Background

Synchronization of estrus and ovulation programs for timed artificial insemination(TAI)has been constantly incorporated on modern reproductive management of beef farms[1,2].These programs can induce the first postpartum ovulation and,consequently,hasten the establishment of pregnancy of suckled beef cows[1,3-5].However,a significant proportion of ovulated and inseminated cows are detected not-pregnant 30 d after insemination　despite the satisfactory ovulation rate(～85%)following protocols for synchronization of ovulation[4,6,7].The uterine environment plays a relevant role among factors that are likely to contribute to the observed failures[8-10].

Early classic studies demonstrated the significant impact of a coordinated and sequential exposure to ovarian steroids on uterine function[11-13].Gene expression of bovine endometrium changes according to the phase of the estrous cycle and is closely controlled by circulating concentrations of estradiol(E2),progesterone(P4)and the expression ratio of their specific receptors[8,14-17].In this regard,proestrus E2 concentration is fundamental in modulation of the uterus for the subsequent luteal phase[8,14,18,19].This E2 priming may be important for induction of endometrial P4 receptors[20,21]to avoid premature luteolysis and short cycles in beef cattle[22].In cyclic dairy heifers,elevated E2 concentrations during proestrus,induce changes in uterine gene expression of E2 and P4 receptors(ESR1 and PGR,respectively),oxytocin receptors,and expression of cyclooxygenase-2,and beta subunit inhibin serpin-14 throughout the subsequent estrus cycle[23].Also,cyclic beef heifers that are exposed to a longer proestrus period exhibit alterations in the pattern of steroids receptors expression in the uterus and other proteins associated with uterine receptivity to pregnancy[19].Therefore,it is reasonable to hypothesize that the modulation of E2 concentration during the synchronized proestrus by means of exogenous E2 supplementation could also alter the uterine gene expression of suckled anestrous beef cows.

有形的时间，通过投射的对象，形成了文化记忆的磁场。怀旧、感伤的历史记忆早在汉代西域就由远嫁和亲的细君公主发出了“愿为黄鹄兮还故乡”的思乡感慨。此后，大抵进入中原的羁旅者，无论何种身份，都留有“故园东望路漫漫”的慨叹之情，这种选择性的描绘，是西域留给大家的一种身份印记，延续到今天，也不自觉地会进入到移居者的话语体系中。

Two pharmacological strategies to manipulate the proestrus phase have been extensively evaluated in cattle breeding programs;exogenous E2 supplementation or equine chorionic gonadotropin(eCG)administration.Firstly,exogenous E2 supplementation using E2 esters enhances the proportion of cows that display estrus[24-26],increases endometrial thickness in lactating dairy cows[27]and improves the pregnancy success of suckled beef cows[6,25,26].Furthermore,Jinks et al.[28]demonstrated that,recipients beef cows with lower E2 concentration at periovulatory phase,receiving in vivo-produced embryo,presented a dramatic reduction on pregnancy establishment(45%vs.65%of pregnancy rate).Secondly,administration of eCG at onset of the proestrus is an efficient alternative to increase final follicular growth,ovulation rate and plasma P4 concentration on subsequent diestrus[5,26,29,30].Such changes may be responsible for the increase in pregnancy rates of anestrous beef cows stimulated with eCG[5,26,29,30].Altogether,both pharmacological strategies to manipulate the proestrus are capable of altering the periovulatory steroidal endocrine profiles,potentially modulating the expression of genes associated with uterine receptivity and ultimately positively influencing pregnancy establishment of suckled anestrous beef cows.

Therefore,based on the importance of the proestrus hormonal milieu on fertility,we hypothesized that supplementation with estradiol cypionate(ECP)and/or eCG at the onset of proestrus alters the ovarian response and the uterine transcriptome of suckled anestrous beef cows.To assess the above mentioned hypothesis,we chose the following approaches.First,taking a comprehensive approach,RNA extracts from endometrial fragments were submitted to Next Generation RNA sequencing followed by functional enrichment analysis to potentially identify and characterize other ECP-regulated biological and molecular processes and pathways.Secondly,following a candidate gene approach,we tested the effect of ECP and/or eCG supplementation on the expression of selected molecules with relevant biological functions in the context of uterine biology,specifically associated with cell proliferation.

Methods

Animals

Animal procedures were approved by the Ethics and Animal Handling Committee of the Faculdade de Medicina Veterinária e Zootecnia,Universidade de São Paulo(CEUA-FMVZ/USP,No.2287/2011).This experiment was conducted during the 2012/2013 spring-summer breeding seasons.A total of 172 suckled anestrous Nelore(Bos indicus)beef cows at 30-60 d postpartum from a commercial farm in the state of Parana,Brazil,were enrolled in this study.Cows were maintained on Brachiaria brizantha pasture with water and mineral supplementation ad libitum.Immediately prior to the initiation of the TAI protocol,information about body condition score from each cow were recorded(BCS;range,1=emaciated to 5=obese;with 0.5 scale)[31].

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Reproductive management and experimental design

After calving,cows were allocated into breeding groups according to calving date.At 30 to 60 d post-partum,females were synchronized using an E2-plus-P4-based TAI protocol.Briefly,suckled cows received an intravaginal P4-releasing insert previously used for 8 d(1 g of P4;DIB®,MSD Animal Health,São Paulo,Brazil)on D-10 along with an intramuscular(IM)administration of 2 mg estradiol benzoate(EB;Gonadiol®,MSD Animal Health,São Paulo,Brazil;Fig.1).The P4 insert were removed eight day later(D-2).All cows received an intramuscular administration of 500 mg of cloprostenol(Ciosin®,MSD Animal Health,São Paulo,Brazil)at the moment of the P4 insert removal.At this moment,cows were blocked by BCS,parity(multiparous vs.primiparous)and the diameter of the largest follicle and then randomly assigned into one of four experimental groups[Control(CON):n=43,Estradiol cypionate(ECP):n=43,eCG:n=44,and ECP+eCG:n=42],in a 2×2 factorial arrangement.Cows from ECP group received an IM injection of 1 mg of ECP(E.C.P.;Zoetis,São Paulo,Brazil),cows from eCG group received an IM injection of 400 IU of eCG(Folligon®,MSD Animal Health),while cows from ECP+eCG group received both treatments and cows from CON group did not receive any treatment.In all groups,ovulation was induced by 10 μg of buserelin acetate(GnRH,Sincroforte,Ourofino Saúde Animal,Cravinhos,São Paulo,Brazil)IM administration 48 h after the P4 insert removal(D 0).Cows were artificially inseminated immediately after GnRH treatment.Inseminations were performed by a single technician using frozen-thawed semen from single Angus sire with proven fertility.The sire used had been previously used in TAI programs and had satisfactory(～50%)pregnancy results.

