| It has been estimated that10%-15%of all clinically recognized pregnancies terminate with spontaneous abortions (SA), which occur mainly by genetic, immune, infectious, anatomic, endocrine, environmental and other factors. Cytogenetic studies have revealed that fetal chromosome abnormalities account for about50%～60%of all spontaneous abortions.Most of these abnormalities comprise numerical chromosomal aberrations86%, whereas a minority of the cases shows structural chromosomal abnormalities6%and chromosome mosaicism8%.Chromosomal abnormalities may arise during the mitotic proliferation of gametes, meiosis of gametes, or the initial several times mitotic of zygotes, and are a major cause of spontaneous abortion or birth defects.All cells in the human body are derived from the two haploid gametes and subsequent cleaving diploid blastomeres. While chromosomal abnormalities and genetic lesions that arise in the gametes are inherited by all daughter cell,chromosomal abnormalities or genetic lesions acquired in one or a few blastomeres during cleavage are inherited in a mosaic pattern with a portion of cells being of normal euploid whereas others may be chromosomally abnormal.The earlier in cleavage a genetic lesion occurs, the greater the number of daughter cell that will inherit the genetic defect. Therefore, maintenance of genomic integrity during gametogenesis,fertilization and cleavage is essential for normal human embryogenesis.A single fertilized eggs develop into an individual,which composed by variety of cells,tissues and organs, through the cell proliferation cycle. The cell cycle divided into G1,S,G2and M,which include prophase, metaphase,anaphase,telophase. Its series of continuous dynamic events were carry out orderly supervising by strict check points in accurate time and space. Chromosome movement through a specialized structure called kinetochore in this process, which locate the centromere. No matter in mitosis or meiosis, the error of chromosome separation leads to offspring cell chromosome number abnormalities. Errors in chromosome segregation at M-phase may be prevented in both mitosis and meiosis by the spindle assembly checkpoint (SAC), an elaborate molecular pathway that monitors the attachment of microtubules to kinetochores. SAC ensure faithfully distributing chromosomes into two daughter cells and continuing cell cycle normally by delays the onset of anaphase until sister chromatids are bivalently attached by microtubules and aligned at the metaphase plate.MAD2and BUB1are the important components of SAC, known as checkpoint proteins.MAD2binded MADl and was recruited to kinetochore, where it changes its conformation to Closed-MAD2to be released into the cytosol for the formation of active MCC(C-MAD2,BUBR1,BUB3and CDC20).MCC interacts with APC/C to render its ubiquitin-ligase activity, securin (human for hPTTG) and cyclin B can’t be targeted degradation. Cell can’t enter into anaphase to provide time correct chromosome inexact connection, prevent chromosome mis-separation.BUB1is a protein kinase combination BUB3.Three main regions can be identified in BUB1:a conserved N-terminal region that contains the kinetochore localization domain, non-conserved region that is required for BUB3binding, and a C-terminal region that contains a catalytic serine/threonine kinase domain. Studies have revealed many roles for Bubl in both mitosis and meiosis, including the localization of other SAC proteins to the kinetochore, SAC signaling, metaphase congression and the protection of sister chromatid cohesion through shugoshin.The role of SAC in chromosome segregation during meiosis and mitotic becomes the hotpoint in cell cycle research recent years.It is important to clarify the potential molecule mechanism in embryonic chromosomal abnormalities and its origin and the role in tumorigenesis. However, what the role SAC play in human early embryonic development and embryonic chromosomal abnormalities is rare reported. To further investigate the role of MAD2and BUB1play in embryonic chromosomal numerical abnormalities,13,16,18,21,22,X,Y special probe were apply to detect chromosome numerical aberrations by FISH,mRNA and protein expression level in human chorionic villus of spontaneous abortion embryos by the means RT-PCR and Western-blot respectively.ObjectiveTo further investigate the possible role of MAD2and BUB1in chromosome segregation through detect their mRNA and protein expression in human chorionic villus of spontaneous abortion between chromosomal numerical abnormalities and normal by the means RT-PCR and Western-blot respectively.Materials and Methods1Study ObjectChorionic villi or fetus tissue sample from99spontaneous abortions, which were detected threatened abortion or embryos stopped developing by ultrasonography, were collected at obstetrics and gynecology clinic of the Third Affiliated Hospital of Zhengzhou University from June2010to January2011.Spontaneous miscarriages embryos was divided into chromosomal abnormalities and normal group by FISH, which using13,16,18,21,22,X,Y special probe.2Methods2.1Specimen collecting and preparingSterile operation was performance and pick up chorionic villi, each tissue sample was divided into fragments:one was treated by a special process