Telocytes Enhanced The Proliferation,Adhesion And Motility Of Endometrial Stromal Cells As Mediated By The ERK Pathway In Vitro

Telocytes(TCs)is special interstitial cell that have recently been identified in the female reproductive system.Endometriosis(EMs)is a benign gynecological disease whose etiology is still not fully clear.Implantation and proliferation of endometrial stromal cells(ESCs)out of the uterus are essential processes in the development of EMs.Herein,we investigate the in vitro changes of ESCs when cocultured with TCs,and the potential mechanisms involved.The current results demonstrated that,vimentin-positive/pan cytokeratin-negative ESCs,and TCs with a characteristic structure and immunophenotype(CD34/vimentin double-positive)were successfully isolated and harvested.Morphologically,direct cell-to-cell contacts were observed between TCs and ESCs.Quantitatively,TCs treatment clearly promotes the viability of ESCs,enhances cell cycle progression at G2/M phase and upregulates p-ERKl/2 and cyclin-D3(all P<0.05).Functionally,ESCs educated by TCs displayed significantly enhanced adhesion ability and accelerated invasion and migration capacity(all P<0.05).However,no significant changes were found in the rate of apoptosis and in the expression of AKT signaling pathway proteins in TCs-educated ESCs(both P>0.05).Therefore,TCs treatment obviously enhanced the in vitro motile and invasive capacity of ESCs,which were mediated by the ERK-cyclin-D3 signaling pathway,likely through direct intercellular contacts and/or juxta-paracrine effects;signaling through this axis therefore increased the likelihood of EMs.The enhanced functions of TCs-educated ESCs not only contribute to a deeper understanding of TCs,but also highlight a new concept regarding the physiopathology and therapy of EMs and associated impaired reproductive function.Method:Adult female BALB/c mice were selected to obtain primary uterine TCs and adult female BALB/c pregnant mice were selected to obtain primary uterine ESCs.1.Mice were killed with an overdose of chloral hydrate;TCs and ESCs were obtained from uterine tissue by enzyme digestion.2.Primary cultured TCs were detected by using methylene blue vital staining,and immunofluorescence cytochemistry(vimentin,CD34 and c-kit).3.Observed and recorded the morphological changes of TC.4.Telocytes media(TCM)were harvested after 24 hrs coculture of TCs with serum-free DMEM/F12.5.Primary cultured ESCs were detected by using methylene blue vital staining,and immunofluorescence cytochemistry(vimentin and PCK).6.ESCs entered into the logarithmic growth phase and direct coltured with TCs,observed and recorded the morphological changes of TC.7.ESCs entered into the logarithmic growth phase and indirect cocluture with TCs(expriment)or DMEM/F12(control).8.The morphological changes of ESC were observed after direct co-culture at Oh,24hrs and 48hrs.Changes in ESCs cell viability,adhesion function,migration and invasion,cell cycle,apoptosis and pathway proteins were detected after 24h of indirect coculture.To explore the morphological and functional changes of ESCs under the action of TCs and the possible mechanism.Results:ESCs with vimentin-positive/pan cytokeratin-negative,and TCs with characteristic structure and immunophenotype(CD34/vimentin double positive)were successfully harvested and isolated.Morphologically,direct cell-to-cell contacts were observed between TCs and ESCs.Quantitatively,TCs treatment obviously promotes viability of ESCs,enhances cell cycle progression at G2/M and upregulation of p-ERK1/2 and cyclin-D3(all P<0.05).Functionally,TCs-educated ESCs displayed significantly enhanced adhesion ability,accelerated invasion and migration capacity(all P<0.05).Conclusions:The current results demonstrated that,through intercellular contacts and/or juxta-paracrine effect,TCs treatment obviously enhanced in vitro motile and invasive capacity of ESCs via extracellular-regulated kinase(ERK)-cyclin-D3 signaling pathway.The enhanced functions of TCs-educated ESCs not only contribute to the deeper understanding of TCs,but also highlight a new thought regarding the physiopathology and therapy of EMs and related impaired reproductive function.