Estrus was determined based on the tail-head mark.At the time of the removal of the P4 insert,the tail-head was marked with chalk(Raidl-Maxi,RAIDEX GmbH,Dettingen/Erms,Germany).Estrus was deemed to have occurred in cattle without a tail-head mark at TAI.

Cows presenting a corpus luteum(CL)on D 6(6 d after GnRH treatment)had the body of the uterus biopsied as previously described[32].Fragments obtained from uterine biopsies were individually allocated in cryotubes and immediately immersed into liquid nitrogen.Day 6 was strategically selected as the moment in which an early embryo is expected to have recently accessed the uterine environments.Pregnancy was diagnosed by transrectal ultrasonography through the detection of a viable embryo(presence of heartbeat)on d 42 post-AI.

在中研院任上，蔡元培本着学术自由、兼收并蓄和吸收消化的原则，延揽一批学有所长的学者到民族学组来研究，其中有著名的法国边疆民族学派代表人物凌纯声、美国批评民族学派代表人物戴裔煊等，共同推动近代民族学研究[15]375。1929年，蔡元培开始筹备中华民族博物馆，由于展品较少，他考虑将在中国的民族学标本重复物品，与德国汉堡民族博物馆等其他国家民族博物馆物品作交换。

Blood sampling for determination of P4 concentrations was performed on D 6,concurrently with the uterine biopsy.Blood　sampleswere　collected　by　coccygeal venipuncture using evacuated tubes containing EDTA(BD,São Paulo,SP,Brazil)and immediately stored in ice.Plasma was separated by centrifugation at room temperature,1,500× g for 15 min,and stored at-20°C.Progesterone concentrations were measured in all samples using a solid-phase radioimmunoassay(Coat-a-count,Siemens,Los Angeles,USA),as validated previously[33].The P4 assay sensitivity was 0.08 ng/mL and the intra-assay coefficient of variation was 8.7%.

Ultrasound examinations

Transrectal ultrasound examinations were carried out on D-10,D-2,D 0 and D 6 to assess cyclic status,growth of the dominant follicle(DF),ovulation,and the presence of CL.Ultrasonography was performed with the aid of a B-mode(gray-scale)ultrasound instrument(8100,Chison Medical Imaging,Co,China),equipped with a multi-frequency linear-array transducer.The anestrous status was defined as the absence of CL in two consecutive ultrasound　examinations performed on D-10 and D-2.Ovulation was defined as the presence of a recently formed CL on D 6 on the same ovary that the DF was observed on D-2 and D 0.The diameter of the DF at the time of P4 insert removal and at TAI,in addition to the diameter of new CL formed,was calculated as the average between measurements of two perpendicular axes of each structure.

Fig.1　Schematic diagram of the synchronization of ovulation protocol in suckled anestrous beef cows.EB=2 mg of estradiol benzoate;P4=progesterone;P4 insert=previously used intravaginal P4 insert containing 1.0 g of P4;GnRH=100 mg of gonadorelin;ECP=1 mg of estradiol cypionate;eCG=400 IU of equine chorionic gonadotropin;PGF2α=0.25 mg of cloprostenol;US=ultrasound examination;BS=blood sample.Cows from ECP group received ECP and cows from eCG group received eCG,while cows from CON did not receive any further treatment and cows from ECP+ECG received both treatments

RNA-seq

Approximately 30 mg of endometrial tissue was macerated in liquid nitrogen using a stainless steel mortar and pestle and immediately mixed with buffer RLT from the PureLink®,RNA Mini kit(Thermo Fisher Scientific,São Paulo,SP,Brazil),as per manufacturer’s instructions.To maximize lysis,tissue suspension was passed at least ten times through a 21-ga needle,and centrifuged at 13,000×g for 3 min for removal of debris,prior to supernatant loading and processing on RNeasy columns.Columns were eluted with 40 μL of RNase free water.Concentration of total RNA on extracts was measured by a spectrophotometer(Nanovue™Plus,Spectrophotometer,GE Healthcare,UK,by the absorbance at 260 nm).Subsequently,samples were treated with 80 μL of DNAse I solution(Life Technologies,São Paulo,SP,Brazil)for 15 min at room temperature during RNA extraction protocol,according to manufacturer’s instructions.RNA samples were stored at-80°C until cDNA synthesis.The cDNA was synthesized by reverse-transcription using High Capacity cDNA Reverse Transcription Kit(Life Technologies)according to manufacturer’s instructions.Briefly,10 μL of master mix containing RT buffer,dNTP mix,random primers,RNase inhibitor and reverse transcriptase were mixed to 1 μg of total RNA and final volume of the reaction was adjusted to 20 μL.Immediately,reactions were incubated at 25°C for 10 min,followed by incubation at 37°C for 2 h,and reverse-transcriptase inactivation at 85 °C for 5 min.Samples were stored at-20 °C.

Blood sampling and hormone measurements

RNAseq

Prior to the RNA-seq analyses,12 samples(n=6/group;ECP and CON)were selected according to previously established criteria by ovarian,occurrence of estrus,pregnancy and endocrine responses.Cows having similar DF diameter at the time of P4 insert removal[ECP(12.1±0.7 mm)and CON(12.1±0.6 mm)]and similar circulating P4 concentration at the time of uterine biopsy[ECP(3.8±0.2 ng/mL)and CON(3.6±0.2 ng/mL)]were considered suitable to further analysis.Additionally,cows were also selected based on pregnancy status 30 d after TAI in order to have both pregnant and non-pregnant cows represented in both experimental groups.Finally,only cows displaying estrus were selected in ECP treated group,whereas only cows that did not display estrus were chosen in the control group.The latter criterion was applied aiming to increase the distinction between two different E2 pre-ovulatory endocrine environments,as cows that display estrus present greater E2 concentration than those not displaying estrus[42].

