金钱鱼促性腺激素释放激素的克隆及其在生殖调控中的功能研究
发布时间：2018-09-11 15:09
【摘要】：促性腺激素释放激素(Gonadotropin-releasing hormone,GnRH)是由下丘脑分泌的一种肽类激素,与其相应受体结合后可以促进垂体合成和释放促性腺激素(gonadotropin hormone,GtH),GtH包括促黄体素(luteinizing hormone,LH)和促卵泡素(follicle stimulating hormone,FSH),对动物生殖具有非常重要的作用。在研究克隆了金钱鱼三种GnRH亚型(sbGnRH,c GnRH-Ⅱand sGnRH)的全长cDNA序列。采用RT-PCR的方法检测了GnRH在各个组织中的表达模式;应用荧光定量PCR技术检测了金钱鱼胚胎发育和性腺发育过程中GnRH的表达模式,同时检测了离体(0.1,1和10mM)和在体(0.001,0.01和0.1mg/g体重)条件下三种GnRH多肽对垂体FSH和LH mRNA表达的影响,以及雌二醇(E2)对下丘脑GnRH mRNA表达的影响。主要结果如下:主要的结果如下:1.金钱鱼促性腺激素释放激素基因克隆及序列分析金钱鱼三种GnRH亚型被克隆,其中sbGnRH的全长为334bp,开放阅读框为288bp,编码95个氨基酸;cGnRH-Ⅱ的全长为284bp,开放阅读框为258bp,编码85个氨基酸;sGnRH的全长为288bp,开放阅读框为273bp,编码90个氨基酸。三种GnRH氨基酸序列的同源性比对发现,金钱鱼cGnRH-Ⅱ是最保守的GnRH类型,s GnRH次之,sbGnRH最不保守。进化树分析发现,金钱鱼的三种GnRH的亲缘关系与鲈形目鱼类最近,与哺乳类最远。2.金钱鱼促性腺激素释放激素基因的时空表达模式在所检测的12种组织(垂体、胃、脑、卵巢、精巢、肾脏、肝脏、肠、脾、鳃、心和肌肉)中,sbGnRH在雌雄金钱鱼的所有组织中都表达,且表达量存在组织差异,而cGnRH-Ⅱ和s GnRH仅在雌雄金钱鱼的脑和性腺中表达,暗示sbGnRH、cGnRH-Ⅱ和sGnRH可能均与生殖调控密切相关,而sbGnRH可能发挥更为广泛的功能;在胚胎发育过程中,三种GnRH都在原肠胚期间高表达,并在原肠胚晚期达到峰值,而在其他胚胎发育时期则表达量很低,暗示着这三种GnRH可能都参与调控原肠胚的发生;在个体发育过程中,随着性腺的成熟,sbGnRH的表达逐渐升高,而c GnRH-Ⅱ和sGnRH的表达量无显著变化,暗示着在金钱鱼的性腺发育和生殖调控主要由sbGnRH来调控。3.GnRHs对FSH和LH mRNA表达的影响在体实验显示,金钱鱼腹腔注射不同剂量(0.001,0.01和0.1mg/g体重)的sbGnRH,3h和6h后,三种剂量的sbGnRH均可显著促进FSH m RNA的表达,低、中剂量(0.001,0.01mg/g体重)sbGnRH可促进LH mRNA的表达,但高剂量(0.1mg/g体重)sbGnRH处理对LH mRNA的表达无显著影响;低、中剂量sGnRH对FSH和LH mRNA表达无显著影响,而高剂量sGnRH可显著促进FSH(3,6h)和LH(3h)mRNA的表达,但对6h的LH mRNA表达无影响;与sGnRH作用类似,低、中剂量cGnRH-Ⅱ对FSH和LH mRNA表达无显著影响,处理3h后高剂量cGnRH-Ⅱ可显著促进FSH和LH mRNA的表达,但是处理6h后则没有影响。离体实验显示,以10μM sbGnRH离体孵育金钱鱼垂体,3h和6h后,FSH和LH mRNA的表达显著升高,但是0.1 and 1μM sbGnRH则对FSH和LH mRNA的表达没有影响。高浓度(10mM)sGnRH仅能在处理3h后显著促进FSH mRNA的表达,处理6h则不影响FSH mRNA的表达,sGnRH对LH mRNA的表达则始终没有影响。高浓度(10mM)cGnRH-Ⅱ仅能在处理6h后显著促进FSH m RNA的表达,处理3h则不影响FSH mRNA的表达,cGnRH-Ⅱ对LH mRNA的表达则始终没有影响。4.雌二醇对GnRH的反馈调节作用金钱鱼腹腔注射4mg/g体重E2,6h后,sbGnRH mRNA的表达显著降低,但cGnRH-Ⅱ和sGnRH的表达无显著变化;不同剂量的(0.1、1和10μM)E2离体孵育金钱鱼下丘脑3、6和12 h,各剂量雌二醇以明显剂量依存关系显著抑制sbGnRH mRNA的表达,但对cGnRH-Ⅱand s GnRH mRNA的表达无显著影响。雌激素受体广谱拮抗剂FULVESTRANT能明显促进下丘脑中sbGnRH mRNA的生成(P0.05)。雌激素受体alpha型拮抗剂MPP也能在浓度为0.01和0.1μM时显著促进下丘脑中sbGnRH mRNA的产生(P0.05),而在1μM时对sbGnRH m RNA的表达没有影响,雌激素受体beta型拮抗剂PHTPP能对下丘脑中sbGnRH mRNA的表达没有影响。说明当高浓度的E2可以显著抑制sbGnRH mRNA基因的表达,但是这种抑制作用可以被FULSTRANRT和MPP解除。研究结果表明,金钱鱼中GnRH至少存在三种亚型,即sbGnRH,cGnRH-Ⅱ和sGnRH,但在生殖调控中,sbGnRH发挥主要作用,E2对sbGnRH的抑制作用主要是通过雌激素受体alpha介导的。
[Abstract]:Gonadotropin-releasing hormone (GnRH) is a peptide hormone secreted by the hypothalamus, which binds to its corresponding receptors to promote pituitary synthesis and release of gonadotropin hormone (GtH). GtH includes luteinizing hormone (LH) and follicle stimulating hormone (follicle stimulating hormone). The full-length cDNA sequences of three GnRH subtypes (sbGnRH, C GnRH - II and sGnRH) of goldfish were cloned. The expression patterns of GnRH in various tissues were detected by RT-PCR, and the expression of GnRH during embryonic and gonadal development of goldfish was detected by fluorescence quantitative PCR. The effects of three GnRH polypeptides on the expression of FSH and LH mRNA in pituitary and the effects of estradiol (E2) on the expression of GnRH mRNA in hypothalamus were