Background

Obesity in females of reproductive age not only increases their risks of developing insulin resistance,diabetes,and other diseases,but is also linked to poor reproductive outcome[1-3].Oocyte quality plays a key role in connecting maternal nutrition with reproductive outcome,and is dramatically impacted by maternal obesity[4,5].A previous report demonstrated that obese women have a lower success rate in both natural and assisted reproduction.Moreover,the higher pregnancy failure rate in obese women returns to normal if they use donor oocytes to replace autologous oocytes[6].Although the molecular mechanisms by which oocytes are impaired by maternal obesity are not completely understood,dysfunctions of the endoplasmic reticulum(ER)or mitochondria have been strongly implicated[7-9].

The ER is the key calcium(Ca2+)storage and release system[10].Obesity changes the ER membrane lipid composition and damages its ability to retain Ca2+,which alters the cytoplasmic homeostasis[11].Mitochondria are the primary effectors of Ca2+absorption and intrinsic apoptosis[12].Prolonged Ca2+release from the ER leads to altered mitochondrial membrane potential and the induction of intrinsic apoptotic pathways[13,14].Mitochondrial defects are thought to be the direct reason for the oocyte developmental deficits in obese females[15].

The ER and mitochondria interact both physiologically and functionally via mitochondria-associated ER membranes(MAM).MAM is involved in a number of physiological processes including Ca2+transfer and cellular apoptosis[16-18].MAM contains several proteins such as inositol 1,4,5-trisphosphate receptor,type 1(IP3R1;a major Ca2+release channel in the ER),inositol 1,4,5-trisphosphate receptor,type 2(IP3R2),mitofusin-2(Mfn2),phosphofurin acidic cluster sorting protein 2(PACS-2;controls the apposition of mitochondria with the ER),sigma-1 receptor(Sig-1R),and calnexin.A recent study demonstrated there to be significantly more MAMs in the livers of obese mice compared with control mice[19].

To date,the association between MAM and altered oocyte quality in obese mice has not been investigated.The current study investigated whether the content of MAM and MAM-related proteins were different in oocytes from obese and control mice.

Methods

All chemicals were purchased from Sigma Chemical Co(St Louis,MO,USA)unless otherwise stated.

虽然现阶段我国面对老年化和无障碍建筑设施方面已经取得了一定的成就，也出台了相关的养老设施标准和养老规范，但是个别条文不适用于全部的养老机构，具有一定的局限性，同时养老机构的管理运营也不完善，尤其针对老年人的软性需求无法满足，因此老年人在使用建筑设施过程中无法理解设计人员的目的。

Obese mice and oocyte harvesting

Female CD-1 mice(3-week-old)were purchased from the Beijing Vital River Experimental Animals Centre(Beijing,China)and housed under 12 h:12 h light:dark cycles at a temperature of 23± 2°C for all experiments.The mice were randomly divided into two groups(five per cage):one group was fed a control diet(CD)and the other was fed a high fat diet(HFD)for 12 week(the formulae of the two diets are shown in Additional file 1:Table S1);water was provided ad libitum.After obesity had been established(Additional file 2:Fig.S1),mice from the two groups were fasted overnight and weighed.Before all experiments,the mice were treated with 8 IU of equine chorionic gonadotropin(eCG)for 46-48 h and then sacrificed by cervical dislocation.Germinal vesicle(GV)oocytes were collected for subsequent experiments.All procedures were performed under the Institutional Animal Care and Use Committee of China Agricultural University.

The MAM-related protein IP3R1 is a major ER Ca2+regulatory protein in oocytes,whereas PACS-2 plays a key role in regulating ER homeostasis and the physical interaction between the ER and mitochondria.Therefore,increased levels of the MAM-related proteins IP3R1 or PACS-2 in oocytes from obese mice might lead to increased ER Ca2+release and[Ca2+]mintake[24].Ca2+is a major regulator of mitochondrial physiology[25].Abnormal increases in[Ca2+]m,even a tiny change,lead to subsequent mitochondrial dysfunction and even apoptosis[9,26].Indeed,the current results showed that[Ca2+]mwas increased in oocytes from obese mice.The intrinsic apoptotic pathways related to oocytes were activated,and in particular the levels of the two important proteins cytochrome C and activated caspase-3 were increased.Regarding cytoplasmic maturation,oocytes from obese mice exhibited a significant reduction in GSH and P-MAPK 3/1 levels(the latter is related to microtubule organization)and a significant increase in ROS.All three of these features indicated compromised cytoplasmic maturation.