1.7 菌种保存 从培养基中刮取少量新鲜菌丝转接到MEA斜面上，室温培养至菌丝覆盖整个斜面后，放入4℃冰箱保存，所有操作均在无菌环境中进行。该方法在保存过程中易出现培养基水分丧失、菌体失活，因此每隔半年须重新转接，只适合短期保存。

Integrity of total RNA extracts was assessed using the Agilent RNA 6000 Nano chip(Bioanalyzer,Agilent Technologies).RNA Integrity Number(RIN)of extracts submitted to RNA sequencing analysis ranged from 8.3 to 8.7.Next,4 μg of RNA were used with the TruSeq RNA Sample Preparation kit(Illumina,San Diego,CA)to prepare the libraries for RNA-Seq.The insert sizes were estimated through the Agilent DNA 1000 chip(Agilent Technologies)and the libraries concentration were measured through Quantitative Real-Time PCR(qPCR)with a KAPA Library Quantification kit(KAPA Biosystems).Samples were diluted,pooled in equimolar amounts and then sequenced at the Centro Genômico Funcional Aplicado a Agropecuária e Agroenergia at the University of São Paulo using a HiScanSQ sequencer(Illumina,San Diego,CA).

Bioinformatics analyses

Raw sequences were trimmed for adaptors and low quality using SeqyClean v1.3.12.(https://github.com/ibest/seqyclean)using 26 Phred quality parameter for maximum average error and a fasta file with contaminantsequencesfrom　the　Univec　database(https://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/).　Only high quality paired-end sequences were kept for further analyses The reads were mapped with Bowtie2 v2.1.0[34]on the masked bovine genome assembly(Bos taurus UMD 3.1,NCBI).The mapping file was sorted using SAMTools v 0.1.18[35]and read counts were obtained using the script from HTSeq-count v0.5.4p2(http://htseq.readthedocs.io/en/release_0.9.1/).The differential expression analysis was performed with package DESeq2[36]from R[37].Using the function estimateSize-Factors,the normalized counts were obtained(baseMean values,which are the number of reads divided by the size factor or normalization constant).The standard deviation along the baseMean values was also calculated for each gene.In order to avoid artifacts caused by low expression profiles and high expression variance,only transcripts that had an average of baseMean＞5 and the mean greater than the standard variation were analyzed.The threshold for evaluating significance was obtained by applying an alpha≤0.10,considering the FDR-Benjamini-Hochberg P-value[38].Integrated analysis of different functional databases was done using the functional annotation tool of the Database for Annotation,Visualization,and Integrated Discovery using as background the genes(DAVID)[39]using as background the set of genes that passed through the differential expression analysis filter.

对设计资料、制造资料、竣工资料、使用登记证、年度检查和上次检验资料进行核查，确定实物与资料相一致。检查压力容器操作员证、安全管理人员证，以保证压力容器操作人员是持证上岗，具有基本的专业素养。

qPCR

The samples employed in qPCR analysis were selected mirroring the general results obtained in regard to ovarian and endocrine responses.Cows receiving ECP should present greater occurrence of estrus,while cows from eCG treatment group should present greater circulation of P4 concentration at the moment of the uterine biopsy.Step-One Plus thermocycler(Life Technologies,Carlsbad,CA)and SYBR Green chemistry were used for quantitative PCR analysis.Primers were designed based on the mRNA sequence of target genes obtained from the RefSeq database,on Genbank(http://www.ncbi.nlm.nih.gov/genbank/).Sequences were masked to remove repetitive sequences with RepeatMasker(http://www.repeatmasker.org/)[40]and then,the masked sequences were used for primer design using the PrimerQuest software(IDT1,http://www.idtdna.com/primerquest/Home/Index).The characteristics of the primers were checked in Oligo Analyzer 3.1 software(IDT1,http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/),while the specificity was compared by BLAST(NCBI,http://blast.ncbi.nlm.nih.gov).The qPCR products obtained from reactions performed with primers not previously validated were submitted to agarose gel electrophoresis and SANGER-DNA sequencing,and identities of target genes were confirmed.Details of primers are provided on Table 1.In order to select reference genes,the GeNorm Microsoft Excel applet was used,as this applet provides a measure of gene expression stability(M)[41].The Glyceraldehyde-3-Phosphate　Dehydrogenase　(GAPDH),Actin,Beta(ACTB)and Ribosomal Protein S18(RPS18)were the most stable genes and were,therefore,selected as reference genes.Determination of qPCR efficiency and Cq(quantification cycle)values per sample were performed with LinRegPCR software(V2014.2;http://www.hartfaalcentrum.nl/index.php?main=files&fileName=LinRegPCR.zip&description=LinRegPCR:%20qPCR%20data%20analysis&sub=LinRegPCR).Quantification was obtained after normalization of the target genes expression values(Cq values)by the geometric mean of the endogenous control expression values.The following genes,associated with regulation of cell proliferation in the uterus,were selected:ovarian steroid receptors[Estrogen Receptor alpha(ESR1),Estrogen Receptor beta(ESR2),P4 receptor(PGR)],growth factors that regulate cellular proliferation[epidermalgrowth　factorreceptor(EGFR),heparinbinding EGF-like growth factor(HB-EGF)and patched homolog 2(PTCH2)],and extracellular matrix[collagen,type IV,alpha 1(COL4A1)and integrin,beta 3(ITGB3)].

Statistical analyses from ovarian,endocrine and gene expression responses

The statistical analyses for ovarian responses were performed using the PROC GLIMMIX of SAS for Windows(SAS 9.3 Institute Inc.,Cary,NC,USA,2003).Continuous variables were presented as mean±standard error of the mean(mean±SEM)and percentage(%)for frequency of occurrence for binomial variables.The continuous response variables were subjected to response scaling test through the solution Guided Data Analysis of SAS.Variables that did not follow these assumptions were transformed accordingly.Binomial variables(i.e.occurrence of estrus and ovulation rate)were analyzed by logistic regression using the SAS GLIMMIX procedure with models fitted to binomial distributions.The explanatory variables considered for inclusion in the models were the treatment with ECP,eCG and interaction of ECP and eCG.The effect of cow within each replicate was included as a random effect.

