Identification Of Biomarkers And Molecular Regulation Mechanisms Of Early Pregnancy In Cattle

Pregnancy recognition and pregnancy establishment after artificial insemination(AI)are crucial for successful pregnancy and calving.Early pregnancy diagnosis can identify pregnant and non-pregnant cattle in time,therefore,it is very useful for both fine management of pregnant cattle and the re-insemination of non-pregnant cattle.Also,it is important for the improvement of fertility.Up to now,identification of candidate genes related to reproductive traits in cattle is relatively limited,and the molecular regulation mechanism of early pregnancy is unclear,and it is urgent to develop an accurate,simple,and fast early pregnancy diagnosis technology in cattle production.Based on comparisons of the physiological and biochemical indicators between the pregnant and the non-pregnant cattle,the m RNA-Seq transcriptome sequencing and the i TRAQ quantitative proteomics techniques were used to identify key candidate genes and proteins involved in early pregnancy.The molecular mechanisms of pregnancy recognition and pregnancy establishment were explored,and,the early pregnancy diagnosis technique based on interferon-stimulated genes(ISGs)was preliminary established.This study provides theoretical and technical supports for our understanding of the molecular mechanisms of pregnancy and the application of new biomarkers for pregnancy diagnosis in cattle,and paves the way for the molecular breeding of high fertility cattle.The research details are as follows:1.Phenotypes of blood parameters and identification of candidate genes during early pregnancy in cattle(1)Comparations of hormone,blood parameters,and cytokines between pregnant and non-pregnant buffaloesThe blood parameters(blood parameters,progesterone,and cytokines)of 29 buffaloes(6 pregnant and 23 non-pregnant buffaloes,respectively)in early pregnancy were comparatively analyzed.The results showed that the absolute value and percentage of mononuclear cells(MONO)in the blood of pregnant buffaloes from 18 to 30 days after AI were significantly higher than those of 0d,and the percentage of MONO in pregnant buffaloes was significantly higher than that in non-pregnant buffaloes at 23 d after AI.The absolute value and percentage of eosinophils(EOS)in pregnant buffaloes at 14 d after AI were significantly higher than that at 0d,but there was no significant difference between pregnant and non-pregnant buffaloes.The progesterone in pregnant buffaloes was significantly higher than that of non-pregnant buffaloes at each sampling time from 14 d to 30 d after AI.Progesterone was negatively correlated with the percentage of lymphocyte(LYMPH),but positively correlated with the absolute value and percentage of EOS.From 0d to 20 d after AI,the expression of IFN-?,TNF-? and IL-1? decreased,while IL-13 and IL-10 increased,but there was no significant difference compared with that on 0d.Expression of ANG-1 in the blood of pregnant buffaloes at 18 d after AI was significantly higher than that on 0d.These results suggest that an increased MONO percentage,progesterone and ANG-1 in blood during early pregnancy may contribute to the establishment of pregnancy.(2)Identification of candidate genes in buffaloes during early pregnancyRNA-Seq analysis was performed via using blood leukocytes of 4 pregnant buffaloes at 0d,14 d,18d,and 20 d after AI.A total of 74 differentially expressed genes(DEGs)were identified.There were 52 DEGs at 18 d,when the blood cell transcriptome changed most significantly.Moreover,the difference of transcriptome at 18 d was synchronous with the change of IFNT.Canonical pathway analysis using Ingenuity Pathway Analysis(IPA)showed that DEGs on 18 d after AI were mainly involved in the IFN signaling pathway(IFI6,IFIT1,IFITM3,ISG15,MX1 and OAS1)and cytoplasmic pattern recognition receptor activation signaling pathway(IRF),etc.The results of functional enrichment analysis showed that HBA1/HBA2,HBB,NUPR1,IFIT1,IFIT3,ITGA4 and ISG15 were key candidate genes involved in regulating embryonic development and the development and function of the reproduction system in early pregnancy.2.Study of early pregnancy diagnosis technology based on candidate genes related to pregnancy recognition in cattleThe expression of ISG15,OAS1 and RSAD2 in peripheral blood of pregnant dairy cows(n=11)and non-pregnant dairy cows(n=8)at 0,14,18,21 and 28 d after AI were detected with fluorescence quantitative polymerase chain reaction(QPCR).Based on further validation in a population(19 pregnant dairy cows and 17 non-pregnant dairy cows),the pregnancy diagnosis effect based on the expression of these three genes separately or in combination was analyzed by receiver operating characteristic curve(ROC).The combination with the highest accuracy was amplified using probe primers anddouble fluorescence quantitative PCR.The results showed that expression of ISG15,OAS1 and RSAD2 onday 18 after AI was significantly higher in pregnant cows than that in non-pregnant cows.Then,the three genes were used separately or in combination for early pregnancy diagnosis,the sensitivity and specificity for the RSAD2 gene was 100% and 88.2%,respectively.The combination of ISG15 with RSAD2 was 94.7% and 100%,respectively.The duplex quantitative QPCR showed that,although the sensitivity of ISG15 alone was 100%,its specificity was only 88.2%(cut-off value 1.402).The sensitivity of RSAD2 alone was 89.5%,and the specificity was 88.2%;however,when the two genes were used in combination,the sensitivity,specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR.These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using ISG15 and RSAD2 was preliminaryly established.3.Study of differentially expressed proteins in bovine serum and identification of key candidate proteins during early pregnancyA combined technologies of isobaric tags for relative and absolute quantitation,(i TRAQ),IPA,QPCR and Western blot were usede for the study.A total of 114 differentially expressed proteins(DEPs)were detected in the serum of dairy cows during the early pregnancy(within 28 d after AI),including 64 DEPs in pregnant cows and 33 DEPs in non pregnant cows,there were 17 DEPs in the serum of both pregnant and non-pregnant cows.The protein expression profiles in serum of pregnant and non-pregnant cows were constructed following the comparion and analysis at different time points after AI.28 DEPs were involved in embryonic development,reproduction system development and function changes.Based on the results of hub protein exploration,some of the key proteins(EFEMP1,CD44,KRT18,NRF2 and BRCA2)were further verified with Western blot.These results showed that the relative expressions of key proteins were consistent with i TRAQ,and it suggested they might be candidate biomarkers for pregnancy diagnosis in dairy cows.4.Mechanisms of key candidate genes mediating early pregnancy in cattleThe proliferation of bovine endometrial epithelial cells and the expression of HO-1 and HOXA10 were detected from 24 h to 96 h after NRF2 inhibition by using si RNA.The results showed that,with the inhibition of the NRF2 expression,cell proliferation was decreased significantly,and the m RNA and protein expression of HO-1 and HOXA10 were significantly decreased.The study further analyzed the expression of genes related to the unfolded protein response(UPR)pathway with IFNT stimulation and si RNA inhibition.Our results showed that,NRF2 and endoplasmic reticulum stress(ERS)-related genes(GRP78,CHOP,PERK and ATF6)were significantly up-regulated after stimulated by IFNT in bovine endometrial epithelial cells,suggesting that IFNT could induce ERS in bovine endometrial epithelial cells and induce UPR by activating ATF6 and PERK.However,si RNA inhibited NRF2 expression,UPR induced by IFNT in bovine endometrial epithelial cells was inhibited,and the expression of CHOP m RNA was significantly up-regulated,the expression of BCL2 and BAX m RNA and the ratio of the expression were significantly decreased,which suggests the cells were induced to apoptosis.These results indicated that NRF2 may participate in IFNT-mediated pregnancy recognition through UPR pathway.