Ghrelin Gene Expression In Female Genital Tract Of Ujimqin Sheep And Construction Of Ghrelin Eukaryotic Expression Vectors

Ghrelin was the ligand of GHSR which found in 1999. Its main function was combined with GHSR to stimulate GH releasing. Recently, Ghrelin was found in procreant organs, suggested Ghrelin could regulate procreant cycles and gestation. For the moment, there weren't any reports about Ghrelin in Ujimqin sheep genital tracts. Estrogen and progesterone could regulate female genital system, but we didn't know whether they could regulate Ghrelin gene expression or not. Based obtained Ujimqin sheep Ghrelin (usGhl) cDNA complete sequence, this paper deeply researched Ghrelin gene location and expression in genital tracts; studied on relationship between Ghrelin gene and estrogen/progesterone in different oestrum and gestation of female Ujimqin sheep genital tracts; validated Ghrelin gene expression content change in cultured fallopian tube epithelium cells by adding estrogen/progesterone into culture medium. Otherwise, we constructed usGhl eukaryotic expression system (pcDNA-usGhl). Animals experiment confirmed pcDNA-usGhl could accelerate animal growth. The results of this paper could be the foundation to research sheep promoted growth.1. Based on Ghrelin sequence in GenBank, we designed special primers to clone usGhl cDNA complete sequence by RT-PCR and RACE technique. The complete sequence contained 634 bp which included an Ujimqin sheep pre-Ghrelin peptide of 116aa. The relationship of Ghrelin cDNA or forecasted mature peptide sequence between Ujimqin sheep and other mammalians were extremely homologous by NCBI blast function and DNAStar software. The result indicated Ghrelin gene was conservative in different kinds of animals.2. We located Ghrelin mRNA in Ujimqin sheep genital tracts tissues (the body of uterus, the cervix of uterus, fallopian tube and vagina) by in situ hybridization technique (ISH). The result showed: Ghrelin mRNA mainly existed in the epithelium and gland; proper layer and muscle layer were less than epithelium and gland; there weren't expression in musculature and connective tissue. Indicated Ghrelin mostly expressed in mucous membrane surface layer cells.3. We confirmed Ghrelin gene was expressed in the body of uterus, fallopian tube, cervix of uterus and vagina for the first time by RT-PCR technique. The expressive content of Ghrelin gene in different genital tracts tissues of Ujimqin sheep was different: the highest was the body of uterus, the next was fallopian tube, the cervix of uterus and vagina included its lowest level. Used RQ-PCR and electrochemistry radiation mensuration researched the relationship between Ghrelin and estrogen, progesterone in oestrus and gestation genital tracts tissue. The content chang of estrogen or progesterone in oestrus and gestation Ujimqin sheep serum accorded to currently rule of critter physiological cycle. Ghrelin gene content in Ujimqin sheep genital tracts tissues had disciplinary change by oestrus or gestation concentration: in the body of uterus, the cervix of uterus and fallopian tube was increased or fell with oestrus or gestation content change in serum of body; the content relationship between Ghrelin gene and oestrus or gestation in vagina was not evident.4. We obtained the original generation and the first generation epithelial cells of Ujimqin sheep fallopian tubes epithelium cells by mechanism, cells sedimentation and density grads centrifugal method. 17-β-estradiol and progesterone could accelerate glue and growth of cultured fallopian tubes epithelial cells. We added different concentration 17-β-estrogen and progesterone into the first generation epithelial cells cultured 24h, 48h, 72h. Then examined Ghrelin gene expression content in cells which treated with hormone by RQ-PCR technique. The result showed that culture time didn't influence Ghrelin gene expression content; certain concentration 17-β-Estradiol or progesterone could promote the expression of Ghrelin gene in cultured fallopian tubes epithelia cell.5. Cultured fallopian tubes epithelia cells were treated with tamoxifen or mifiprestone first, then added estrogen or progesterone corresponding concentration cultured 24h, 48h, 72h. Ghrelin gene expression content of treated cells were detected by RQ-PCR. The results showed: tamoxifen and mifiprestone could restrain growth and intensity increased in cultured fallopian tubes epithelial cells; tamoxifen or mifiprestone could obviously debase Ghrelin gene expression content in cultured fallopian tubes epithelia cells compared with cultured epithelia cells with no tamoxifen or mifiprestone. If increased tamoxifen or mifiprestone concentration in culture medium without FBS, Ghrelin content was gradually debased; culture time didn't influence Ghrelin gene expression content. Suggested that estrogen and progesterone could promote the expression of Ghrelin. Estrogen and progesterone exerted their function by combining with their corresponding receptors.6. We constructed usGhl eukaryotic expression system (pcDNA-usGhl). Ghrelin gene plasmid could express in mice, and maintained about 20d. Ghrelin recombined plasmid could accelerate mice growth and debased feed and avoirdupois ratio. PcDNA-usGhl DNA didn't insert mice chromosome in our experiment. There weren't novel intumescence or pathological changes in mice heart, liver, kidney, brain or muscle. The results indicated pcDNA-usGhl plasmid we constructed was successful.