Table 1　Gene name,accession number,forward and reverse primer sequences used for qPCR analysis

aTranscript variants X1 to X7

Gene Name　Gene ID　Sequence ID　Forward primer sequence(5′-3′)Reverse sequence(5′-3′)　Primer efficiency,%Amplicon length,bp Progesterone receptorPGR　NM_001205356.1　GCCGCAGGTCTACCAGCCCTA　GTTATGCTGTCCTTCCATTGCCCTT　96.9　199 Estrogen receptor 1　ESR1　NM_001001443.1　CAGGCACATGAGCAACAAAG　TCCAGCAGCAGGTCGTAGAG　99.1　82 Estrogen receptor 2　ESR2　NM_174051.3　TCACGTCAGGCACGCCAGTAAC　CACCAGGTTGCGCTCAGACCC　99.5　155 Patched 2a　PTCH2　XM_005197904.1　CATCCTGCTGCTGTGTACTT　ATCGCCAGGACCAGTACTAT　99.9　87 Epidermal growth factor receptor EGFR　XM_002696890.3　ATGCTCTATGACCCTACCAC　TTCCGTTACAAACTTTGCCA　97.6　178 Heparin-binding EGF-like growth factor HB-EGF　NM_001144090.1　CATCCACGGAGAATGCAAATAC　CAGCAGACAGACGGATGATAG　98.6　181 Collagen,type IV,alpha 1 COL4A1　NM_001166511.1　CACGGCTACTCTTTGCTCTAC　GAAGGGCATGGTACTGAACTT　96.48　102 Integrin,beta 3(platelet glycoprotein IIIa,antigen CD61)ITGB3　NM_001206490.1　GGGAGAGTGCTATGGTTAGA　CTTCACAAGACACCCAAGAG　92.09　142 Actin Beta　ACTB　NM_173979.3　GGATGAGGCTCAGAGCAAGAGA　TCGTCCCAGTTGGTGACGAT　93.7　77 Glyceraldehyde-3-Phosphate Dehydrogenase GAPDH　NM_001034034.2　GCCATCAATGACCCCTTCAT　TGCCGTGGGTGGAATCA　99.99　69 Ribosomal Protein S18　RPS18　AY786141.1　TGGAGAGTATTGCGCCTTCTC　CACAAGTTCCACCACACTATTGG　97.9　79

The qPCR data were tested for normality of residuals and homogeneity of variances followed by ANOVA using the GLIMMIX procedure of SAS fitting log normal distribution.The explanatory variables considered for inclusion in the models were the treatment with ECP,eCG and interaction between ECP and eCG.Final results are presented in natural log(Ln)scale(because of the log normal distribution considered)as normalized values of a specific gene transcript by the mean level of the transcript from Control(No-ECP and No-eCG treated animals).Downregulation of expression in a specific experimental group may be represented by negative values relative to control because of Ln scale.To avoid negative values,the mean used for data normalization was divided by the fifth negative exponent.All data were compared with the relative mean expression level of the control group.

Statistical difference was considered when P＜0.10.Graphs were plotted with Sigmaplot(version 11.0;Systat Software,Inc.San Jose,CA,USA).

Results

Ovarian,pregnancy and endocrine responses

Animals receiving different hormonal therapies at the proestrus presented different rates of occurrence of estrus between P4-releasing device removal and TAI,final follicular growth,ovulatory responses and subsequent CL function(Table 2).There were no interactions between ECP and eCG treatment on response variables,except for the CL diameter 6 d after the TAI(P=0.06).Larger CLs were observed in cows treated with eCG,especially in cows not treated with ECP.The ECP treated cows presented a greater frequency of occurrence of estrus[ECP=64.7%(55/85)vs.No-ECP=44.8%(39/87);P=0.008]and presented greater pregnancy per TAI[ECP=47.1%(40/85)vs.No-ECP=33.3%(29/87);P=0.07].Cows treated with eCG presented greater rate of final follicular growth[eCG=1.2±0.1 mm/d vs.NoeCG=0.9±0.1 mm/d;P=0.01],resulting in a greater DF diameter at TAI[eCG=13.5±0.3 mm vs.NoeCG=12.6±0.3 mm;P=0.03].Also,a greater proportion of cows receiving eCG displayed estrus[eCG=62.8%(54/86)vs.No-eCG=46.5%(40/86);P=0.03]and ovulated[eCG=96.5%(83/85)vs.No-eCG=82.6%(71/86);P=0.008].A greater P4 concentration at the moment of uterine biopsy(D 6)was observed in cows receiving eCG at the onset of the proestrus[eCG=4.8±0.2 ng/mL vs.No-eCG=3.9±0.2 ng/mL;P=0.001].However,there was no influence of eCG treatment on the pregnancy per TAI[eCG=43.0%(37/86)vs.No-eCG=37.2%(32/86);P=0.42].

Table 2　Overall occurrence and effects of treatment with estradiol cypionate(ECP)and/or equine chorionic gonadotropin(eCG)at onset of the proestrus on follicular and luteal development in an estradiol/progesterone-based synchronization protocol on anestrous suckled beef cows

1Suckled anestrous beef cows received an previously used intravaginal insert containing 1.0 g of progesterone(P4)and 2.0 mg of estradiol benzoate on the first day of the estrus/ovulation synchronization protocol(D-10).The P4 insert was removed eight days later(D-2),and cows from ECP group received an IM treatment of 1 mg of ECP,cows from eCG group received an IM injection of 400 IU of eCG,while cows from ECP+ECG received both treatments and cows from CON did not receive any treatment at this moment.All cows received GnRH IM and were timed artificially inseminated(TAI)48 h after the P4 insert removal(D 0).Different letters within the same row indicate the presence of difference between groups(P＜0.05)when an interaction between eCG and ECP was observed 2BCS=Body condition score collected at insertion of the P4 insert 3DF=Dominant follicle 4TAI=timed artificial insemination 5DF growth between the P4 insert removal and TAI divided by two 6Estrus determined based on the tail-head mark 7Number of cows with a new CL formed 6 d after the TAI divided by the number of animal synchronized