Not applicable.

Quantitative real-time PCR(qRT-PCR)and semi-quantitative reverse transcription PCR(RT-PCR)

Total RNA was extracted from collected oocytes using an RNeasy micro-RNA isolation kit(Qiagen,Valencia,CA,U.S.)following the manufacturer’s instructions.The RNA concentrations were measured using a Nanodrop ND-1000 Spectrophotometer(Biolab,Scoresby,Victoria,Australia).Reverse transcription was conducted to generate cDNA libraries using a QuantiTect Reverse Transcription Kit(Qiagen)according to the manufacturer’s instructions.QRT-PCR and RT-PCR were performed using an ABI 7500 real-time PCR instrument or a Veriti 96-well Thermal Cycler(Applied Biosystems,Foster City,CA,U.S.).The sequences of all primers used are listed in Additional file 3:Table S2.The results were analyzed using the 2-ΔΔCtmethod.

2013年，湖北省水利厅继续深入贯彻中央和省关于加快水利改革发展的决策部署，强化工作举措，加快水利发展，各项工作保持又好又快的发展态势。

Western blotting

综上可知，公路工程具有极强的复杂性，多方面的影响因素使得质量安全问题频繁发生。要求施工单位可以重视这一质量安全问题，并认清当前质量安全监督与管理中存在的阻碍，从而采取有效对策，改进其质量安全监督与管理工作，让公路工程建设项目的质量与安全都可以获得良好的保障，推进公路工程建设的健康发展，实现维护人们出行安全、维护社会安定的目的。

Oocytes were washed in PBS three times,collected,and homogenized in sodium dodecyl sulfate buffer.They were then boiled for 5 min and immediately cooled on ice.The proteins were separated using SDS-PAGE with a 5%stacking gel and a 6%(for proteins greater than 90 kDa)or 10%(for proteins less than 90 kDa)separating gel and transferred to nitrocellulose membranes.After blocking with Tris-buffered saline with Tween-20(TBST)containing 5%BSA,the membranes were incubated with the following primary antibodies:anti-IP3R2,anti-IP3R1(1:1,000;Thermo Fisher Scientific,Rockford,IL,U.S.),anti-PACS2(1:500;EMD Millipore,Temecula,CA,USA),anti-actin(1:200;Santa Cruz Biotechnology,Santa Cruz,CA,U.S.),anti-p-MAPK3/1,anti-MAPK3/1(1:2,000),anti-cytochrome C(1:5,000),anti-caspase 3(1:1,000;Cell Signaling Technology,Beverly,MA,U.S.),and anti-GAPDH(1:500;CW Biotech,Beijing,China)in TBST overnight at 4°C.The membranes were incubated with horseradish peroxidase(HRP)-conjugated secondary antibodies(1:5000,CW Biotech)for 1 h at 25°C.Subsequently,the blots were detected using a chemiluminescence kit(Thermo Fisher Scientific).All results were quantified using ImageJ software(National Institutes of Health,Bethesda,MD,U.S.).

Measuring mitochondrial Ca2+([Ca2+]m)

[Ca2+]mlevels were assessed using Rhod-2AM(Invitrogen/Molecular Probes,Carlsbad,CA,U.S.)according to a previous study[20].Briefly,zona pellucid was enzymatically removed.The oocytes were then processed in maturation medium with 3 μmol/L Rhod-2AM for 30 min,rinsed three times,and incubated without Rhod-2AM at 37°C and 5%CO2for 30 min.Subsequently,they were analyzed using a confocal laser scanning microscope(Nikon A1R,Tokyo,Japan)and quantitatively processed using NIS-Elements AR(Nikon Instruments,Tokyo,Japan).

Measuring intracellular ROS and GSH levels

Intracellular ROS and GSH levels were measured as described previously[21].Oocytes were incubated in M2supplemented with 1 mmol/L 2′,7′-dichlorodihydrofluorescein diacetate(H2DCFDA)for measuring ROS　or10　　μmol/L　4-chloromethyl-6,8-difluoro-7-hydroxycoumarin(CellTracker Blue)for measuring GSH for 30 min at 37°C and washed three times.The fluorescence was examined under an epifluorescence microscope with a filter at 460-nm excitation for ROS and 370-nm excitation for GSH(DP72,Olympus,Tokyo,Japan).All data were analyzed using ImageJ software.

