| The seasonal breeding is one of the main bottlenecks in limiting the sheep reproduction effectivenesswhich are typically seasonal estrous animals. So far, the regulation mechanism of breeding-season estrousmainly focused on the photo period and primarily verified the patten of seasonal reproduction. However,except for the light and temperature et al., we found the estrous Hazakh sheep in non-breeding seasoninduced by the high nutrition level in practice. The regulation of nutririon level change on the estrous innon-breeding season is important for promoting the production effectivity of sheep. For this propose, thisstudy works under non-breeding season condition to select micro RNAs and identify target genes functioncontrolled by nutrition level of estrous stall-feed sheep. The contents are as following:Object: Under different nutrition treaments, the different expressed micro RNAs in sheep are screened,the target genes are predicted by the bioinformation technology, subsequently, the target genes in thephotoperiod and related pathways are analysed. This maybe a workable method to establish the signalpathways of nutrition on regulating estrous.Methods:(1) Building different nutrition level kazakh sheep, the sheep estrous condition is observedin non-breeding season.(2) In non-breeding season, 3 estrous(EN) and 3 anestrous(AN) sheep wereslaughtered and collected the hypothalamus(H), pituitary(P), and ovary(O), in all, 6 mi RNA libraries areconstructed and analyzed by the bioinformation methods. Part of mi RNAs were choosen to test the qualityof mi RNA libraries by q RT-PCR. The mi R-200a/b/c were choosen to do the tissue expression spectrumanalysis.(3) Cell level was tested. ? By using Dual-luciferase vectors, the mi R-200 b interfering the targetgene GNAQ was tested.? Hypothalamus nerve cells transfect mi R-200 b test, the downstream genes(ITPR,PRKCB, GNA1, CREB, MAP3K1,GPR54, KISS1 and Gn RH)of GNAQ were selected to detect by Q-PCR.?The ovarian granulosa cells transfect mi R-200 b test, the downstream genes(COMT, HSD3B1,CYP1A1 andPKA) of GNAQ were selected to detect by Q-PCR.Results:(1) The estrous rate in high nutritional group was significantly higher than the control group.(2) ? Compared the two hypothalamus(HEN vs HAN) libaries, 8 known and 140 novel mi RNAs weredifferent expressed. ? Compared the two pituitary(PEN vs PAN) libaries, the 14 known and 104 novelmi RNAs were different expressed. ? Compared the two ovary(OEN vs OAN) libaries, the 9 known and104 novel mi RNAs were different expressed. The GO analysis suggested that, the target genes functionwere the cell process, the development process, the growth process, the metabolism process, biologyregulation process, cell conjugation, catalyze activity, reproduction and enzyme activity, et al. The KEGGpathway analysis found the MAPK signal pathway(which is related with the CLOCK genes), Gn RH signalpathway(which was connected with the estrous hormone), insulin signal pathway, glycine, serine andthreonine metabolism, glycerol phospholipid metabolism which were associated with energy metabolism.The quality of the mi RNA libraries was eligible. The tissue expression profile analyses of mi R-200a/b/c inKazakh sheep found that these mi RNAs were expressed in the pituitary, uterus and fallopian tubes.(3) ?The results were shown by the dual-luciferase: the GNAQ-3’UTR-psi-CHECKTM-2 andpri-mi R-200b-pc DNA3.1(+) transfection group in hela cell was 77.5% of that in the psi CHECKTM-2transfection group. The other two transfected groups were without statistical significance compared to thepsi CHECKTM-2 transfection group. ? We found that in mi R-200 b interference group, GNAQ genesignificantly decreased, ITPR, PRKCB, GNA1, MAP3K1 genes significantly raised and estrous related geneGPR54, KISS1 and Gn RH significantly increased. ?Results showed that in mi R-200 b interference group,GNAQ gene expression increased, its downstream COMT, HSD3B1, CYP1A1 and PKA genes expressiondecreased.Conclusion:(1) Depending on the targets and estrous pathways, from two kinds of nutrients of thetyrosine and folic acid, we can find the different estrous pathways from the light regulation: The PI3 K geneis taken as the core, the downstream gene is MEK1 in the PI3K-AKT signal pathway, or the MMP14 genein the leukocyte transendothelial migration which subsequently effects on MEK1. MEK1 gene effects onthe FSH and LH through the Gn RH signal pathway. MEK1 could also effect on the PLA2G4 A in Fc gammaR-mediated phagocytosis pathway and then effects on the ALOX5 gene in ovarian steroidogensis pathwayin order to impact the secretion of E2 and leptin. On the upstream of PI3 K, the Rap1 effects on the PI3 K inthe leukocyte transendothelial migration pathway, the dopamine receptor gene effects on the Rap1 in theRap1 signal pathway, the dopamine effects on the dopamine receptor gene in the dopamine synapse and itis effected by the tyrosine upstream in this signal pathway. The pathway which starts with the tyrosine isdifferent from the known photo-regulated estrous pathways. Additionally, the folic acid effects on theSMAD7 in the endocytosis pathways, then effects on the SMAD2/3 and SMAD4, in order to impact on theE2 secretion and finally influences on the estrous. The above pathways set a foundation to explore theseasonal nutrition regulation estrous network in future.(2) The mi R-200 b target GNAQ gene by cell leveltest which also show that the mi R-200 b may regulate the estrous process by targeting the GNAQ. |
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