Itens　Treatments1 Pvalue No ECP　ECP No eCG　eCG　No eCG　eCG　ECP　eCG　ECP×eCG Number of cows　43　44　43　42　-　-　-BCS at onset of the synchronization2　3.1±0.1　3.0±0.1　2.9±0.1　3.0±0.1　0.12　0.71　0.43 DF diameter at insert removal,mm3　11.0±0.4　11.2±0.4　10.8±0.4　11.3±0.4　0.90　0.38　0.77 DF diameter at TAI,mm4　12.6±0.4　13.6±0.4　12.7±0.4　13.4±0.4　0.90　0.03　0.68 Daily DF growth,mm/d5　0.9±0.1　1.3±0.1　0.9±0.1　1.1±0.1　0.52　0.01　0.25 Occurrence of estrus,%6　37.2　52.3　55.8　73.8　0.008　0.03　0.77 Ovulation rate,%7　81.4　95.5　83.7　97.6　0.54　0.008　0.71 CL diameter at d 6 after TAI,mm　17.8±0.6b　20.1±0.5a　18.6±0.6ab　18.7±0.6ab　0.54　0.04　0.06 Plasma P4 at d 6 after TAI,ng/mL　3.8±0.3　5.1±0.3　4.1±0.3　4.6±0.3　0.94　0.001　0.18 Pregnancy per TAI,%　30.2　36.4　44.2　50.0　0.07　0.42　0.95

Tissue processing,RNA isolation and cDNA synthesis

KEGG pathway and Gene ontology(GO)term analyses were performed with DAVID(Table 3).Functional enrichment analysis using DAVID revealed two KEGG pathways overrepresented by the ECP-upregulated transcripts:pathways in cancer(5 genes;P＜0.01)and small cell lung cancer(3 genes;P＜0.05).On the other hand,ECP downregulated transcripts indicated the enrichment of three pathways:Parkinson’s disease(3 genes;P=0.06),oxidative phosphorylation(3 genes;P=0.06)and Alzheimer’s disease(3 genes;P=0.09).More specifically,ECP-upregulated transcripts associated with pathways in cancer were[gene symbol(fold change;adjusted P value on RNA-seq);respectively]:LAMC3(1.55;P=0.10),PTCH1(1.51;P=0.09),PTCH2(1.52;P=0.03),PIK3R3(1.22;P=0.10),and PIAS1(1.18;P=0.09),whereas ECP downregulated transcripts associated with oxidative phosphorylation were ATP5F1(1.18;P=0.01),ATP5J(1.24;P=0.06),and NDUFB3(1.37;P=0.01).Additionally,analysis of GO terms identified that ECP upregulated transcripts over represented epidermis development[ADAM9 and ENSBTAG00000017455(uncharacterized protein)].On the other hand,ECP downregulated GO terms indicated the enrichment of five biological processes:generation of metabolic precursors and energy(GPI,NDUFB3,ATP5F1,IDH3B,ATP5J),Translation　(RPS2,EEF1D,ENSBTAG00000013866,ENSBTAG00000011263),and mRNA processing,mRNA metabolic process and RNA splicing with 3 common genes(GEMIN7,SNRPD2,STRAP).

Sequences　ofallreads　were　deposited　in　the Sequence Read Archive(SRA)of the NCBI(http://www.ncbi.nlm.nih.gov/sra/;Additional file 3:Table S3)and,an overview of these data has been deposited in NCBI’s Gene Expression Omnibus(GEO)and is accessible through GEO Series accession number GSE67807.

Functional enrichment analysis of RNA-seq data-DAVID results

RNA sequencing produced a total of～334 million reads with an average of 27.5 million reads for each group.Six biological replicates were analyzed for each phenotype(please see Statistical Analyses section above)with the reads ranging from 17 to 26 million per sample after filtering(Additional file 1:Table S1).Approximately～65%of the total reads uniquely mapped to the UMD 3.1 reference genome(https://www.ncbi.nlm.nih.gov/genome?term=bos%20taurus).Only the uniquely mapped reads were considered in the analysis.From the remaining,approximately 20%of the reads were not uniquely mapped,and 15%unmapped reads.After applying the variance and minimal value of baseMean filtering,a total of 15,161 genes were included on the differential expression analysis.A total of 310 out of the 15,161 analyzed genes showed differential expression(adjusted P-value＜0.1),of which 73 and 62 were upregulated in the endometrium of ECP and CON samples,respectively(see Volcano plot,Fig.2 and Additional file 2:Table S2).Differentially expressed genes(DEG)with the greatest expression profiles were RPS2[ribosomal protein S2],GABARAP[GABA(A)receptor-associated protein],up-regulated in the CON endometrium,and PEPD[peptidase D],SG100g[calcium binding protein G]and CEACAM1[carcinoembryonic antigen-related cell adhesion molecule 1],up-regulated in the ECP group.Heatmap on Fig.3 shows the 50 genes with the lowest p-adjusted values.It is possible to observe the similarity of gene expression patterns among individuals within each group,as indicated by the shades of green(for low expression)or red color(high expression).

Fig.2　Volcano plot obtained from DESeq analysis.Volcano plot shows that the vertical lines axe is log2-fold change and the horizontal axis is the statistical significance(P value≤ 0.10).Genes with P value≤0.10 are marked with blue dots

Fig.3　Heat map obtained from DESeq2 analysis.Each column represent one sample showing the intensity of expression profile per gene.The colors in the map display the relative standing of the reads count data;GREEN indicates a count value that is lower than the mean value of the row while red indicates higher than the mean.The shades of the color indicate distance from each data point to the mean value of the row

FAPESP(2012/14731-4)to MFSF.

According to selection criteria described previously,uterine tissue used in the qPCR analysis derived from cows that presented different estrus responses[CON(10.1%),ECP(90.9%),eCG(66.7%)and ECP+ECG(83.3%)]and P4 concentration at uterine biopsy[CON(3.4±0.2 ng/mL),ECP(3.7±0.2 ng/mL),eCG(5.3±0.4 ng/mL)and ECP+ECG(5.0±0.6 ng/mL)].

There were no interactions(P＞0.10)between ECP and eCG on the expression of the transcripts evaluated.ECP treatment induced greater endometrial abundance of PTCH2(P=0.07)and COL4A1(P=0.02)genes,whereas it reduced EGFR(P=0.09)gene expression(Figs.4 and 5).The ECP treatment did not affect geneexpression of ESR1(P=0.90),ESR2(P=0.61),HB-EGF(P=0.80)and ITGB3(P=0.57).On the other hand,eCG treatment induced greater endometrial abundance of HBEGF(P=0.06),ESR2(P=0.09),and ITGB3(P=0.05)genes,whereas reduced the gene expression of ESR1(P=0.05).Supplementation with eCG did not alter expression of EGFR(P=0.34),PTCH2(P=0.31)and COL4A1(P=0.19).Additionally,expression of PGR was not altered by either ECP(P=0.51)or eCG(P=0.25)treatments.

