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张晓茜,贾功雪,王玉军,宋玉坤,阿布力孜.吾斯曼.2022.高原鼠兔褪黑素受体在下丘脑-垂体-性腺轴中的表达分析.动物学杂志,57(3):350-358.
高原鼠兔褪黑素受体在下丘脑-垂体-性腺轴中的表达分析
Expression of Melatonin Receptors in Hypothalamic-Pituitary-Gonadal Axis of Plateau Pika Ochotona curzoniae
投稿时间:2021-12-21  修订日期:2022-05-02
DOI:10.13859/j.cjz.202203003
中文关键词:  高原鼠兔  下丘脑-垂体-性腺轴  季节性繁殖  MTNR1A  MTNR1B
英文关键词:Plateau Pika (Ochotona curzoniae)  Hypothalamic-pituitary-gonadal axis  Seasonal reproduction  MTNR1A  MTNR1B
基金项目:中国科学院战略性先导科技专项(No. XDA2005010406),青海省重大科技专项(三江源国家公园研究院专项资金),中国科学院青年创新促进会项目(No. 2021432),青海省“昆仑英才·高端创新创业人才”项目
作者单位E-mail
张晓茜 新疆农业大学动物科学学院 乌鲁木齐 830052中国科学院西北高原生物研究所中国科学院高原生物适应与进化 重点实验室青海省动物生态基因组学重点实验室 西宁 810008 qianvet@126.com 
贾功雪 中国科学院西北高原生物研究所中国科学院高原生物适应与进化 重点实验室青海省动物生态基因组学重点实验室 西宁 810008中国科学院大学 北京 100049 jiagongxue@nwipb.cas.cn 
王玉军 中国科学院西北高原生物研究所中国科学院高原生物适应与进化 重点实验室青海省动物生态基因组学重点实验室 西宁 810008中国科学院大学 北京 100049 yjwang@nwipb.cas.cn 
宋玉坤 新疆农业大学动物科学学院 乌鲁木齐 830052 songyukun@cau.edu.cn 
阿布力孜.吾斯曼 新疆农业大学动物科学学院 乌鲁木齐 830052 abulizi68@126.com 
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中文摘要:
      褪黑素在季节性繁殖动物的生殖细胞发育与性腺功能调控中发挥重要作用,然而褪黑素如何通过下丘脑-垂体-性腺(HPG)轴实现其调控功能目前仍不清楚。因此,本研究选取典型的长日照动物——高原鼠兔(Ochotona curzoniae)作为研究对象,使用酶联免疫吸附法测定繁殖期与非繁殖期雌雄鼠兔的血清褪黑素水平昼夜变化,利用实时荧光定量PCR分析褪黑素受体基因Mtnr1a与Mtnr1b在下丘脑、垂体与性腺中的表达水平,并通过免疫荧光染色进一步确认两种受体在性腺不同类型细胞中的定位。结果显示,非繁殖期雄性鼠兔的血清褪黑素含量始终高于繁殖期,且呈现不同的昼夜变化模式;雌性鼠兔的血清褪黑素含量远低于雄性鼠兔,且在繁殖期与非繁殖期并不存在显著差异。Mtnr1a与Mtnr1b在下丘脑、垂体与性腺中均有表达,雄性鼠兔在下丘脑与垂体中表现出繁殖期与非繁殖期基因表达的显著差异,而雌性鼠兔基因表达的差异主要出现在垂体与性腺中。两种受体蛋白在雄性性腺生殖细胞和支持细胞中均有分布,但MTNR1A更局限于精原细胞内,MTNR1B则在管腔内生殖细胞中表达。雌性性腺中MTNR1A在卵母细胞胞质内有表达,但更集中表达于颗粒细胞;MTNR1B在颗粒细胞以及卵母细胞核质内均有表达,且在生长卵泡的卵泡膜细胞中呈现高表达。以上结果表明,褪黑素调控雌雄高原鼠兔季节性繁殖的模式并不相同,其作用不仅限于通过下丘脑-垂体-性腺轴的间接调控,也可能通过性腺内靶向受体直接影响生殖细胞与体细胞命运。
英文摘要:
      [Objectives] Melatonin is an important neuroendocrine amine hormone that plays a critical role in regulating gonadal function and germ cell development in seasonal breeding animals. However, the regulatory pathway of melatonin affecting seasonal reproduction through hypothalamic-pituitary-gonadal (HPG) axis is still unclear. [Methods] In the present study, Plateau Pika (Ochotona curzoniae), a representative long-day animal, was used. Adult Pikas from reproductive and non-reproductive stages were captured in May and November, respectively, and assigned to four groups: reproductive male, reproductive female, non-reproductive male and non-reproductive female. Blood samples were collected from Plateau Pikas by posterior orbital venous plexus approach every 2 h within 24 h, and serum melatonin concentrations were detected by enzyme linked immunosorbent assay. Quantitative real-time PCR was used to determine relative expression levels of Mtnr1a and Mtnr1b with reference to Gapdh in hypothalamus, pituitary and gonad of Plateau Pika. The expression and location of MTNR1A and MTNR1B in pika’s gonad were confirmed by immunofluorescence staining with a germ cell specific marker DDX4 and a DNA specific dye Hoechst 33342. The concentration of melatonin and relative expression levels of Mtnr1a and Mtnr1b were compared among groups by one-way ANOVA. [Results] Results showed that serum melatonin concentration of non-reproductive male Pikas was always higher than that of reproductive males (P < 0.05), and the two groups showed distinct diurnal dynamics. Serum melatonin concentration of female pikas was lower than that of males all the time (P < 0.05), although there was no significant difference in female pikas between reproductive and non-reproductive stages (Fig. 1). The mRNA expressions of Mtnr1a and Mtnr1b were detected with similar changing pattern. There were significant seasonal differences in Mtnr1a and Mtnr1b expressions in male hypothalami and pituitaries (P < 0.05), and seasonal differences were also found in Mtnr1a and Mtnr1b expressions in female pituitaries and ovaries (P < 0.05) (Fig. 3). Immunostaining indicated that the two receptors were widely distributed in germ cells and Sertoli cells of testis. Nevertheless, MTNR1A was more extensively expressed in spermatogonia, while more expression of MTNR1B was observed in advanced germ cells at reproductive stage. In the ovary, MTNR1A was found in oocyte cytoplasm and granulosa cells preferentially. MTNR1B expression was observed in both oocyte nucleus and cytoplasm as well as in granulosa cells. Strikingly, distinct high expression of MTNR1B was found in theca cells of growing follicles (Fig. 4). [Conclusion] In conclusion, melatonin showed gender difference effects on seasonal reproduction in Plateau Pikas, implying that its control pattern is not limited to indirect regulation through HPG axis, but it also acts directly on melatonin receptors in gonad to control the fate of germ cells and somatic cells.
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