investigated in vitro (0.1, 1 and 10 mM) and in vivo (0.001, 0.01 and 0.1 mg/g body weight). Three GnRH subtypes were cloned, of which the length of sbGnRH was 334 bp, the open reading frame was 288 bp, encoding 95 amino acids; the length of cGnRH-II was 284 bp, the open reading frame was 258 bp, encoding 85 amino acids; the length of sGnRH was 288 bp, the open reading frame was 273 bp, encoding 90 amino acids. Evolutionary tree analysis showed that the three GnRH types of goldfish were closest to perch and farthest from mammals. 2. Temporal and spatial expression patterns of gonadotropin-releasing hormone genes in twelve tissues (pituitary, stomach, brain, egg) were detected. In the nest, testis, kidney, liver, intestine, spleen, gill, heart and muscle, sbGnRH was expressed in all tissues of male and female goldfish, and there were tissue differences in the expression of sbGnRH, while cGnRH-II and sGnRH were only expressed in the brain and gonad of male and female goldfish, suggesting that sbGnRH, cGnRH-II and sGnRH may be closely related to reproductive regulation, and sbGnRH may play a more important role. GnRH is highly expressed during gastrula embryogenesis and peaks at late gastrula embryogenesis, but very low at other embryonic development stages, suggesting that all three GnRH may be involved in regulating gastrula embryogenesis. During ontogenesis, the expression of sbGnRH increases gradually with the maturation of gonads. However, the expression of C GnRH-II and sGnRH did not change significantly, suggesting that the gonadal development and reproductive regulation of goldfish were mainly regulated by sbGnRH. 3. The effect of GnRHs on the expression of FSH and LH mRNA in vivo showed that the three doses of sbGnRH after intraperitoneal injection of different doses (0.001, 0.01 and 0.1 mg/g body weight) were all significant. Low and medium dose (0.001,0.01 m g/g body weight) of sbGnRH could promote the expression of LH mRNA, but high dose (0.1 m g/g body weight) of sbGnRH had no significant effect on the expression of LH mRNA; low and medium dose of sGnRH had no significant effect on the expression of FSH and LH mRNA, while high dose of sGnRH could significantly promote the expression of FSH (3,6 h) and LH (3 h) mRNA, but had no significant effect on the expression of LH (6 h) mRNA. Similar to sGnRH, low and medium dose cGnRH-II had no significant effect on the expression of FSH and LH mRNA. High dose of cGnRH-II could significantly promote the expression of FSH and LH mRNA after 3 hours of treatment, but had no effect after 6 hours of treatment. High concentration (10m M) sGnRH could only significantly promote the expression of FSH mRNA after 3 hours of treatment, but did not affect the expression of FSH mRNA after 6 hours of treatment. High concentration (10m M) cGnRH-II could only significantly promote the expression of FSH M R after 6 hours of treatment. The expression of FSH mRNA was not affected after 3 hours of treatment, but the expression of LH mRNA was not affected by cGnRH-II. Estradiol had no effect on the