Table 3　KEGG Pathways and Gene ontologies(GO category)of mRNA transcripts differentially expressed in cows treated with estradiol cypionate(ECP).

Enrichment analysis was performed with DAVID tools(https://david.ncifcrf.gov/tools.jsp)

Category　Term　Count　P value　Genes Upregulated in ECP KEGG PATHWAY　Pathways in cancer　5　0.008　PIK3R3,PTCH1,PTCH2,LAMC3,PIAS1 KEGG PATHWAY　Small cell lung cancer　3　0.020　PIK3R3,LAMC3,PIAS1 GO TERM BP_FAT　Epidermis development　2　0.095　ADAM9,ENSBTAG00000017455 Downregulated in ECP KEGG PATHWAY　Parkinson’s disease　3　0.062　NDUFB3,ATP5F1,ATP5J KEGG PATHWAY　Oxidative phosphorylation　3　0.064　NDUFB3,ATP5F1,ATP5J KEGG PATHWAY　Alzheimer’s disease　3　0.090　NDUFB3,ATP5F1,ATP5J GO TERM BP_FAT　Generation of precursor metabolites and energy　5　0.004　GPI,NDUFB3,ATP5F1,IDH3B,ATP5J GO TERM BP_FAT　Translation　4　0.059　RPS2,EEF1D,ENSBTAG00000013866,ENSBTAG00000011263 GO TERM BP_FAT　mRNA processing　3　0.071　GEMIN7,SNRPD2,STRAP GO TERM BP_FAT　mRNA metabolic process　3　0.087　GEMIN7,SNRPD2,STRAP GO TERM BP_FAT　RNA splicing　3　0.041　GEMIN7,SNRPD2,STRAP

Discussion

The present study investigated the impact of hormonal manipulation of proestrus on ovarian response and on uterine transcriptome 6 d post-TAI.The most relevant observations from this study are:1)ECP treatment improves occurrence of estrus and pregnancy per AI,whereas eCG treatment enhances final follicular growth,size of ovulatory follicle,ovulation rate and subsequent P4 concentration,2)the endometrial transcriptional profile is regulated by ECP supplementation and cell proliferation was one of the overrepresented gene ontology terms;3)selected candidate genes with altered expression further support an ECP effect on cellular proliferation and tissue morphology.

在新一轮的辽宁振兴中，人才政策受到了各方的关注，在毕业生的留辽政策方面，政府在住房、贷款、创业、就业等多方面给予了扶持；在外地人才的回流方面，政府也在多方筹措，提供更多的政策、资金方面的支持；同时不断优化营商环境，加大放权力度，转变服务理念，优化办事流程。在历史文化街区的发展问题上，政府部门可以更加细化政策，明确相关条件与资质，说明政府的政策与扶持，这将对历史文化街区新一轮的发展、变革提供了有力的机会和保障。

Synchronized cows displaying estrus before TAI exhibited larger dominant follicles,greater E2 concentration during the proestrus/estrus,greater luteal function on the subsequent estrus cycle,and greater conception rate when compared to cows that did not display estrus[6,25,42-44].In agreement,exogenous ECP treatment at the onset of proestrus improved the proportion of suckled beef cows displaying estrus,determining greater pregnancy outcomes following TAI than non-ECP treated cows[25,26],similar to what was observed in the present study.Furthermore,the eCG treatment at onset of the proestrus was effective to increase conception rates in suckled beef cows[1,5,29,30].Also corroborating with the present results,eCG-treated cows presented greater final follicular growth,follicular diameter at TAI,ovulation rate and plasma P4 concentration on subsequent diestrus[5,26,29,30].Therefore,the hormonal therapies established in the present study may be considered a profertility model for suckled anestrous beef cows and potentially allow the establishment of two distinct periovulatory endocrine milieus,that are associated with an uterine environment of better receptivity.Specifically,itwasexpected　thatECP-treated　cows present greater periovulatory E2 concentration due to the exogenous estradiol administration.Additionally,those cows treated with eCG also presented greater concentrations of E2 during proestrus/estrus due to endogenous estradiol from a healthy larger DF at TAI,in addition to presenting greater concentrations of P4 during early diestrus.

Unexpectedly,transcriptome analysis of D 6 endometrium from cows treated or not with ECP did not reveal dramatic differences of gene expression patterns.Our model was unique in selecting for cows displaying estrus behavior in ECP-treated group versus not displaying estrus behavior in the control group.Estrus behavior is associated with higher pregnancy rates[6,25,43,44].Global transcriptome analysis of D 14 endometrium from high fertility heifers compared to low fertility ones did not reveal substantial differences[45].Another study using a similar criterion for high and low fertility revealed that D 7 endometrium presented 417 DEG,however,most of the DEG exhibited fold change between 1.0 and 2.0[46].These results are in agreement with our data showing that endometrial gene expression is not dramatically different between groups with contrasting fertility;however,it is important to point out that half of the samples came from pregnant animals,whereas the other half came from non-pregnant cows in both ECP or control groups.

选择SPSS 19.0统计学软件对数据进行处理，计量资料以均数±标准差表示，组间比较采用t检验，计数资料以率(%)表示，组间比较行χ2检验，预后影响因素采用Cox回归分析，术后生存情况采用Kaplan Meier描述，组间比较采用Log-rank检验，P<0.05为差异有统计学意义。

Fig.4　Comparison of gene expression between suckled anestrous beef cows receiving 1 mg of estradiol cipionate(ECP)and/or 400 IU of equine chorionic gonadotropin(eCG)at onset of the proestrus[CON(n=11),ECP(n=11),eCG(n=12)and ECP+ECG(n=11)].The amounts of ESR1,ESR2,PGR,and PTCH2 transcripts are expressed in relation to control(CON)untreated cows.Expression values were normalized by the geometric mean of GAPDH,ACTB,and RPS18.The P values refer to comparisons made for each gene between groups(effects of ECP,eCG and interaction between ECP and eCG)

Estradiol levels are higher after ECP administration[28,42,47]and estrus behavior is correlated with estradiol levels[42,48];however,we did not quantify estradiol plasma concentrations.It was observed in ovariectomized cows that estradiol benzoate injection alters global gene expression of the endometrium when compared to a control group or progesterone treatment;whereas a combined estradiol and progesterone group shows data closer to estradiol treatment,suggesting that estradiol counteracts progesterone effects[49].In our model,progesterone is the dominant steroid hormone at the time of sample collection;however its impact on gene expression is likely influenced by the previous exposure to estradiol.

