| The experiment to explore the expression of genes characteristic analysis the follicular developmentamong lamb, adult sheep with superovulation lamb. clear to the position and ovarian tissue expressionpattern of the lamb and adult sheep and superovulation lamb follicular by anti-Mullerian hormone(anti-Mullerian hormone, AMH), follicle-stimulating hormone (follicle-stimulating hormone receptor,FSHR), luteinizing hormone receptor (luteinizing hormone receptor, LHR), in order to grasp the regulationof follicular development in sheep. It will be applied in superovulation can play a supporting role.comparative analysis the different developmental patterns of lambs and adult sheep ovarian follicles,analysis the expression of the different periods in different related genes changes in follicle, understand themechanism of lambs follicular maturation and development regulatory, improve production efficiency. Theexperiment was divided into four parts in the following:Section1Molecular Cloning, Sequence Analysis and Prokaryotic Expression ofAMH Gene of OvineAnti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a member ofthe transforming growth factor β superfamily of growth and dfferentiation factors. In mouse, AMHparticipates in two critical selection points of follicle develpment: it inhibits the recruitment of primordialfollicles into the pool of growting follicles and also decreases the responsiveness of growing follicles toFSH. However, little is known of the physiology of AMH in monovular species. This study aimed to obtainAMH full-length cDNA of monovular specie ovine and conduct the sequence analysis. In this study, thetemplate cDNA which was reversely transcribed from the total RNA of sheep granulosa cells, wassubjected to polymerase chain reaction (PCR) with the specific primers of AMH. The full-length cDNA ofovine amh (oAmh) were achieved with pMD19-T cloning and sequencing. The N-terminal260aminoacides of ovine AMH encoding sequence was subcloned into the pET15b expession vector withNdeI/BamHI enzyme sites, and then transformed into BL21(DE3) E.coli. The induced products by IPTGwere analyzed with SDS-PAGE and Western blot. The oAmh includes1797bp (Genbank No. KC986978)with the open reading frame of1728bp and575coding amino acides cotaining a24amino acide leaderpeptide. A comparison of the ovine and other mammals AMH proteins reveals a highly conservedC-terminal domain that shows marked homology with transforming growth factor-β family. The predictedprotein sequence shared95.8%identidy with bovine’s. SDS-PAGE analysis showed that the expressedprotein in BL21(DE3) was about30kDa. Western blot revealed that the expressed protein was a His tagfusion protein. Cloning and successful expression of the ovine AMH built the foundation for further studyon its biological function.Section2Immunocytochemical study of anti-Müllerian hormone in ovarian follicles from lambsTo investigate the temporal and spatial expression patterns of AMH in one-month-old lamb and eweovarian.Histological and immunohistochemistry of AMH were analysised of ovarians from one-month-oldlambs and ewes. The mRNA expression of AMH on the granulosa cells from in vitro maturation (IVM) ofcumulus-oocyte complex (COC) were detected using quantitative RT-PCR. Many medium-sized vesicularfollicles are present of the untreated lamb ovary. The ovaries were over-stimulated in that all the surfaceswere filled with large follicles without any small ones, and without luteinization or ovulation. In addtion, alarge number of primordial follicles is present on the gonadotropin-unstimulated lambs ovary. Theimmunoreactivity of AMH was limited to granulosa cells in growing follicles, and was localised in thegranulosal cells of preantral and small antral follicles, as well as in the cumulus oophorus, and noimmunoreactivity was observed in most of the primordial follicles, except in transitory follicles containedan oocyte surrounded by a single-layer mixture of flattened and cuboidal granulosal cells. The granulosalcell had been differentiated two types, expressing AMH and unexpressing AMH cell lines. The expressionof AMH is no significant difference in the granulosal cell from IVM of COC between the lamb andewe.These observations suggest that AMH plays a key role in the modulation of the responsiveness of follicles progress through the gonadotropin-responsive to gonadotropin-dependent stage. A betterunderstanding of AMH function is needed to uncover the mechanism which participate in managing thereserve of primordial and small growing follicles.Section3Sheep Lamb FSHR gene multiple splice forms in ovarian and its expression characteristicanalysis in granulosa cellsThe phenomenon of different isoforms according to pre-mRNA splicing through different ways,explore the difference FSHR gene splicing isoforms in lambs and adult sheep ovary.usingimmunohistochemistry to detect the protein expression profiles of sheep ovarian FSHR and RT-PCR andWestern blotting to detect changes related protein expression of ovarian granulosa cell FSHR gene.It hasimportant significance for research capabilities. Get sheep shearing in the form of a variety of FSHR genecoding sequence, immunohistochemistry showed that in normal lambs and superovulation lambs FSHRexpressed mainly in ovarian granulosa cells and there are positive signals in the primordial follicles, normaladult sheep no positive signals in the original follicles, in large dominant follicle FSHR expression in asignificant reduction.Section4Immunocytochemical study of LHR in ovarian follicles from lambsBy detecting LHR protein expression in lambs and adult sheep ovaries, lay the theoretical foundationfor the developmentally regulated follicle. Immunohistochemical staining for sheep ovary LHR locatestaining and Western blotting analysis related proteins. LHR mainly in normal lambs and superovulationlambs and adult sheep highly expressed in large antral follicles in ovarian granulosa cells, the expression ofpositive signals superovulation lamb more than adult sheep LHR positive sexual number. |
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