expression of LH mRNA. 4. The expression of sbGnRH mRNA was significantly decreased after intraperitoneal injection of 4 mg/g body weight E2 and 6 hours, but the expression of cGnRH-II and sGnRH had no significant change. Estradiol significantly inhibited the expression of sbGnRH mRNA in the hypothalamus at 3,6 and 12 h, but had no significant effect on the expression of cGnRH-II and s GnRH mRNA. The estrogen receptor antagonist FULVESTRANT could significantly promote the production of sbGnRH mRNA in the hypothalamus (P 0.05). The expression of sbGnRH mRNA in hypothalamus was significantly increased at concentrations of 0.01 and 0.1 mu M (P 0.05), but not at 1 mu M. The estrogen receptor beta antagonist PHTPP had no effect on the expression of sbGnRH mRNA in hypothalamus. The results showed that there were at least three subtypes of GnRH in goldfish, namely, sbGnRH, cGnRH-II and sGnRH. However, sbGnRH played a major role in reproductive regulation, and the inhibitory effect of E2 on sbGnRH was mainly mediated by estrogen receptor alpha.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2237051
[Abstract]:Gonadotropin-releasing hormone (GnRH) is a peptide hormone secreted by the hypothalamus, which binds to its corresponding receptors to promote pituitary synthesis and release of gonadotropin hormone (GtH). GtH includes luteinizing hormone (LH) and follicle stimulating hormone (follicle stimulating hormone). The full-length cDNA sequences of three GnRH subtypes (sbGnRH, C GnRH - II and sGnRH) of goldfish were cloned. The expression patterns of GnRH in various tissues were detected by RT-PCR, and the expression of GnRH during embryonic and gonadal development of goldfish was detected by fluorescence quantitative PCR. The effects of three GnRH polypeptides on the expression of FSH and LH mRNA in pituitary and the effects of estradiol (E2) on the expression of GnRH mRNA in hypothalamus were investigated in vitro (0.1, 1 and 10 mM) and in vivo (0.001, 0.01 and 0.1 mg/g body weight). Three GnRH subtypes were cloned, of which the length of sbGnRH was 334 bp, the open reading frame was 288 bp, encoding 95 amino acids; the length of cGnRH-II was 284 bp, the open reading frame was 258 bp, encoding 85 amino acids; the length of sGnRH was 288 bp, the open reading frame was 273 bp, encoding 90 amino acids. Evolutionary tree analysis showed that the three GnRH types of goldfish were closest to perch and farthest from mammals. 2. Temporal and spatial expression patterns of gonadotropin-releasing hormone genes in twelve tissues (pituitary, stomach, brain, egg) were detected. In the nest, testis, kidney, liver, intestine, spleen, gill, heart and muscle, sbGnRH was expressed in all tissues of male and female goldfish, and there were tissue differences in the expression of sbGnRH, while cGnRH-II and sGnRH were only expressed in the brain and gonad of male and female goldfish, suggesting that sbGnRH, cGnRH-II and sGnRH may be closely related to reproductive regulation, and sbGnRH may play a more important role. GnRH is highly expressed during