基于第二条基本原则，对所要教学的内容确定度量和度量单位的形式，如前所述，形式主要分为两类：一类是通过抽象得到的，是人思维的结果；一类是借助工具得到的，是人实践的结果．

Fig.5　Comparison of gene expression between suckled anestrous beef cows receiving 1 mg of estradiol cipionate(ECP)and/or 400 IU of equine chorionic gonadotropin(eCG)at onset of the proestrus[CON(n=11),ECP(n=11),eCG(n=12)and ECP+ECG(n=11)].The amounts of EGFR,HBEGF,ITGB3,and COL4A1 transcripts are expressed in relation to control(CON)untreated cows.Expression values were normalized by the geometric mean of GAPDH,ACTB,and RPS18.The P values refer to comparisons made for each gene between groups(effects of ECP,eCG and interaction between ECP and eCG)

We have observed previously that the endometrial tissue of cows ovulating larger follicles expressed markers of proliferative activity earlier than cows ovulating smaller follicles[8].Similarly,gene expression changes suggesting reduction of proliferative activity and transition to a biosynthetic phenotype were also hastened in cows with larger ovulatory follicles.Larger follicles led to increased estradiol concentrations during proestrus and greater progesterone concentrations during early diestrus[8,14].Functional enrichment analysis using DAVID identified gene ontology terms associated with regulation of cell proliferation such as pathways in cancer and small cell lung cancer.Similarly,endometrial gene expression at D 7 in one estrous cycle prior to embryo transfer revealed enrichment of GO-terms cell cycle and antiapoptosis　in　cows　that　successfully　established pregnancy[50].It is noteworthy that the above mentioned studies obtained samples from non-lactating cyclic cows[8]or heifers[50],whereas in the present study all cows were lactating and in anestrus.Additionally,assessment of the expression of proliferationrelated　candidategenesshowed　thatPTCH2and COL4A1 were induced by ECP treatment,whereas EGFR expression was suppressed.PTCH2 is a membrane receptor,member of the Hedgehog signaling pathway[51],and has been associated with proliferation-related disorders such as endometriosis and ovarian carcinoma[52],playing a role as a tumor suppressor gene[53].COL4A1 encodes a type IV collagen protein that is an integral component of basement membranes[54].In the endometrium,the breakdown of the basement membrane as well as increased expression of COL4A1 have been related with inhibition of angiogenesis and reduced tumor growth[55].In addition,the oncogene EGFR,which is associated with growth of placental tissue[56],was downregulated by ECP-treatment suggesting a suppression of the endometrial ability to respond to mitogenic stimuli.The collective interpretation of these data is that estrogenic stimulus given by ECP induced a nonproliferative status on D6 endometrium.Such findings are consistent with our previous report,in which ovulation of a larger follicle(associated with greater proestrus and estrus plasma concentrations of estradiol)inhibited proliferation in both luminal and glandular epithelial cells on D 7 endometrium　[8].Importantly,such regulation occurred despite similar plasma concentrations of P4 between animals that received an did not receive ECP.

The most remarkable eCG-induced changes in gene expression were associated to E2 signaling.Indeed,transcript abundance was greater for ESR2 and lesser for ESR1 in eCG-treated cows than No eCG-treated cows,suggesting the establishment of a transition phase,from proliferative to secretory.The recognized proliferative role of estrogens in the female reproductive tract appears to be mediated by ESR1[57].After estrus,ESR1 abundance decreases and reaches nadir endometrium concentrations during the mid-luteal phase of the estrous cycle[58].In contrast,uterine ESR2 expression is positively associated with the increasing P4 concentration observed from early to mid diestrus.The greater abundance of ESR2 expression found in eCG treated cows could be justified by the positive effect of eCG on P4 concentration during early diestrus.Altogether,these results suggest that the endometrium of suckled anestrous cows at Day 6 receiving either ECP or eCG is still transitioning from a proliferative to a secretory state,as previously reported[8].

Conclusions

Supplementation with ECP or eCG at onset of the synchronized proestrus of suckled anestrous beef cows significantly influence the ovarian responses;however,the impact on global uterine gene expression is discrete,presenting few DEG that are associated with ceasing cell proliferation.Such phenotype is consistent with the beginning of the secretory phase of the endometrium,required to support conceptus growth and survival.

Additional files

Additional file 1:Table S1.Number of reads from all samples from suckled cows receiving(ECP)or not(CON)1 mg of ECP at the onset of the proestrous(DOCX 14 kb)

Additional file 2:Table S2.Differential gene expression results.BaseMean is the average of all samples expression profile after normalization;lfcSE-standard error from log2FoldChange;padj-P-value adjusted after correction of BH-FDR for multiple tests(DOCX 26 kb)

Additional file 3:Table S3.Bio-samples and Experiment accession numbers of the Raw reads resulted from the RNAseq of endometrial biopsis in the SRA data base(DOCX 20 kb)

Abbreviations

清华大学美术学院副教授蒋红斌“以设计思维为核心的企业创新组织”，从企业创新角度阐述了冰箱行业产品结构升级的必要性。他认为，设计思维来自于生活之美，而非生硬的技术，技术应该与生活对话，在使用中及时发现问题，以用户应用体验驱动行业创新，企业才能达到最佳的创新状态。

Acknowledgements

We would like to thank the Empyreo Farm(Jacarezinho-PR)for allowing the use of their animals and facilities during the trial.These experiments were supported by Firmasa-Pecuária com Tecnologia.This research was funded,in part,by São Paulo Research Foundation(FAPESP;2012/14731-4).We are thankful to Professor Marcos Roberto Chiaratti from Departamento de Genética e Evolução,CCBS,Universidade Federal de São Carlos-UFSCar for the statistical analysis of qPCR data.We also want to thank Bruno Moura Monteiro,Julia G.Soares and Milena L.Oliveira for technical support.

