Paracrine Regulation Of Leptin And GDNF On Mouse Oocyte Maturation

ObjectivesTo investigate whether leptin or glial cell line-derived neurotrophic factor(GDNF)could regulate mouse oocyte maturation via paracrine manner.Materials and Methods1.DNA microarray analysisMice were injected at 21 days of age with Humegon and forty-eight hours later,some animals were treated ip with Pregnyl to induce ovulation.Ovaries were dissected from animals for RNA extraction. Samples were hybridized to the Affymetrix mouse MGU74v2 arrays A,B,and C according to standard Affymetrix protocols.2.Quantitative real-time RT-PCRWe performed quantitative real-time RT-PCR to show the ovarian transcripts for candidate factors (leptin and GDNF)and their receptors in mice treated with PMSG followed 48 h later by an injection of hCG..3.Immunostaining was performed to show the localization of protein(leptin and GDNF)in mouse ovary.4.Follicle cultures were performed to investigate the effect of leptin and GDNF on GVBD.5.Either cumulus-oocyte complexes(COCs)or denuded oocytes were cultured with or without leptin and GDNF to evaluate the effect of leptin and GDNF on first polar body extrusion.6.To assess the effect of leptin and GDNF on the cytoplasmic maturation of preovulatory oocytes, COCs were cultured with leptin followed by in vitro fertilization was performed.Results1.Microarray results:the expression of Ob-Ra mRNA was stimulated after treatment with hCG, whereas the transcript level of leptin showed minor changes.Different from leptin,the expressions of both GDNF and Gfral mRNAs were stimulated after hCG.treatment.2.Following the DNA microarray results,treatment with hCG increased Ob-Ra transcript levels followed by a peak at 5 h in whole ovaries and the expression of leptin mRNA was not regulated by gonadotropin.In oocytes,the level of Ob-Ra transcripts was increased shortly after hCG stimulation, whereas the level of Ob-Rb transcripts was not changed.Although clear stimulation of GDNF rnRNA was found in cumulus and mural granulosa cells,levels of this transcript were negligible in oocytes. In contrast,the GfralmRNA was predominantly expressed in oocytes and cumulus cells from mice primed with PMSG for 48 h.After hCG treatment,the Gfral transcript level was increased in both cumulus cells and mural granulosa cells,whereas the Gfraltranscript level remained stable in oocytes.3.Immunohistochemistry results:strong leptin staining was observed in interstitial cells,theca cells and oocytes of both preovulatory and small antral follicles at 5 h after hCG treatment.In the preovulatory follicles,leptin was detected in granulosa cells and cumulus cells,however,granulosa cells of pre-antal follicles and small antral follicles showed no staining of leptin.GDNF staining was observed in cumulus,mural granulosa,and theca cells,but not in oocytes of preovulatory follicles at 4 h after hCG treatment.In addition,small antral follicles were not stained with GDNF.4.Treatment with leptin induced GVBD in preovulatory oocytes,and enhanced first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes.The leptin-induced GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase(MAPK)extracellular signal regulated kinase(ERK)kinases(MEK)1/2 inhibitor.Treatment of cumulus-oocyte complexes and denuded oocytes with GDNF enhanced first polar body extrusio.However,treatment with GDNF did not affect GVBD.5.Including leptin in the maturation medium promoted fertilization of oocytes and the in vitro development of zygotes to preimplantation embryos.Pretreatment with GDNF did not increase the proportions of MII oocytes that developed into the two-cell or blastocyst-stage embryos.Conclusion1.LH/hCG stimulates the Ob-Ra transcripts,in mouse oocytes.Leptin is produced by cumulus cell, granulosa cells,theca cells and interstitial cells of ovaries and its transcript level is not regulated during gonadotropin treatment.2.Leptin promotes the GVBD and first polar body extrusion of mouse oocytes in in vitro culture. Ob-Ra/ MEK 1/2 signaling is involved in the process of leptin induced mouse oocyte nuclear maturation.Furthermore,leptin enhances cytoplamic maturation of mouse oocytes.3.GDNF is secreted by cumulus,granulosa and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge.The GDNF receptor Gfral-Ret is identified in mouse oocyte,suggesting a paracrine role of GDNF on mouse oocyte maturation by acting on Gfral-Ret receptor complex in oocytes.4.GDNF promotes the first polar body extrusion in in vitro culture but treatment with GDNF did not affect GVBD and cytoplasmic maturation of oocytes.