siRNA microinjection

siRNAs were microinjected into oocytes as described previously[20].To knockdown Itpr1 or Pacs-2,10 μmol/L of each siRNA smart pool(a mixture of four siRNAs)or NC siRNA(GE Healthcare Life Sciences,South Logan,UT,U.S.)was used and the microinjection was completed within 30 min.All oocytes were arrested at the GV stage and cultured in maturation medium supplemented with 4 mol/L hypoxanthine(HX)to deplete the endogenous targeted mRNAs and proteins.The sequences of Itpr1 siRNAs were as follows:siRNA-1:5′-GAAAUGG AGUGAUAACAAA-3′;siRNA-2:5′-GGACGAGGCU GGAAAUGAA-3′;siRNA-3:5′-GUACAUGAGUUCUU CUAUA-3′;siRNA-4:5′-GAGAUGAACUGGCAGAA GA-3′.The sequences of Pacs-2 siRNAs were as follows:siRNA-1:5′-GCUCAGGCCUUACUUCGAA-3′;siRNA-2:5′-CCAACAGCCUAGACAAUGA-3′;siRNA-3:5′-GG ACGAUCCUGGGCUACAA-3′;siRNA-4:5′-UCUCUCG GAUACAGCGAUA-3′.The sequences of NC siRNAs were as follows:siRNA-1:5′-UAGCGACUAAACACAU CAA-3′;siRNA-2:5′-UAAGGCUAUGAAGAGAUAC-3′;siRNA-3:5′-AUGUAUUGGCCUGUAUUAG-3′;siRNA-4:5′-AUGAACGUGAAUUGCUCAA-3′.

Statistical analyses

Fig.1　HFD induced obesity leads to increased content of MAM in mouse oocytes.a Characteristic TEM images of oocytes from control or obese mice.Magnification,30,000×;scale bars,500 nm.The lower panels showed a higher magnification of the selected areas in the top panels.ER,endoplasmic reticulum;M,mitochondria;N,nucleus.b,c The length of the ER tethered to the mitochondria was normalized to the ER length(b)or mitochondrial circumference(c).N=30(five sections per mice,six mice per group)for each group across three replicates.d The subcellular structures of a larger area of oocytes from both groups are depicted.Magnification,15,000×;scale bars,1 μm.Graphs show means± SEM.*P＜0.05,**P＜0.01,CD vs.HFD;t-test

Each experiment was repeated at least three times.A representative image of each experiment is shown.All data are presented as means±SEM and were analyzed using t-tests with SigmaPlot(Systat Software,San Jose,CA,U.S.U.S.).Differences were deemed statistically significant as follows:*P＜0.05,**P＜0.01.

Results

HFD-induced obesity increases the amount of MAM in mouse oocytes

The ER and mitochondria were in closer proximity to each other in oocytes from obese mice than in those from control mice(Fig.1a).The proportion of the physical apposition relative to the total ER length(Fig.1b)or mitochondrial circumference(Fig.1c)was significantly greater in oocytes from obese mice.For a better view of the alterations to the MAMs in oocytes from obese mice,a simple corresponding subcellular structural diagram of the ER,mitochondria,and lipid droplets was generated(Fig.1d)within a larger field of vision compared with that shown in Fig.1a.

Oocytes from obese mice express more MAM-related genes and proteins

To assess any changes in MAM-related proteins in oocytes from obese mice,the mRNA and protein levels of these proteins in oocytes were measured.Notably,oocytes from obese mice exhibited significantly higher IP3R1,IP3R2,and PACS-2 expression at both the mRNA(Itpr1,Itpr2,and Pacs-2)(Fig.2a)and protein levels(Fig.2b).

在现代企业经营模式不断优化和完善的过程当中，吸收合并是一种重要的合并方法。但是相关的工作人员在进行会计处理时，必须要严格遵守相应的原则和制度，选择最科学合理的会计处理方法，减小企业经营风险，促进其发展。

Oocytes from obese mice exhibit higher[Ca2+]mlevels and increased apoptosis

Knocking down PACS-2 alleviates the metabolic decline in oocytes from obese mice

Fig.2　Oocytes from obese mice exhibit altered MAM-related gene and protein expression.a QRT-PCR showing Itpr1,Itpr2,Pacs-2,Mfn2,Sigmar1,and Canx mRNA levels.N=240 for each group across three replicates.b Western blotting showing IP3R1,IP3R2,and PACS-2 protein levels.The lower histograms show quantification of the upper panels.N=450 for each group across three replicates.Graphs show means±SEM.*P＜0.05,**P＜0.01,CD vs.HFD;t-test