gastrula embryogenesis and peaks at late gastrula embryogenesis, but very low at other embryonic development stages, suggesting that all three GnRH may be involved in regulating gastrula embryogenesis. During ontogenesis, the expression of sbGnRH increases gradually with the maturation of gonads. However, the expression of C GnRH-II and sGnRH did not change significantly, suggesting that the gonadal development and reproductive regulation of goldfish were mainly regulated by sbGnRH. 3. The effect of GnRHs on the expression of FSH and LH mRNA in vivo showed that the three doses of sbGnRH after intraperitoneal injection of different doses (0.001, 0.01 and 0.1 mg/g body weight) were all significant. Low and medium dose (0.001,0.01 m g/g body weight) of sbGnRH could promote the expression of LH mRNA, but high dose (0.1 m g/g body weight) of sbGnRH had no significant effect on the expression of LH mRNA; low and medium dose of sGnRH had no significant effect on the expression of FSH and LH mRNA, while high dose of sGnRH could significantly promote the expression of FSH (3,6 h) and LH (3 h) mRNA, but had no significant effect on the expression of LH (6 h) mRNA. Similar to sGnRH, low and medium dose cGnRH-II had no significant effect on the expression of FSH and LH mRNA. High dose of cGnRH-II could significantly promote the expression of FSH and LH mRNA after 3 hours of treatment, but had no effect after 6 hours of treatment. High concentration (10m M) sGnRH could only significantly promote the expression of FSH mRNA after 3 hours of treatment, but did not affect the expression of FSH mRNA after 6 hours of treatment. High concentration (10m M) cGnRH-II could only significantly promote the expression of FSH M R after 6 hours of treatment. The expression of FSH mRNA was not affected after 3 hours of treatment, but the expression of LH mRNA was not affected by cGnRH-II. Estradiol had no effect on the expression of LH mRNA. 4. The expression of sbGnRH mRNA was significantly decreased after intraperitoneal injection of 4 mg/g body weight E2 and 6 hours, but the expression of cGnRH-II and sGnRH had no significant change. Estradiol significantly inhibited the expression of sbGnRH mRNA in the hypothalamus at 3,6 and 12 h, but had no significant effect on the expression of cGnRH-II and s GnRH mRNA. The estrogen receptor antagonist FULVESTRANT could significantly promote the production of sbGnRH mRNA in the hypothalamus (P 0.05). The expression of sbGnRH mRNA in hypothalamus was significantly increased at concentrations of 0.01 and 0.1 mu M (P 0.05), but not at 1 mu M. The estrogen receptor beta antagonist PHTPP had no effect on the expression of sbGnRH mRNA in hypothalamus. The results showed that there were at least three subtypes of GnRH in goldfish, namely, sbGnRH, cGnRH-II and sGnRH. However, sbGnRH played a major role in reproductive regulation, and the inhibitory effect of E2 on sbGnRH was mainly mediated by estrogen receptor alpha.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
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