Funding

但是这样说，只是道出了一个方面的道理，并不一定是要人人都走这样的极端。因为从另一方面看，一些激烈参与社会生活、推动社会波澜的人，也有高屋建瓴的气魄，有力挽狂澜的力量。像雨果，常在国会演讲，参与党派斗争，被流放等等，结果也是一个精神和文学的巨人。

Cell proliferation-related gene expression

CAPES PEC-PG 15068-12-9 to AMGD.

CNPq 142,387-2015-0 to MS.

唐玉烟见青辰望着地面眼珠乱转，面上还挂着笑，不知道对方在琢磨些什么。她皱了皱眉，停止了言语。青辰察觉了自己的失态，尴尬了两声，然后道：“你真厉害，一个女孩子，年龄那么小，就能做出这些事，真的很不容易。”

CNPq 481,199/2012-8 and FAPESP-2011/03226-4 to MB.

ACTB:actin,Beta;ADAM9:ADAM metallopeptidase domain 9;ANOVA:Analysis of variance;ATP5F1:ATP Synthase,H+Transporting,Mitochondrial Fo Complex Subunit B1;ATP5J:ATP Synthase,H+Transporting,Mitochondrial Fo Complex Subunit F6;BCS:Body contition score;CEACAM1:Carcinoembryonic antigen-related cell adhesion molecule 1;CL:Corpus luteum;COL4A1:Collagen,type IV,alpha 1;CON:Control;Cq:Quantification cycle;DAVID:Database for Annotation,Visualization,and Integrated Discovery;DEG:Differentially expressed genes;DF:Dominant follicle;E2:Estradiol;eCG:Equine chorionic gonadotropin;ECP:Estradiol cypionate;EDTA:Ethylenediaminetetraacetic acid;EEF1D:Eukaryotic translation elongation factor 1 delta;EGFR:Epidermal growth factor receptor;ESR1:Estrogen receptor alpha;ESR2:Estrogen receptor beta;GABARAP:GABA(A)receptor-associated protein;GAPDH:Glyceraldehyde-3-Phosphate Dehydrogenase;GEMIN7:Gem nuclear organelle associated protein 7;GEO:NCBI’s gene expression omnibus;GnRH:Gonadotropin-releasing hormone;GO:Gene ontology;GPI:Glucose-6-Phosphate Isomerase;HBEGF:Heparin-binding EGF-like growth factor;IDH3B:Isocitrate dehydrogenase 3(NAD(+))Beta;IM:Intramuscular;ITGB3:Integrin,beta 3;LAMC3:Laminin Subunit Gamma 3;LN:Natural logarithm;NDUFB3:NADH:Ubiquinone Oxidoreductase Subunit B3;P4:Progesterone;PEPD:Peptidase D;PGR:Progesterone receptor;PIAS1:Protein Inhibitor Of Activated STAT 1;PIK3R3:Phosphoinositide-3-Kinase regulatory subunit 3;PTCH1:Patched 1;PTCH2:Patched homolog 2;qPCR:Quantitative real time PCR;RIN:RNA integrity number;RNAseq:RNA sequencing;RPS18:Ribosomal Protein S18;RPS2:Ribosomal protein S2;SEM:Standard error of the mean;SG100G:Calcium binding protein G;SNRPD2:Small nuclear ribonucleoprotein D2 polypeptide;SRA:Sequence read archive;STRAP:Serine/Threonine Kinase Receptor Associated Protein;TAI:Timed artificial insemination

The funding bodies had no participation on the study,collection,analysis,interpretation of data or in writing the manuscript.

Availability of data and materials

Sequences of all reads generated in the RNAseq were deposited in the Sequence Read Archive(SRA)of the NCBI(http://www.ncbi.nlm.nih.gov/sra/;Additional file 3:Table S3)and,an overview of these data has been deposited in NCBI’s Gene Expression Omnibus(GEO)and is accessible through GEO Series accession number GSE67807.

Authors’contributions

MFSF contributed to experimental design,animal management,samples preparation for RNAseq and qPCR analysis,statistical analysis of transcripts and was a major contributor in writing the manuscript.RSR,and MFM contributed to animal management.AMGD,GP,and MS performed samples preparation for RNAseq and qPCR analysis,performed qPCR analysis,and contributed in writing the manuscript.SSCA,GG,and LLC performed the RNAseq and the bioinformatics analysis.MDG,FSM,and PSB contributed to experiment design and writing the manuscript.MB was the PI and contributed to experiment design and writing the manuscript.All authors read and approved the final manuscript.

Ethics approval

Animal procedures were approved by the Ethics and Animal Handling Committee of the Faculdade de Medicina Veterinária e Zootecnia,Universidade de São Paulo(CEUA-FMVZ/USP,No.2287/2011).

Consent for publication

Not Applicable.

党史研究属于史学研究的范畴，它是以真实的叙述和准确的分析为基础的。因此，它常常成为那些不把创新视为理所当然的研究人员的借口。党史研究中创新意识和创新思维的缺失，是阻碍学术顺利发展和理论素养有效提高的绊脚石。创新不是改变人类的意志的客观历史事实，也没有改变正确的角度，分析、判断和结论，而是在研究角度的很多方面，如研究内容、研究方法探索新领域，形成一个新的主题，符合社会发展规律，用新的、正确的认识，满足群众的需求，用新的可靠史料填空或更正旧的不准确史料，以适应社会的需要，促进学术的发展和进步。

Competing interests

The authors declare that they have no competing interests.

Author details

1Departamento de Reprodução Animal,FMVZ-USP,São Paulo,SP,Brazil.

2Departamento de Genética e Biologia Evolutiva,IB-USP-,São Paulo,SP,Brazil.3Laboratório de Biotecnologia Animal,ESALQ-USP,Av Pádua Dias,Piracicaba,SP 11,Brazil.4Universidade Federal do Pampa,Uruguaiana,RS,Brazil.5Universidade de São Paulo,Faculdade de Medicina Veterinária e Zootecnia,Departamento de Reprodução Animal,Avenida Duque de Caxias Norte,225,Pirassununga,SP Zip Code 13635900,Brazil.

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