摘要
初情期是动物首次出现发情排卵的年龄,是开始获得繁殖能力的标志。初情期的启动,是由下丘脑GnRH神经元控制。从目前的研究报道看,初情期启动的机制实际上是GnRH脉冲式释放的调控,是非类固醇介导的中枢机制,调控因素包括神经肽、神经递质、神经类固醇。主要参与调控GnRH释放的神经肽有鸦片肽、NPY、促生长激素神经肽(Galanin)、促皮质激素释放激素(CRF)；神经递质有去甲肾上腺素(NE)、多巴胺、5-羟色胺、褪黑激素、Y氨基丁酸(GABA),但其具体调控机制还需进一步研究探讨。目前,关于初情期启动相关基因受体NPY-Y1、GPR54、Ob-Rb基因的研究报道较少,特别是关于他们对初情期启动的调控机制几乎没有报道。本研究系统的研究初情期启动相关基因受体NPY-Y1、GPR54、Ob-Rb基因在下丘脑-垂体-卵巢内的组织定位及其组织发育性变化,目的是为深入研究探讨NPY-Y1、GPR54、Ob-Rb在初情期启动中扮演的角色。同时本实验还采用mRNA差异显示(mRNA DD)技术对猪初情期启动的关键因子做进一步的研究,以筛选与猪初情期启动相关的重要基因。主要研究结果如下：
1.根据GenBank发表的序列设计NPY-Y1、GPR54、Ob-Rb基因特异性引物,采用反转录聚合酶链式反应(RT-PCR)扩增出NPY-Y1、GPR54、Ob-Rb基因的部分cDNA序列,扩增产物克隆入pGEM-T easy载体中进行序列测定。结果：与发表的猪NPY-Y1、GPR54、Ob-Rb基因序列(NO.AF106081,NO.DQ459346,NO.AF092422)同源性均为100%,说明所扩增的序列为本实验所要的,为后续研究奠定了基础。
2.用原位杂交技术检测苏姜猪母猪初情期前后NPY-Y1 mRNA在下丘脑、垂体和卵巢的分布定位。结果：在苏姜猪初情期前后下丘脑、垂体和卵巢三种组织中,均检测到NPY-Y1 mRNA阳性杂交信号。下丘脑的阳性杂交信号主要集中在丘脑室旁核和下丘脑的弓状核、室旁核、室周核等处；在垂体中分布相对密集；各级卵泡颗粒细胞和内膜细胞中均有阳性杂交信号,间质中也有少量的杂交信号。在三种组织中NPY-Y1 mRNA杂交信号均初情期较弱,初情期前相对较强。结果可以初步证明NPY-Y1在下丘脑-垂体-卵巢轴对雌性生殖的调节中具有关键的调控作用,并且对下丘脑-垂体-卵巢轴具有调控作用。
3.用原位杂交技术检测苏姜猪母猪初情期前后GPR54 mRNA在下丘脑、垂体和卵巢的分布定位。结果：在苏姜猪初情期前后下丘脑、垂体和卵巢三种组织中,均检测到GPR54 mRNA阳性杂交信号。下丘脑的阳性杂交信号主要集中在弓状核和腹内侧核中,其它区也有一定的表达；在垂体中分布相对密集；各级卵泡颗粒细胞和内膜细胞中均有阳性杂交信号,间质中也有少量的杂交信号,大卵泡的阳性率明显高于小卵泡。在三种组织中GPR54 mRNA杂交信号均初情期较强,初情期前相对较弱些。结果可以初步证明GPR54在下丘脑-垂体-卵巢轴对雌性生殖的调节中具有关键的调控作用,并且对下丘脑-垂体-卵巢轴具有调控作用。
4.用免疫组织化学的方法测定苏姜猪母猪初情期前后下丘脑、垂体、卵巢内Ob-Rb的分布及定位。结果：Ob-Rb阳性颗粒广泛分布于下丘脑,尤其是弓状核；垂体中Ob-Rb阳性颗粒主要集中于垂体前叶细胞；卵巢中Ob-Rb阳性颗粒出现在各级卵泡的颗粒细胞,而卵母细胞没有阳性颗粒。结果可以初步证明Ob-Rb在下丘脑-垂体-卵巢轴对雌性生殖的调节中也具有关键的调控作用。
5.用荧光定量PCR技术检测苏姜猪初生、60、120、初情期、180日龄五个不同发育阶段的下丘脑、垂体和卵巢三种组织中NPY-Y1、GPR54、Ob-Rb mRNA的表达丰度变化。结果：(1)下丘脑、垂体与卵巢内NPY-Y1 mRNA表达量从初生到初情期表达量逐渐下降,在初情期达到最低,初情期后呈上升趋势。下丘脑内各个时期NPY-Y1 mRNA表达量均存在显著差异(P<0.05),垂体内120日龄与180日龄之间NPY-Y1 mRNA表达量差异不显著(P>0.05),其它时期NPY-Y1 mRNA表达量均显著差异(P<0.05),卵巢内初情期与180日龄之间NPY-Y1 mRNA表达量差异不显著(P>0.05),其它时期NPY-Y1 mRNA表达量均显著差异(P<0.05)。(2)下丘脑、垂体与卵巢内GPR54 mRNA表达量均从初生到初情期表达量逐渐上升,在初情期达到最高,初情期后呈下降趋势。下丘脑内初情期与初生、180日龄的GPR54 mRNA表达量差异均达到显著水平(P<0.05),垂体内初情期与初生、60日龄、180日龄的GPR54 mRNA表达量差异均达到显著水平(P<0.05),卵巢内初情期与初生、60日龄、120日龄、180日龄的GPR54 mRNA表达量差异均达到显著水平(P<0.05)。(3)下丘脑、垂体与卵巢内Ob-Rb mRNA发育性变化模式与NPY-Y1基本相同,从初生到初情期表达量逐渐下降,在初情期达到最低,初情期后呈上升趋势。下丘脑内120日龄、初情期与180日龄之间Ob-Rb mRNA表达量差异不显著(P>0.05),其它时期NPY-Y1 mRNA表达量均显著差异(P<0.05),垂体内60日龄与120日龄之间Ob-Rb mRNA表达量差异不显著(P>0.05),其它时期NPY-Y1 mRNA表达量均显著差异(P<0.05),卵巢内初情期与180日龄之间Ob-Rb mRNA表达量差异不显著(P>0.05),其它时期Ob-Rb mRNA表达量均显著差异(P<0.05)。结果初步显示了三种受体在猪下丘脑-垂体-卵巢生殖轴中不同发育时期的变化,并且证明发育时期中NPY对GnRH释放起到是抑制作用,而leptin对GnRH起释放作用,低剂量的leptin可促进GnRH-LH的分泌,高浓度则抑制其分泌,这为以后的研究提供了可靠的科学数据。
6.采用mRNA差异显示技术、Reverse Northern技术以及半定量分析技术,对不同发育时期猪下丘脑差异表达的ESTs进行分离,共得到cDNA差异片断20条,半定量分析显示：其中5条差异条带在出生时呈表达增强相,11条差异条带在初情期时呈表达增强相,其余4条在性成熟时呈表达增强相。测序结果与GenBank数据库比对,二个cDNA与已知基因同源,其余大部分都是功能未知的EST序列。经Reverse Northern验证,有9个cDNA呈阳性,可用于下一步探针定位及表达的研究。
