Effect Of Activin A With Different Concentration On Behavior Of Cytotrophoblast Cells

PrefaceEmbryo implantation includes a series of complicated event. First, blasto-cyst adheres on surface of epithelium of receptive endometrium. After adhesion, blastocyst invades stroma of uterine epithelium through trophoblast. In the third week after fertilization, some cytotrophoblast cells (CTB) in villi proliferate and breakthrough syncytiotrophoblast, forming cytotrophoblst cell column. CTB in distal end of cytotrophoblast cell column interconnects and forms cytotrophoblast-ic shell which gets in touch with maternal deciduas and anchors placenta on uterine wall. In early stage of implantation, some extravillous cytotrophoblast cells that are called endovascular cytotrophoblast enter lumina on top of uterine spiral artery. They take part in modification of deciduas blood vessel, increase blood flow of placenta and confirm normal material exchange between mother and fetus.Hypertensive disorder complicating pregnancy is a special disease in pregnancy. It is main reason for morbidity and death rate of pregnant women and neonatal. Dysfunction of CTB in placenta and deficiency of vascular remodeling are widely accepted as one etiology of hypertensive disorder complicating pregnancy. Activin is a member of TGF - β superfamily. It has many functions, Dys-regulation or disruption of activin signaling is associated with multiple pathological states including reproductive disorders and carcinogenesis.In this experiment, concentration range of activin A was 0. 1 - 100ng/ml. Secretion of matrix metalloproteinases ( MMP) 2,9 and apoptosis were detected by gelatin zymography and TUNEL technique. Proper concentration that caused different behavior of cell were being tried to find out in order to provide marker molecule for early diagnosis of embryo implantation during pregnancy and pro-vide experimental evidence for etiology research of hypertensive disorder complicating pregnancy.Methods1. The source of specimenTrophoblast tissue in normal pregnancy 7 to 8 weeks was gotten from department of gynaecology and obstetrics in Beijing Haidian Hospital. All patients that were chosen were excluded chronic diseases such as hypertension and diabetes and infectious diseases such as hepatitis and tuberculosis. No special drug was given before termination of pregnancy.2. Primary culture of humane cytotrophoblast cellsVillus in normal pregnancy 7 to 8 weeks was verified age under anatomical lens. Villus was sheared and put in 0. 15% trypsin for 50 to 90 minutes. Then it was centrifuged and 25IU/ml DNase I was added for 10 to 15 minutes. It was centrifuged again, filtered, settled by 1.25% -2.5% BSA density gradient for 1 hour and the CTB were collected. Supplemented cells by serum - free FD medium with lOng/ml EGFxljxg/ml insulin,2mM glutamine and 0. 1%BSA were inoculated in plate with Cell matrix and cultured in humid incubator with 5% CO2 at 37^. 24 hours later.3. Gelatin Zymography and TUNELCytotrophoblast cells were divided into seven groups. After dealt with ac-tivin A (concentration was respectively 0x0. 1%1%1OX25>5OX1OO ng/ml)for 24 hours, they were analyzed by Gelatin Zymography or TUNEL.4. Statistic analysisOne - way ANOVA was used by SPSS12.0. Apoptosis rate or MMPs power of CTB dealt with different concentration of activin A was analyzed. The result had the meaning if p <0. 05.Results1. Detection of related index of Activin A stimulating CTB to secrete MMPsThe result of gelatin zymography indicated that activin A with proper concentration range (1-10 ng/ml ) could stimulate humane CTB to secrete MMp -2. During this concentration range, it could also stimulate humane CTB to secrete PRO - MMP -2 and PRO - MMP -9.2. Detection of related index of Activin A inducing apoptosis of CTB The result of TUNEL found that with increase of concentration of activin A, power of Activin A inducing apoptosis of CTB increased. Apoptosis rate was increased from 3. 80% with 0 ng/ml to 64. 67% with 100 ng/ml. When concentration of activin A reached 10 ng/ml, apoptosis of CTB began to increase. When concentration was higher than 50 ng/ml, power of Activin A inducing apoptosis of cytotrophoblast cells increased significantly.DiscussionThe only difference is concentration, in which way activin A makes CTB produce different cell behavior? The first possible reason is that affinity between activin A and ActR II is 7 times than that between activin A and ActR II B. And whatever activin A bound to which type E receptor, it can continue interacting with ALK4 and form complex and accomplish signaling. So, in lower concentration , activin A mainly forms complex with ActR II , affects Smad protein activity further and starts transcription factor related to MMPs secretion in order to make CTB secrete MMPs. While in higher concentration, activin A also combines with ActR IIB and starts transcription factor related to apoptosis to induce CTB apoptosis. The second possible reason is that when Activin concentration differences of as little as 3 - fold are quantitatively correlated with levels of nuclear SMAD complexes, change of this concentration may start different transcription factor and produce different biological effect. This experiment mainly supported the first possibility.Controlled invasion of trophoblast into the maternal endometrium is one of the critical events of Embryo implantation. Extracellular matrix (ECM) , inte-grin, proteolyses and many other molecules are involved. Lots of members in the family of MMPs play important role during the procedure. MMP -2 and MMP -9 belong to gelatinase. Both of them can dissolve basement membrane and degrade ECM. This is good for CTB invasion and embryo implantation. In this experiment, it was found that activin A would stimulate CTB to secrete MMP -2 and MMP - 9 in concentration of 1 - lOng/ml that accorded with concentration of activin A in peripheral serum of normal pregnant women. This indicated that during pregnancy, activin A might increase invasion power of CTB by stimulating MMPs secretion.Apoptosis plays important role in pregnancy establishment, development and maintenance. However, increased apoptosis or decreased apoptosis of placenta cells may lead to pathological pregnancy. This experiment detected activin A inducing CTB apoptosis with concentration of 0 to lOOng/ml completely by TUNEL method and found that when the concentration was more than lOng/ml CTB apoptosis was induced and when the concentration was more than 50ng/ml CTB apoptosis was induced significantly increased. This concentration was the same with that in patients with preeclmpsia found by Madazli, which indicated that activin A with high concentration in patients with preeclmpsia was enough to cause apoptosis of CTB.ConclusionActivin A with different concentration leads to different behavior of CTB. In low concentration, it stimulates CTB to secrete MMPs. In high concentration, it induces apoptosis of CTB and cannot stimulate CTB to secrete MMPs. Keeping proper concentration of Activin A is considerable in pregnancy establishment.