1 mRNA，而雌性缺乏包含 DM 结构域序列的 dmrt1 外显子。这项研究通过提供同时影响关键基因（cyp19a1 和 dmrt1）的表观遗传学和选择性剪接的证据，为阐明这个大型雄性先熟雌雄同体鱼类的雌雄性腺中的遗传机制提供了新的认识，同时也为阐明遗传机制提供了新的认识。