Fig.3　Oocytes from obese mice exhibit higher[Ca2+]mand increased apoptosis.a[Ca2+]mlevels in oocytes from control and obese mice.Scale bars,50 μm.b Quantitative analysis of[Ca2+]m.N=30 per group across three replicates.c Western blotting for activated caspase-3,cytochrome C,P-MAPK-3/1,and MAPK-3/1.d Statistical analysis of activated caspase-3 and cytochrome C levels.e Statistical analysis of P-MAPK-3/1 and MAPK-3/1 levels.N=360 per group across three replicates.f ROS(green)and GSH(blue)levels in control and obese mice.Scale bars,100 μm.g Quantitative analysis of ROS and GSH levels.N=30 per group across three replicates.Graphs show means±SEM.*P＜0.05,**P＜0.01,CD vs.HFD;t-test

Mitochondrial physiology is very sensitive to alterations in[Ca2+]m,and abnormal increases in Ca2+could activate　the　signaling　pathwaysrelated　to　oocyte apoptosis.Indeed,the expression of cytochrome C and activated caspase-3 was greatly increased in oocytes from obese mice(Fig.3c,d).In addition,the phosphorylation of extracellular-regulated protein kinase 3/1(P-MAPK 3/1)(Fig.3c,e)and GSH levels(Fig.3f,g)were significantly decreased and ROS levels were significantly increased(Fig.3f,g)in oocytes from obese mice.

Funding

siRNA treatment decreased both the mRNA(Fig.4a,b)and protein(Fig.4c,d)expression of IP3R1.The level of MAM was not influenced by IP3R1 knockdown(Fig.4e,f),but the[Ca2+]mwas reduced(Fig.4g,h),the levels of cytochrome C was reduced and the levels of P-MAPK 3/1 increased(Fig.4i,j).This was accompanied by decreased ROS levels and increased GSH levels(Fig.4k,l).

[Ca2+]mlevels were increased in oocytes from obese mice at baseline(Fig.3a).Quantitative analysis revealed that the mean[Ca2+]mintensity was significantly higher in oocytes from obese mice compared with those from control mice(Fig.3b).

Pacs-2 siRNA successfully reduced PACS-2 expression at both the mRNA(Fig.5a,b)and protein levels(Fig.5c,d).The level of MAM was significantly decreased by PACS-2 knockdown(Fig.4e,f).Knocking down PACS-2 led to significantly reduced[Ca2+]m(Fig.5e,f)and decreased cytochrome C and increased P-MAPK 3/1 expression(Fig.4i and Fig.5g).This was accompanied by significantly decreased ROS and increased GSH levels(Fig.5h,i).

Discussion

Previous studies reported that obesity leads to functional defects in oocytes from both mice and humans[21].Obesity-induced cellular dysfunction is related to ER stress and mitochondrial dysfunction[22].However,recent studies demonstrated that the ER and mitochondria interact directly via MAM[16].Thus,MAM may contribute to these oocyte defects.

Fig.4　Knocking down IP3R1 prevents the metabolic decline in oocytes from obese mice.a The mRNA levels of Itpr1 after knocking down IP3R1.b The relative mRNA levels of Itpr1.N=90 per group across three replicates.c Representative immunoblots showing IP3R1 protein levels after IP3R1 knockdown.d The relative protein levels of IP3R1.N=300 per group across three replicates.e Characteristic TEM images of oocytes from NC,siIP3R1 and siPACS-2 treatment.Magnification,60,000×;scale bars,500 nm.f The length of the ER tethered to the mitochondria was normalized to the ER length.N=30(five sections per mice,six mice per group)for each group across three replicates.g[Ca2+]mlevels after IP3R1 knockdown;scale bars,50 μm.h Quantitative analysis of[Ca2+]m.N=30 per group across three replicates.i Representative immunoblots of increased P-MAPK 3/1 and decreased cytochrome C levels after IP3R1 or PACS-2 knockdown(left column,NC siRNAs;middle column,Itpr1 pooled siRNAs;right column,Pacs-2 pooled siRNAs).j Quantification of the immunoblots.N=300 per group across three replicates.k ROS and GSH levels after IP3R1 knockdown;scale bars,100 μm.l Statistical analysis of ROS and GSH levels after IP3R1 knockdown.N=30 per group across three replicates.Graphs show means±SEM.*P＜0.05,**P＜0.01,CD vs.HFD;t-test

The current study revealed that MAM were enriched in oocytes from obese mice.This is consistent with the results of a previous study revealing that MAM are enriched in the hepatocytes of obese mice[19].Ideally,MAM should be studied using enriched fractions of MAM from oocytes.However,a previous study that used～7×107cells or 350-400 g liver tissues to isolate enriched fractions of MAM revealed that we would need at least 140,000 oocytes(one oocyte is equivalent to 500 granule cells)to obtain a sufficient amount of enriched fractions for study,which is the equivalent of～4000 mice per group;this is not possible[23].Therefore,we decided to assess whether the levels of MAM-related proteins were changed in oocytes from obese mice.When we measured the mRNA and protein levels of the proteins related to MAM in oocytes,the data revealed a significant increase in IP3R1,IP3R2,and PACS-2 levels.

