半滑舌鳎rimoc1基因在雌雄鱼中的差异表达

Differential expression of the rimoc1 gene in male and female Chinese tongue sole (Cynoglossus semilaevis)

中文关键词: 半滑舌鳎  rimoc1  性别生长二态性  表达模式  启动子活性  RNA干扰

英文关键词: Cynoglossus semilaevis  rimoc1  Sexual growth dimorphism  Expression pattern  Promoter activity  RNA interference

中文摘要:

      动物中普遍存在性别生长二态性，这种现象在海水鱼中更为明显。半滑舌鳎(Cynoglossus semilaevis)性成熟雌鱼体重显著高于雄鱼，雌性个体的生长速度比雄性快2~4倍。本研究旨在探究rimoc1基因在半滑舌鳎雌、雄鱼中的表达差异及与性别和生长的关系。实时定量和原位杂交结果显示，rimoc1在雄性组织中未检测到表达，而在雌性组织中表达量最高的是卵巢和肌肉，在卵巢中表达相对稳定，在孵化后1.5年(1.5 yph)和3 yph的水平有所增加。在细胞系中通过siRNA干扰敲除rimoc1后，发现生长相关基因igf1、性别分化相关基因sox9b和foxl2的表达水平显著降低，而sox9a的表达升高。启动子活性分析显示，rimoc1启动子可能受到C/EBPdelta、Sox2和c-Jun等转录因子的调控。推测rimoc1可能在半滑舌鳎的性别分化和生长中发挥重要作用，为深入研究半滑舌鳎的性别生长二态性提供了基础。

英文摘要:

      Chinese tongue sole (Cynoglossus semilaevis) is a traditional, nutrient rich and economically valuable fish that is important for mariculture in China. It has small visceral masses, few spines, with tender and tasty flesh. Studies on the sexing mechanism of fish were carried out since the revelation of sexual reversal and hermaphroditism in the eel. Sex is the most common biological phenomenon and there are significant differences in morphology, reproductive strategies, and behavior between females and males of many organisms. Sole is a typical representative of the heterogeneous growth of males and females: adult females are approximately 2–4 times larger than males. This has constrained the sustainable development of the sole culture industry. Why do females grow faster than males? Do sex differentiation-related genes play a role in sex determination and size heteromorphism in sole? Answering these questions has important implications and applications in the genetic improvement and development of sole farming. Rimoc1 encodes a protein in the inner mitochondrial membrane that is involved in regulating the activity of complex Ⅰ of the mitochondrial respiratory chain that affects mitochondrial respiratory function. Rimoc1 interacts with the NDUFAF1 subunit of complex Ⅰ in the inner mitochondrial membrane and enhances its affinity for NADH. This increases the catalytic activity of complex Ⅰ and increases the rate of electron transfer in the mitochondrial respiratory chain. In addition, RIMOC1 interacts with proteins in other mitochondrial respiratory chain complexes (such as complex Ⅲ and complex Ⅳ) and may play a role in regulating the overall stability and function of the mitochondrial respiratory chain. This study aimed to investigate the expression differences of the rimoc1 gene in male and female Chinese tongue sole and determine its potential relationship with sex and growth. Rimoc1 expression was highest in the ovaries and muscles of females; it was not expressed in male tissues. Interestingly, its expression was relatively stable in the ovaries, but increased at 1.5- and 3-years post-hatching. Knocking out rimoc1 through siRNA interference in cell lines significantly reduced the expression levels of growth-related gene igf1, and sex differentiation-related genes sox9b and foxl2, while sox9a expression increased. Promoter activity analysis further demonstrated that the rimoc1 promoter strongly responded to transcription factors C/EBPdelta, Sox2, and c-Jun. This suggested their potential role in regulating rimoc1. Sex determination in semislipped sole occurs at approximately 50 days, while gonadal differentiation begins at 60 days. However, differentiation at the cellular level is relatively delayed, with ovarian cell differentiation usually occurring at 90–120 days with the emergence of an ovarian cavity followed by continued oocyte differentiation and sexual maturity at approximately 2 years of age. Thus, high rimoc1 expression at 90 days is consistent with the differentiation of naturally growing ovarian cells in semi-smooth tongue sole. Analysis of promoter activity showed that rimoc1 was transcriptionally active at 981 bp upstream and had strong initiation activity. The activity was enhanced following cotransfection with three transcription factors (C/EBPalpha, Sox2, and C-Jun), although only the C/EBPalpha group was significantly different. This suggested that these three transcription factors exert an enhancing effect on rimoc1 transcription. It is known that C/EBPalpha plays a major role in keratinocyte and adipocyte differentiation. However, its regulation of rimoc1 in hemipterous sole requires further investigation. There are few reports on the function of rimoc1 in sexual differentiation. Therefore, we performed the first functional investigation of rimoc1 by siRNA-mediated RNA interference in an ovarian cell line. Quantitative polymerase chain reaction showed that the expression levels of igf1, sox9a, sox9b, and foxl2 changed at different degrees after rimoc1 gene interference compared to the control group; sox9a expression was up-regulated, while the expression of igf1, sox9b, and foxl2 was down-regulated. Fish igf1 is closely related to reproductive function and gonadal development. Tilapia igf1 expression was detected from the early stages of oogenesis till oocyte maturation. At the same time, Igf1 is involved in regulating growth hormones and growth. Sexual growth dimorphism begins to appear in one-year old half-sauropod sole. The quantification of different developmental stages shows that rimoc1 expression starts to increase at 180 days until its peak expression at 1.5 years of age. It is worthwhile investigating whether rimoc1 has a potential regulatory function in growth. Sox9 is a key transcription factor that plays an important role in regulating the proliferation and differentiation of various cell types. It has a critical role in embryonic development, with expression occurring throughout this period. Its level is maintained in adult tissues and plays an important role in the repair of postnatal damage in endodermal and ectodermal organs. It has a role in sex differentiation, with sox9a and cyp19a together forming a regulatory pathway for sexual differentiation. We hypothesize that rimoc1 is involved in ovarian development and oogenesis by regulating the steroid hormone pathway. Sox9b is highly expressed in the gonads of early semi-smooth sole, and rimoc1 knockdown results in its reduced expression. This suggests that Rimoc1 and Sox9b act together to regulate Foxl2 and play a role in sex determination and differentiation, ovarian development and maintenance, embryonic development, and immune regulation in the animal. The foxl2 gene positively regulates ovarian differentiation in mice, and its sustained expression inhibits abnormal differentiation of ovarian cells to testicular cells during growth and development, thereby maintaining the female sex. Meanwhile foxl2 deletion can lead to poor ovarian development, and even female sterility. Rimoc1 knockdown results in its reduced expression, suggesting that rimoc1 may be closely associated with ovarian development in hemipelagic sole. Therefore, a deeper understanding of the function and regulatory mechanisms of rimoc1 may be the key to resolving sexual growth dimorphism. Our results suggest that rimoc1 plays a crucial role in sex differentiation and growth of Chinese tongue sole. These findings provide a foundation for further investigations into the sexual growth dimorphism of this species.