for FISH test numerical chromosomal analysis and another was frozen quickly by liquid nitrogen and stored at-80℃refrigerator used for RT-PCR and Western-blot test.This study was approval by ethics committee of the third affiliated hospital of zhengzhou university. Informed consent was obtained from all participants.2.2Detection chromosome numerical aberrations in chorionic villus tissue of spontaneous abortion by FISH2.2.1Tissue preparation for FISH procedureDuring sterile operation was performance, pick up fresh chorionic villus5～10mg in sterile tube,clearing blood clots and mucus by physiological saline,cutting to chip, hypotonic and fixed by fixing agent(methanol:glacial acetic acid=3:1),next digesting by glacial acetic acid for1minute and methanol stop it,discard clear supernatant liquid and drop it to clean slides.2.2.2Detection chromosome number of embryonic villus by FISHFresh slide spreads were digesting by pepsin,then they were denatured for5minute in70%formamide/2×saline sodium citrate(SSC)at74.5℃and dehydrated in a graded-20℃pre-cooled ethanol series. The10μl probes mix was then applied to air-warmed slides and put slides in a moist chamber for hybridization overnight at42℃. Following hybridization, the slides were washed in50%formamide/2×SSC for2minute two times at42℃, rinsed in2×SSC,0.1%NP-40/2xSSC for3min each and dehydrated through70%ethanol. The slides were counterstained in4,6-diamidino-2-phenylindole and analyzed for simultaneous viewing of nuclear fluorescent signals.2.3Detection MAD2, BUB1mRNA expression in embryonic chorionic villus of spontaneous abortion by RT-PCRMAD2, BUB1primers was synthesis by Shenggong biological engineering (Shanghai)Co.,LTD.Total RNA of embryonic chorionic villus or foetus tissue was extracted and the RNA reverses transcription to cDNA. The cDNA amplification by RT-PCR. The products were analyzing use gel imager scan after15g/L agarose gel electrophoresis (ethidium bromide staining).The ratio of aim gene to β-actin gene as the MAD2,BUB1mRNA relative expression.2.4Detection MAD2, BUB1protein expression in embryonic chorionic villi of spontaneous abortion by Western-blot Total protein of embryonic chorionic villus was extracted by RIPA. Concentration of total protein was dected by BCA means. MAD2,BUB1protein relative expression in embryonic chorionic villi of spontaneous abortion was detecting by Western-blot. The ratio of aim protein to P-actin protein as the MAD2,BUB1relative expression.3Statistical treatmentSPSS17.0was used for statistics.Gestational weeks between two groups compare by corrected Chi-square test. Compare maternal age, gravidity and spontaneous abortion times between two groups by Mann-Whitney U test. Gestational age and the rate of embryonic chromosomal abnormalities were comparing by corrected Chi-square test, while Kruskal Wallis test was applied for testing differences between the times of spontaneous abortion and embryonic chromosomal abnormality. All data were calculated by x±s and the two-sample t-test was applied for testing differences of expression MAD2、BUB1and correlation between the study groups. All tests applied were two-tailed, and a P-value of5%or less was considered statistically significant.Results1General information of spontaneous abortions99spontaneous abortions embryos were divided into chromosomal abnormalities (49cases) and normal group (50cases) by FISH, which using13,16,18,21,22,X,Y special probe. Maternal age range was21～41years old, gestational age ranged from6to16gestation weeks, gravidity1-9times, spontaneous abortion times1-4.There were no statistical difference between chromosomal abnormalities and normal group in gestation weeks, maternal age, gravidity and spontaneous abortion times (t=0.101,P=0.92;Z=1.881,P=0.06;Z=1.852,P=0.06;Z=0.226,P=0.82).2MAD2, BUB1mRNA relative expression in chorionic villus of spontaneous abortion MAD2mRNA relative expression in chorionic villus of embryos with chromosomal abnormality was0.89±0.19and significantly higher than that0.79±0.21in embryos with normal chromosome(t=-2.317,P=0.023). There was no significantly difference (t=-1.333,P=0.186) BUB1mRNA relative expression between Chorionic villus of embryos with chromosomal abnormality(1.04±0.23) than that with normal chromosome(0.97±0.25).3MAD2,BUB1relative expression in chorionic villus of spontaneous abortionMAD2protein relative expression in chorionic villus of embryos with chromosomal abnormality was0.46±0.06and significantly higher than that0.42±0.08in embryos with normal chromosome(t=-2.373,P=0.020).There was no significantly difference(t=1.510,P=0.134)BUB1relative expression between chorionic villus of embryos with chromosomal abnormality0.35±0.05than that with normal chromosome0.33±0.07.ConclusionsOverexpression of MAD2in embryos with chromosomal abnormality may prolong duration of mitosis by delaying satisfaction of the spindle assembly checkpoint. MAD2overexpression may play an important role in ensure faithfully distributing chromosomes into two daughter cells and maintenance of genomic integrity.BUB1,which is upstream genes of SAC, can’t play a centre role in prolong duration of mitosis, however its normal expression is vital for SAC function. |
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