以上4个维度中，将各维度每个条目定量化转为得分值以后，再把同一维度下的各个条目得分值加权平均计算各个维度的得分总值。结果显示，分值从高到低分别为：人际沟通能力、工作环境适应能力、中医诊疗能力、设备操作使用能力，见表5。

Transmission electron microscopy(TEM)

Fig.5　Knocking down PACS-2 alleviates the metabolic decline in oocytes from obese mice.a The mRNA levels of Pacs-2 after PACS-2 knockdown.b The relative mRNA levels of Pacs-2.N=90 per group across three replicates.c Western blotting showing PACS-2 levels after PACS-2 knockdown.d The relative protein levels of PACS-2.N=300 per group across three replicates.e[Ca2+]mafter PACS-2 knockdown;scale bars,50 μm.f Quantitative analysis of[Ca2+]m.N=30 per group across three replicates.g Quantitative analysis of the immunoblots shown in Fig.4(i).N=300 per group across three replicates.h ROS and GSH levels after PACS-2 knockdown;scale bars,100 μm.i Quantitative analysis of ROS and GSH levels.N=30 per group across three replicates.Graphs show means±SEM.*P＜0.05,**P＜0.01,CD vs.HFD;t-test

Abbreviations

夫妻性生活没了趣味，杨力生的精神彻底失去了寄托。在买卖忙碌时，杂事全忘到脑后，倒还过得去。当闲来无事时，就上网找人聊天。今天聊，明天聊，聊来聊去，竟然聊出了一场“祸灾”。二○○九年四月底，他和一位二十岁刚出头的姑娘“接上了头”。二人是越聊越热乎，起初只是语音和打字聊，最后干脆视频聊起来。

Conclusions

In conclusion,it is possible that enriched MAM could increases[Ca2+]m,which is associated with increased apoptosis and compromised cytoplasmic maturation of oocytes from obese mice.This finding suggests a novel therapeutic target for obesity-induced oocyte defects.

Additional files

Additional file 1:Table S1.Formulas of D12450B(CD)and D12492(HFD).(DOCX 27 kb)

Additional file 2:Figure S1.Female CD-1 mice exhibit obesity after being fed a HFD for 12 weeks.(a)Representative image of female CD-1 mice fed a CD or HFD diet for 12 weeks.(b)The mean bodyweight of mice in the HFD group was higher than that of mice in the CD group.N=105 for HFD group and N=112 for CD group across three replicates.(c)The mean bodyweight of mice in both groups changed over time.N=105 for HFD group and N=112 for CD group across three replicates.(d)The proportion of abdominal adipose weight in both groups.N=18 for each group across three replicates.Graphs show means±SEM.*P＜0.05,**P＜0.01,t-test.(TIFF 1850 kb)

Additional file 3:Table S2.List of the primers used for qRT-PCR.(DOCX 17 kb)

To assess whether the increased[Ca2+]m,activated apoptosis pathway,and compromised cytoplasmic maturation in oocytes from obese mice were influenced by the MAM-related proteins IP3R1 or PACS-2,RNAi was used to knock them down.Knocking down either IP3R1 or PACS-2 in oocytes reduced the[Ca2+]m,relieved apoptosis,and simultaneously decreased the compromised cytoplasmic maturation in oocytes from obese mice.The results also showed that knocking down PACS-2,but not IP3R1,reduced the MAM content.This may because PACS-2 is an integral MAM protein that regulatesthe　ER-mitochondrialphysicalinteraction,whereas IP3R1 only regulates ER-mitochondrial Ca2+fluxes.Therefore,knocking down IP3R1 may just affect the function of MAM rather than their morphology.Knockdown of PACS-2 did reduce MAM content and improve oocyte quality.

