金錢魚促性腺激素釋放激素的克隆及其在生殖調控中的功能研究
發(fā)布時間：2018-09-11 15:09
【摘要】：促性腺激素釋放激素(Gonadotropin-releasing hormone,GnRH)是由下丘腦分泌的一種肽類激素,與其相應受體結合后可以促進垂體合成和釋放促性腺激素(gonadotropin hormone,GtH),GtH包括促黃體素(luteinizing hormone,LH)和促卵泡素(follicle stimulating hormone,FSH),對動物生殖具有非常重要的作用。在研究克隆了金錢魚三種GnRH亞型(sbGnRH,c GnRH-Ⅱand sGnRH)的全長cDNA序列。采用RT-PCR的方法檢測了GnRH在各個組織中的表達模式;應用熒光定量PCR技術檢測了金錢魚胚胎發(fā)育和性腺發(fā)育過程中GnRH的表達模式,同時檢測了離體(0.1,1和10mM)和在體(0.001,0.01和0.1mg/g體重)條件下三種GnRH多肽對垂體FSH和LH mRNA表達的影響,以及雌二醇(E2)對下丘腦GnRH mRNA表達的影響。主要結果如下:主要的結果如下:1.金錢魚促性腺激素釋放激素基因克隆及序列分析金錢魚三種GnRH亞型被克隆,其中sbGnRH的全長為334bp,開放閱讀框為288bp,編碼95個氨基酸;cGnRH-Ⅱ的全長為284bp,開放閱讀框為258bp,編碼85個氨基酸;sGnRH的全長為288bp,開放閱讀框為273bp,編碼90個氨基酸。三種GnRH氨基酸序列的同源性比對發(fā)現(xiàn),金錢魚cGnRH-Ⅱ是最保守的GnRH類型,s GnRH次之,sbGnRH最不保守。進化樹分析發(fā)現(xiàn),金錢魚的三種GnRH的親緣關系與鱸形目魚類最近,與哺乳類最遠。2.金錢魚促性腺激素釋放激素基因的時空表達模式在所檢測的12種組織(垂體、胃、腦、卵巢、精巢、腎臟、肝臟、腸、脾、鰓、心和肌肉)中,sbGnRH在雌雄金錢魚的所有組織中都表達,且表達量存在組織差異,而cGnRH-Ⅱ和s GnRH僅在雌雄金錢魚的腦和性腺中表達,暗示sbGnRH、cGnRH-Ⅱ和sGnRH可能均與生殖調控密切相關,而sbGnRH可能發(fā)揮更為廣泛的功能;在胚胎發(fā)育過程中,三種GnRH都在原腸胚期間高表達,并在原腸胚晚期達到峰值,而在其他胚胎發(fā)育時期則表達量很低,暗示著這三種GnRH可能都參與調控原腸胚的發(fā)生;在個體發(fā)育過程中,隨著性腺的成熟,sbGnRH的表達逐漸升高,而c GnRH-Ⅱ和sGnRH的表達量無顯著變化,暗示著在金錢魚的性腺發(fā)育和生殖調控主要由sbGnRH來調控。3.GnRHs對FSH和LH mRNA表達的影響在體實驗顯示,金錢魚腹腔注射不同劑量(0.001,0.01和0.1mg/g體重)的sbGnRH,3h和6h后,三種劑量的sbGnRH均可顯著促進FSH m RNA的表達,低、中劑量(0.001,0.01mg/g體重)sbGnRH可促進LH mRNA的表達,但高劑量(0.1mg/g體重)sbGnRH處理對LH mRNA的表達無顯著影響;低、中劑量sGnRH對FSH和LH mRNA表達無顯著影響,而高劑量sGnRH可顯著促進FSH(3,6h)和LH(3h)mRNA的表達,但對6h的LH mRNA表達無影響;與sGnRH作用類似,低、中劑量cGnRH-Ⅱ對FSH和LH mRNA表達無顯著影響,處理3h后高劑量cGnRH-Ⅱ可顯著促進FSH和LH mRNA的表達,但是處理6h后則沒有影響。離體實驗顯示,以10μM sbGnRH離體孵育金錢魚垂體,3h和6h后,FSH和LH mRNA的表達顯著升高,但是0.1 and 1μM sbGnRH則對FSH和LH mRNA的表達沒有影響。高濃度(10mM)sGnRH僅能在處理3h后顯著促進FSH mRNA的表達,處理6h則不影響FSH mRNA的表達,sGnRH對LH mRNA的表達則始終沒有影響。高濃度(10mM)cGnRH-Ⅱ僅能在處理6h后顯著促進FSH m RNA的表達,處理3h則不影響FSH mRNA的表達,cGnRH-Ⅱ對LH mRNA的表達則始終沒有影響。4.雌二醇對GnRH的反饋調節(jié)作用金錢魚腹腔注射4mg/g體重E2,6h后,sbGnRH mRNA的表達顯著降低,但cGnRH-Ⅱ和sGnRH的表達無顯著變化;不同劑量的(0.1、1和10μM)E2離體孵育金錢魚下丘腦3、6和12 h,各劑量雌二醇以明顯劑量依存關系顯著抑制sbGnRH mRNA的表達,但對cGnRH-Ⅱand s GnRH mRNA的表達無顯著影響。雌激素受體廣譜拮抗劑FULVESTRANT能明顯促進下丘腦中sbGnRH mRNA的生成(P0.05)。雌激素受體alpha型拮抗劑MPP也能在濃度為0.01和0.1μM時顯著促進下丘腦中sbGnRH mRNA的產(chǎn)生(P0.05),而在1μM時對sbGnRH m RNA的表達沒有影響,雌激素受體beta型拮抗劑PHTPP能對下丘腦中sbGnRH mRNA的表達沒有影響。說明當高濃度的E2可以顯著抑制sbGnRH mRNA基因的表達,但是這種抑制作用可以被FULSTRANRT和MPP解除。研究結果表明,金錢魚中GnRH至少存在三種亞型,即sbGnRH,cGnRH-Ⅱ和sGnRH,但在生殖調控中,sbGnRH發(fā)揮主要作用,E2對sbGnRH的抑制作用主要是通過雌激素受體alpha介導的。
[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.
【學位授予單位】：廣東海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4
本文編號：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.
【學位授予單位】：廣東海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4
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