Puberty is a term that animal first time to oestrus and ovulates, which is a sign of the attainment of full fertility. The activation of hypothalamic GnRH neuron controlled the onset of puberty. Recent research showed that the activation of hypothalamic GnRH secretion is a key event of the onset of puberty, and this event works mediated with steroid, which includes Neuropeptide, Neurotransmitters and Neurosteroid. The chief neuropeptide regulate GnRH secretion includeed Opium Peptide, NPY, Galanin, CRF, NE, DA,5-HTA, Melatonin and GABA. But the regulated mechanism required further research. Presently, the research on puberty promoter of NPY-Y1, GPR54 and Ob-Rb is few, especially on their regulated mechanism. This paper first systemic studied the developmental expression and localization of NPY-Y1, GPR54, Ob-Rb promoter gene in hypothalamic-pituitary-ovary axis, and analysed their function in the onset of puberty. The mRNA differential expression of the hypothalamic organism in different development period were also separated to screen key genes correlated to the onset of puberty using RFDD-PCR technique,
1. NPY-Y1, GPR54, Ob-Rb gene of pig were obtained using reverse transcription polymerase chain reaction amplification (RT-PCR), specific primers were designed according to NPY-Y1, GPR54, Ob-Rb cDNA from GenBank. The RT-PCR product was subcloned into pGEM-T easy vector and the correctness of the sequences was confirmed by sequencing. The results of sequencing analysis showed that their homology with NPY-Y1, GPR54, Ob-Rb cDNA from GenBank (Accession NO. AF106081, NO. DQ459346, NO. AF092422) were all up to 100%. The data indicateed that the NPY-Y1, GPR54, Ob-Rb cDNA were cloned correctly, which would play a great role in research of their physiological functions and mechanism.
2. Before and after puberty, the disposition and localization of NPY-Y1 mRNA in hypothalamus, pituitary and ovary were detected by ISH technology. The detection of masculine hybridization signals of NPY-Y1 mRNA in hypothalamus, pituitary and ovary of Sujiang suggested that NPY-Y1 mRNA was expressed in hypothalamus including hypothalamic paraventricular nucleus (PVN), arcuate nucleus, paraventricular nucleus, periventricular nucleus and so on. NPY-Y1 positive particles of pituitary were concentrating distributed. NPY-Y1 positive particles of ovaries appeared in all types of follicles and intima cells, few in mesenchymal cell. In different tissues, which showed weak NPY-Y1 mRNA hybridization signal in puberty, much strong than before puberty. All results showed NPY-Y1 is key gene required for regulating function of the hypothalamic-pituitary-ovary axis.
