The Study On The Apoptosis Of Yak Placentomes From Pregnancy, Pre And Post-Parturation And Dex Induced Abortion Cases

Objective: It would offer a new insight for us to understanding the physiological function andmechanism of abortion by discussing the relationship between apoptosis and the interruptionof pregnency during ealier gestation and investigating the morphological changes ofplacentome and apoptosis at cellula and molecular level. These works would play animportant role in yaks reproduction and disease prevention. On the orther hand, it also providesome usefull data concern to the reproduction mechanism of human being and other animals.Methords:(1)47yaks of4～8years old during pregnancy, pre and post parturation, afterdexamethasone(DEX) intramuscular injection were used to collect the placentomes. Aferfixed with4%(W/V)paraformaldehyde, paraffins embedding tissue sections and HE dyeingtechnology was used to investigate the basic structure and the apoptotic morphologicalcharacteristics of placentome cells. Stereoscopy methord was used to analyse the changes ofsome stereological parameters of yak placentomes in each stage of gestation. In situfluorescence labeling(DAPI) and DNA terminal labaling(TUNEL) techniques were also usedto confirm the apoptotic cells in yak placentomes. The expression of Fas,FasL,Bcl-2and Baxproteins was detected by immunohistochemistry methord.(2) Placental villi were collected from5months pregnant yak plcentomes. After enzymicdigested, villous trophoblast cells were separated and cultured in vitro. The influence ofdifferent concentrated DEX on apoptosis of trophoblast cells was inspected using in situfluorescence labeling(DAPI) techniques. The expression of Fas,FasL,Bcl-2and Bax proteinswas detected by FITC labeled fluorescence scecond antibody using immunocytochemistrytechniques.Results:(1) yaks placentomes experienced different physiological process as generation,development and maturation during pregnancy. Different morphological characteristics wereobserved during different stage of gestation. About in45days of gestation, yaks placentomes demonstrated typical epitheliochorial placental structure. Fetal villous epithelium was consistof columnar trophoblast cells and gaint trophoblast cells. Meternal crypt was covered with alayer of intact cubic epithelium. With the progress of gestation, fetal villi branched and deeplypenetrated into the meternal caruncula crypts. The volume density of binucleate cells wasincreased in placentomes. In6months of gestation, obviously degenerated changes such ascell vacuolization can be observed in caruncula crypt epithelium. At pre-parturition, most ofthe crypt epithelium were disappear. obviously degenerated changes was also found in yakplacentomes of DEX induced abortion case.(2) Apoptotic cells were found in yak placentomes. These cells displayed obvious changesafter HE, DAPI and TUNEL dyeing. Though the number was little, the apoptotic cellsdistributed in the placentomes by a spatiotemporal specific manner. The positive cells ofTUNEL reaction mainly located in the fetal villious trophoblast, meternal caruncula cryptepithelium during whole gestation and in the caruncula stroma during early stage of gestation.With the progress of gestation, the number of positive cells was increased in these two layersof epithelium, especially in the fetal villious epithelium of pre and post-parturation and DEXinduced abortion case.(3) the expression of Fas protein was strong in the fetal villi during early and middle stage ofgestation. The positive index of Fas reached the maximum in the third month of gestation.Then Fas protein was decreased in fetal villi. In caruncula stroma, brown stained and singlelydistributed Fas positive cells were clearly observed in most time of gestation. The expressionof FasL protein was more stable in fetal villi, most positive reation was presented in somemononuclear, binuclear and multinuclear trophoblast cells. In caruncula crypts, FasLexpression was more weak and the positive index was declined with the progress of gestation.Bcl-2protein was expressed mainly in villious and caruncula crypt epithelium,the quantity ofBcl-2protein was declined with the progress of gestation.but the positive index in fetal villi ofpost-parturation was higher than pre-parturation. Bax protein was expressed mainly in fetalvillious epithelium and the quantity was increased during whole gestation. In DEX inducedabortion cases, both Bcl-2and Bax proteins were all strong expressed, especailly Bax protein.(4)10-9mol/L DEX significantly promoted the apoptosis of in vitro cultured trophoblast cells.The effection of DEX on expression of Fas and FasL was unconspicuous at the concentration of10～10mol/L. but Bcl-2and Bax protein expression was obviously increased after DEXtreatment,especially Bax protein.Conclusion:(1) Yaks placentomes experienced different physiological process as generation,development and maturation during pregnancy. The changes of morphological characteristicswere taken placed to accommodate the physiological requirements of cyotrophy supplementand placental maturation during pre-parturation.(2) A small number of apoptotic cells maintained the normal replacement and dynamicbalance of placental tissue. The process of placental maturation during pre-parturation wasaccompanied with the obviously increase of apoptotic cells. In DEX induced abortion cases,the number of apoptotic cells was increased. The status of apoptosis related proteinsexpression indicated that Fas and FasL probably involved in the process of fetal villi growingand the mechanism of immune privilege during pregnancy. The apoptosis of the trophblastcells was related to the Bcl-2and Bax protein. The strong expression of Bcl-2and Bax proteinin early stage of gestation perhaps reflected a higher cell replacement with the placentalgeneration. The increase of the number of apoptotic cells in DEX induced abortion cases wasresulted by the change of Bcl-2and Bax expression.(3) in vitro experiment indicate that DEX can induced the apoptosis of cultured yak placentaltrophoblast cells and the mechanism of the apoptosis probably related to the expression ofBcl-2and Bax protein.