| 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.
|
PDF Full Text Request