3. Before and after puberty, the disposition and localization of GPR54 mRNA in hypothalamus, pituitary and ovary were also detected by ISH technology. The detection of masculine hybridization signals of GPR54 mRNA in hypothalamus, pituitary and ovary of Sujiang suggested that GPR54 mRNA was expressed in hypothalamus including arcuate nucleus, ventromedial nucleus, there also detecting few hybridization signals of GPR54 mRNA in other section. GPR54 positive particles of pituitary were concentrating distributed. GPR54 positive particles of ovaries appeared in all types of follicles and intima cells, few in mesenchymal cell, and the large follicular showed much strong GPR54 mRNA hybridization signal than little follicular. In different tissues, there showed much strong GPR54 mRNA hybridization signal in puberty than before puberty. All results showed GPR54 is another key gene required for regulating function of the hypothalamic-pituitary-ovary axis.
4. With immunohistochemical method, the distribution and localization of the expression of Ob-Rb gene were detected in hypothalamus, pituitary and ovaries. The results showed that Ob-Rb positive particles were distributed in hypothalamus generally, especially arcuate nucleus. Ob-Rb positive particles of pituitary were concentrated into pituitarium anterius cells. And Ob-Rb positive particles of ovaries appeared in granular cell, granule cells of all types of follicles, but megagametocyte didn't discover the existence of the positive particles. All this showed that Ob-Rb is capital gene required for regulating function of the hypothalamic-pituitary-ovary axis.
5. In order to investigated quantitative expression of NPY-Y1, GPR54, Ob-Rb mRNA in hypothalamus, pituitary, ovaries of Su Jiang porcine on newborn,60,120, puberty and 180 days, Fluorescent Quantitation PCR technology was used. The results showed that:(1) the expression abundance of NPY-Y1 mRNA was gradually descending from birth to puberty and after puberty the expression abundance was ascending tendency in all three tissues. In hypothalamus tissue, the expression abundance of NPY-Y1 mRNA between each stage were all significant difference (P<0.05). In pituitary tissue, the expression abundance at the stage of 120 days was no significant difference with the stage of 180 days (P>0.05), between other each stage were significant difference(P<0.05). In ovaries tissue, the expression abundance at the stage of puberty was no significant difference with the stage of 180 days (P>0.05), between other each stage were significant difference(P<0.05); (2) the expression abundance of GPR54 mRNA was gradually ascending from birth to puberty, and after puberty the expression abundance was descending tendency in all three tissues. In hypothalamus tissue, the expression abundance at the stage of puberty was significant difference with the stage of birth and 180 days (P<0.05), In pituitary tissue, the expression abundance at the stage of puberty was significant difference with the stage of birth,60 days and 180 days (P<0.05), In ovaries tissue, the expression abundance at the stage of puberty was significant difference with the stage of birth,60 days,120 days and 180 days (P<0.05).(3) The expression abundance of Ob-Rb mRNA was gradually descending from birth to puberty, and after puberty the expression abundance was ascending tendency in all three tissues. In hypothalamus tissue, the expression abundance at the stage of 120 days, puberty were no significant difference with the stage of 180 days (P>0.05), other each stage were significant difference(P<0.05). In pituitary tissue, the expression abundance at the stage of 60 days were no significant difference with the stage of 120 days (P>0.05), other each stage were significant difference(P<0.05). In ovaries tissue, the expression abundance at the stage of puberty were no significant difference with the stage of 180 days (P>0.05), other each stage were significant difference(P<0.05). This study explored the express regularity of the three receptor in hypothalamic-pituitary-ovary axis in different development period, and identified that NPY-Y1 expressing inhibites GnRH releasing, low dose leptin expressing promotes GnRH-LH Releasing, high dose leptin expressing inhibited GnRH-LH releasing. That will provide reliable science data on future research.
6. The ESTs of mRNA differential expressing of the hypothalamic organs in different development period were separated using RFDD-PCR technique. The results showed 20 differential cDNA bands were found. The semiquantitative analysis indicated that 5 cDNA bands were expressing strengthly when at the stage of birth. While 11 cDNA bands were expressing up-regulation when at the stage of puberty, other 4 cDNA bands were expressing up-regulation when at the stage of sex maturation. Sequencing these cDNA and blasted with Genbank showed 2 cDNA bands homologous consequence with it, the other were all uncharted EST sequence. Validated using Reverse Northern technique, we have found 9 cDNA bands would as probes on next expressive research.
引文
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