2.A、C组创造性思维水平比较：2组流畅力、开放力、变通力、独创力及标题5个维度差异均有统计学意义(P<0.01)，见表1。

[Ca2+]m:Mitochondrial Ca2+;CD:Control diet;ER:Endoplasmic reticulum;HFD:High fat diet;IP3R1:Inositol 1,4,5-trisphosphate receptor,type 1;IP3R2:Inositol 1,4,5-trisphosphate receptor,type 2;MAM:Mitochondriaassociated ER membranes;Mfn2:Mitofusin-2;PACS-2:Phosphofurin acidic cluster sorting protein 2;P-MAPK 3/1:Phosphorylation of extracellularregulated protein kinases 3/1;Sig-1R:Sigma-1 receptor

Acknowledgements

Oocytes were collected and fixed with a fixative buffer(1%　paraformaldehyde,2.5%　glutaraldehyde　in 0.1 mol/L PBS,pH 7.4)for 3 h at 4°C.They were then washed in 0.1 mol/L PBS,post-fixed with 1%osmium tetroxide(Ted Pella,Redding,CA,U.S.)and 1.5%potassium ferricyanide.Samples were dehydrated with a graded alcohol series,placed in 100%acetone,and embedded in 100%　812 resin.Ultrathin slices(70 nm)generated by an ultramicrotome were stained with 2%uranyl acetate and viewed under a TEM(H-7650,Hitachi,Tokyo,Japan).The main structures in a larger area of oocytes from the CD and HFD groups were depicted using Imaris software(Bitplane,Concord,NH,U.S.).

大数据和云计算方面，随着技术要求的提高，离线的单机模式的存储已经不能满足大数据和云计算的要求，GIS平台也已经由传统的单机版到web版然后发展到云环境版，比如知名的ESRI、SuperMap等公司的各种在线产品。在地质灾害监测中，也发展了基于WebGis、手机App和云计算的平台，将数据存储在网络中。蒋锐、宋焕斌等(2011)[16]提出一种基于SensorWeb的系统架构，联合多个矿山的局部监测网络，将监测信息汇集到管理部门，进行大数据的统一化管理。佘东、朱晓彦等(2013)[17]通过一种省级的云计算平台，提前或及时的预测地质灾害的发生，其本质上是一套WebGis+手机APP系统。

Knocking down IP3R1 prevents the metabolic decline in oocytes from obese mice

This work was supported by the National Basic Research Program of China[grant number 2014CB138500].

Availability of data and materials

SD大鼠，雌雄各半，体质量（200±20）g，实验动物生产许可证号为SCXK（冀）2015‐1003，由河北医科大学实验动物中心提供。

Not applicable.

Authors’contributions

L-HZ designed the research;L-HZ,T-FL,and LG performed the research;L-HZ analyzed the data;L-HZ wrote the paper.All authors read and approved the final manuscript.

Ethics approval and consent to participate

力争农村水利发展再上新台阶。确保年内解决240万农村人口饮水安全问题。大规模开展农田水利基本建设，全年新增有效灌面积98万亩（6.53万hm2）。加快“全域灌溉”和水利现代化灌区示范建设。加快病险水库除险加固，确保年内完成694座小型病险水库除险加固任务。积极推进现代渔业建设，力争全省水产品总产量达到130万t，渔业经济总产值突破325亿元，农民人均渔业收入490元。

对于手刹车，一些车辆，特别是轿车往往性能很好。而在检测台上测出的数据却很小，达不到标准。这是因为这些汽车的轴重很轻，前轮阻尼很小，手刹车时，后轮抱死，后轴被检测台滚子卷上来，整个车身后移，对滚子的力矩变小，检测台测得的数据就小了。我们曾试验将前轮固定，避免车辆后移，结果此类车手刹制动数据全部达标。因此在此类车的测试中应采用固定前轮的方法，或将后轮抱死车身，对后移的汽车数据放宽才是合理的。

The present study was approved by the Institutional Animal Care and Use Committee of China Agricultural University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

1State Key Laboratory of Agrobiotechnology,College of Biological Sciences,China Agricultural University,Yuanmingyuan west Rd 2,Beijing 100193,China.2Key Laboratory of Animal Genetics,Breeding and Reproduction,College of Animal Science and Technology,China Agricultural University,Beijing,China.

引入法。借鉴国外成熟的烹饪技术，有效地对中国菜进行改进，打造出中西合璧的菜品，比如虾仁食料，将其创新出中菜西做或者西菜中做的美味佳肴。

一是因地制宜，布设雨水情监测点。根据各地实际情况，以小流域为单元，在山洪灾害防治区内建立自动监测与简易监测相结合的雨水情监测站网，实时监测暴雨山洪。

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