Effect Of DES On The Migration Of Gubernacular Cells In Vitro Through MMP

Objective:The primitive testis is a pararenal gonad during embryonic period and gradually descends to the scrotum by the traction of gubernaculum.Environmental estrogens could affect the development of fetal gubernaculum and hinder the descent of the testis.Previous study about the effects of EEs on gubernacular cells mainly focused on the proliferation and apoptosis of such cells,but rarely on the cellular migration.Actually,the migration of gubernacular cells was active throughout the whole process of testicular descent.Previous study of our teamwork had proved that DES,as an environmental estrogen,might interfere with the cytoskeleton remodeling and development of gubernacular cells and finally affect the descent of the testis.It had been proved that MMP could promote migration in other non-gubernacular cells such as epithelial cells in cornea and conjunctiva,as well as metastasis of carcinoma cell.However,it’s still unclear that whether DES interfered with gubernacular cell migration by hindering the MMP synthesis.The purpose of this study was to investigate the effects of EEs on the cellular migration and MMP2/14 expression of mouse gubernacular cells in vitro,so as to explore the possible mechanism.Materials and Methods:The gubernacula were removed from 3-day-old mice and cultured.The sub-cultured cells were randomly divided into the normal control group,the solvent control group（DMSO group）and the experimental group（DES group）.And then the DES group was administered1.5X10^-8mol/L of diethylstilbestrol dissolved in DMSO.Each group of cells was treated for the same period of time.After immunofluorescence staining,the expression and location of MMP2/14 were observed under an inverted microscope.Furthermore,the transcription and expression of MMP2/14 gene were investigated by RT-PCR and western-blot respectively.Finally,an experimental group（MMP inhibitor group）was added in wound healing assay and transwell migration assay,and the cellular migration in different groups were observed.Results:1.Immunofluorescence:MMP2 and MMP 14 were highly expressed in the cytoplasma of gubernacular cells.2.Wound Healing Assay:The wound healing rate of the DES and MMP inhibitor groups was significantly lower than the control group in 12h（P<0.01）.There was no significant difference between the wound healing rate of the DMSO group and the control group in 12h（P>0.05）.After 24h,the computer software could not find out any wound line while the wound lines of the MMP inhibitor group and DES group were still visible to the naked eye.The wound lines of the DMSO group and the control group were not visible to the naked eye.3.Transwell Migration Assay:There was no gubernacular cell found in the lower chamber of the MMP inhibitor group and the DES group.However,a number of the gubernacular cells in the lower chamber of DMSO group and the control group were found.The cellular density in the lower chamber of the DMSO group and the control group was not significantly different（P>0.05）.4.Reverse Transcription-Polymerase Chain Reaction:Compared with the control group,the transcription of MMP2 and MMP14 gene was reduced in the DES group（P<0.01）.There was no significant difference between the DMSO group and the control group（P>0.05）.5.Western-blot:Compared with the control group,the expression of MMP2 and MMP14 was reduced in the DES group（P<0.01）.There was no significant difference between the DMSO group and the control group（P>0.05）.Conclusion:1.MMP2 and MMP14 were highly expressed in the cytoplasma of gubernacular cells,which had been proved by immunofluorescence staining.2.MMP2 and MMP14 participated in the migration of gubernacular cells and they were reliable biomarkers for cellular migration.3.DES could significantly suppress the transcription and expression of MMP2 and MMP14 gene.4.DES might suppress the migration of gubernacular cells through down-regulation of MMP2 and MMP14.