| Recent studies have demonstrated that brain-derived neurotrophic factor（BDNF）plays crucial roles in animal reproduction, including follicular development,oocyte maturation and embryo development. Deletion of BDNF and its receptor TrkBcould lead to female animal infertility. MicroRNAs (miRNAs) are endogenous noncoding small molecule RNAs that participated target gene regulation by repressingtranslation or degradation mRNA.However, little is known about the role of miRNAsin BDNF mediated porcine oocyte maturation in vitro.Differentially expressedmiRNAs were screened from BDNF-stimulated pig COCs maturation in vitro24hand48h using Real-time PCR. The results as follows:1. The expression of BDNF and its receptor TrkB in porcine COCs.The presence of BDNF and its receptor TrkB in porcine COCs was investigatedby RT-PCR and immunofluorescence.The results that showed BDNF and TrkBtranscripts were expressed in porcine COCs. BDNF and its receptor TrkB proteinswere mainly expressed in oocytes and cumulus cells.2. Effects of different concentrations of BDNF on porcine oocyte maturation invitro.Effects of BDNF on cumulus cell expansion and the first polar body release werestudied. Significant increased cumulus cell expansion and the first polar body releaserate were observed in response to50-200ng/mL exogenous BDNF，respectively，incomparison with the control. The first polar body release rate was significantlyincreased when COCs were cultured in medium supplied with50ng/mL BDNFcombined with gonadotropin.and EGF.3. Detection of differentially expressed miRNAs in pig COCs maturation invitro. About30miRNAs expression profilings were identified from BDNF-stimulatedpig COCs maturation in vitro24h and48h using Real-time PCR. The miRNA-205was found to be significantly down-regulated and miRNA-29a up-regulated inmaturation24h group compared with the control.4. Prediction and verification of miRNA-205target genes.PTX3was idendified as one of the target of miRNA-205by a series biologicalanalysis. COCs expansion marker gene including PTX3, TAFAIP6, and HAS2, were allup-regulated by BDNF stimulation using Real-time PCR.The expression of PTX3inporcine oocyte maturation was negatively correlated with that of miRNA-205thatconfirmed the above predictin.By summarizing the above results，the present study firstly demonstrated theexpression of BDNF and its receptor TrkB in porcine COCs maturation in vitro andthat BDNF could promote cumulus cell expansion and the first polar body release.The expression of PTX3was up-regulated but miRNA-205down-regulated.Thus, wesuppose that BDNF may regulate oocyte maturation by fine tuning the expression ofmiRNA-205and PTX3. |
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