草鱼不同月龄性腺组织学观察及性别特征基因cyp19a1a和amh的表达分析

Gonadal histology and expression analysis of sex characteristic genes in grass carp at different ages

关键词:  草鱼  性别分化  组织切片  基因表达

Key words:  Grass carp  Sexual differentiation  Tissue slicing  Gene expression

摘要:

为探讨草鱼(Ctenopharyngodon idella)性腺发生、性别分化及发育规律，本研究对1、2、3、4、5、6、12、24、36和48月龄草鱼性腺组织结构以及性别特征基因cyp19a1a和amh的表达差异进行了分析。组织切片结果显示，2月龄时，首次在生殖嵴中观察到原始生殖细胞，标志原始性腺形成。3月龄时，雌性草鱼性腺中观察到卵巢腔和卵巢小叶。4月龄时，观察到卵原细胞，表明其在3月龄时出现解剖学分化，4月龄时出现细胞学分化。4月龄雄性草鱼在性腺中观察到输精导管，5月龄时观察到精原细胞，表明其在4月龄出现解剖学分化，5月龄出现细胞学分化。12、24、36和48月龄草鱼卵巢分别处于发育的第Ⅰ、Ⅱ、Ⅲ和Ⅳ期，精巢则分别为发育的第Ⅱ、Ⅱ、Ⅲ和Ⅳ期。荧光定量结果显示，雌性特征基因cyp19a1a在卵巢中的表达量总体呈先上升后下降再上升的趋势，2月龄时显著上调(P<0.05)，3、6和48月龄时处于峰值。雄性特征基因amh在精巢中表达量总体呈先上升后下降的趋势。2月龄时显著上调(P<0.05)，5月龄时达到峰值。综上，草鱼性腺发育启动时间约为2月龄，雌雄性腺分化时间分别约为3月龄和4月龄，至4龄时雌雄性腺发育成熟。本研究结果丰富了草鱼的繁殖生理学资料，也为其性别调控技术研究奠定了基础。

Abstract:

Grass carp belongs to Cypriniformes, Cyprinidae, and Cyprinus family, and is typically referred to as the four famous domestic fishes, black carp, silver carp, and bighead carp, respectively. The annual production of Chinese grass carp is consistently among the highest, with an output of 5.905 million tons in 2022, accounting for 24.3% of total of freshwater fish production. Reproduction is the core process of fish reproduction. Normal gonad development is a prerequisite for fish reproduction. At present, there are few reports on the sex differentiation of Chinese grass carp. Studying the differentiation and development of grass carp gonads can provide a typical model for gonadal development of cyprinid fish. In contrast, the start and completion time of gonadal differentiation are one of the key factors inducing sex reversal in fish using hormones. Research on gonadal differentiation and development in grass carp can also lay the foundation for the establishment of a unisexual population of grass carp. Sex-related genes play important roles in biological processes such as sex determination, gonadal differentiation, and reproduction and development in fish. In most differentiated teleost fish, genes such as dmrt1, sox9, foxl2, amh, and cyp19a1a, are considered the key genes involved in sex regulation. cyp19a1a plays a key role in the gonadal differentiation and ovarian development of teleost fish by aromatizing androgens into estrogen via aromatase activity. This gene is specifically expressed in the gonads and is used served as a characteristic gene for sex differentiation towards females. Anti-Müllerian hormone (amh) regulates the structure of reproductive organs and the differentiation and development of reproductive cells. In fish such as carp, rainbow trout, and grass carp, the expression level of the amh gene in the testes is significantly higher than that in the ovaries, and is often used as a characteristic gene for male differentiation. To explore the sex differentiation and developmental patterns of grass carp gonads, this study analyzed the tissue structure and expression differences of sex characteristic genes cyp19a1a and amh in the gonads of grass carp at 1, 2, 3, 4, 5, 6, 12, 24, 36, and 48 months of age using tissue sectioning technology and real-time fluorescence quantification technology. The results of tissue sectioning showed that grass carp exhibited reproductive ridges at 1-month-old. At 2 months of age, primitive germ cells were first observed in the reproductive crest, marking the formation of their primitive gonads. At 3-month- old, ovarian cavities and lobules were observed in the gonads of female grass carp. At 4-month-old, oogonia was observed, indicating anatomical differentiation at 3-month-old and cytological differentiation at 4-month-old. At the age of 4 and 5 months, ductus deferens and spermatogonia were respectively observed in male grass carp gonads, indicating anatomical differentiation at 4 months and cytological differentiation at 5 months. The ovaries of 24, 36, and 48-month-old grass carp were in the first, second, third, and fourth stages of development, respectively, while the testes were in the second, second, third, and fourth stages of development, respectively. At 48 months of age, the gonads of grass carp have matured. The fluorescence quantitative results showed that the expression level of the female characteristic gene cyp19a1a in the ovary showed an overall trend of first increasing, then decreasing, and then increasing again. The expression level of this gene was significantly upregulated at 2 months of age (P<0.05), and reached its peak at 3, 6, and 48 months of age. During these three periods, anatomical changes were observed in the female gonads in the tissue sections, with a large proliferation of oocytes and the gonads tending to mature. The expression level of the male characteristic gene amh in the testes showed an overall trend of first increasing and then decreasing. At 2 months of age, the expression level of this gene was significantly upregulated (P<0.05). At 5 months of age, it reached the peak when there were a large proliferation of spermatogonia in the gonads, and then remained low expression until gonadal maturity. In summary, this study confirmed that the initiation time of grass carp gonadal development is approximately 2 months old, and the differentiation time of male and female gonads is approximately 3 and 4 months old, respectively. By the age of 6 months, both male and female grass carp gonads completely differentiated. These results not only enrich the reproductive physiology data of grass carp but also provide a reference basis for the study of hormone-induced sex reversal technology in grass carp.