Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors.

Xiuzhi Tian,Wusiman Abulizi,Pengyun Ji,Zhenzhen Zhang,Jing Wang,Xuguang Wang,Dongying Lv,Lu Zhang,Feng Wang,Changjiu He,Zhengxing Lian,Guoshi Liu

doi:10.3390/ijms18040834

Abstract

(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus–oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10−3, 10−5, 10−7, 10−9, and 10−11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10−7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

Highlights

Compared to in-vivo-derived embryos, the efficiency of in vitro embryo development is still low [1], which is mainly because the in vitro handling and culture conditions are far from perfect when oocytes and embryos are exposed to in vivo conditions
Melatonin Receptors in Sheep Oocytes, Cumulus Cells, and Granulosa Cells The expression of MT1 and MT2 receptor was confirmed by using immunofluorescence and Western Tbhloet eaxnparleysssiisonwiotfhMspTe1ciafincdaMntTib2ordeiceespitnorGwVa(sgceornmfiirnmaledvebsyiclues)inagndimMmIIu(nmofelutaoprhesacseenIcIe) aonodcytes, andWoGoecVsytteaernsn,dabnMldotIGIaVcnuaamlnydsuiMsluwIsI icctuehlmlsspuielnucsitfihcceelalgsnritnaibntohudelioegssraainncueGllolVss.ailoeanvneldelvoMeflIoMIf(MTm1Tet1raerpcehecapespteotorIrsIs)was signwifiacsasnigtlnyifihciagnhtleyrhiinghGerVincuGmV uculumsucluelslsceallnsdangdragnraunluolsoasacecelllslsccoommppaarreeddtotothtahtaitnitnhrteheroeteheorthceelrl cell typetsy,pwesh, iwlehtihleetheexpexrepsrseisosniolnelveevleol fofththeeMMTT22rreecceeppttoorriinnMMIIIIoooocycytetsews wasahsighhigehsteasmt aomngoanlgl caellll ctyeplletsypes (Figu(Friegsu1reasn1dan2d). 2)
The identification of MT1 and MT2 in granulosa cells, cumulus cells, and oocytes in sheep were accordance with the observations of El-Raey et al found in cows [9], and it confirmed previous reports that melatonin was important for the regulation of reproduction in animals [36]

Summary

Introduction

Compared to in-vivo-derived embryos, the efficiency of in vitro embryo development is still low [1], which is mainly because the in vitro handling and culture conditions are far from perfect when oocytes and embryos are exposed to in vivo conditions. To solve this problem, many efforts have been made. The focus has been on melatonin, which is a potent antioxidant and free radical scavenger [2,3] due to its beneficial effects on oocytes maturation and embryo development [4,5,6,7,8,9,10,11,12,13,14]. The specoioficcyotebmjeacttuivraetsioonf etihtheecruvriraeintst dsitruedctyawntieorxeid(ia)nttoacdteivteitrymoirnmeewdihaetethdebryMitsTr1ecaenpdtoMr aTct2ivaarteioenx. pTrheessed in shspeecpifoicoocbyjteecsti,vceusmofutlhuesccuerrllesn,tasntuddgyrwaneurelo(is)atocedleltse;r(miii)nteowinhvetehsetirgMatTe1wanhdatMthT2e aorpeteimxparlesmseedlaitnonin concsehneetrpatoiocnytisest,ocpumroumluosteceinllsv, iatrnodogorcayntuelomsaatcuerllast;io(ini) atondinevmesbtirgyaotedwevhealtotphme eonpti;manaldm(ieiil)attoonainssess the ecfofneccetnotframtioenlaitsontoinproonmtohtee einxpvrietrsosioooncyotfeemmabturyraotidonevaenldopemebnryt-oredleavteldopgmeneenst;ianncdu(miiiu) ltuosacsseellsss and oocythteese;f(feivct) otof minevlaetsotniignaoten tthhee eexfpfercetssoiofnthoef eamntbargyoondiesvteolofpmmeelnatt-ornelianteadngdentheseienfcfeucmt uolfums ceellalstoanndin on cAMoPocaynteds;c(GivM) tPo (icnyvcelsitciggauteanthoesienfefemctoonfotphhe oasnptahgaotne)isitnoof omcyeltaetsonainndacnudmthueluesffceecltlso.f melatonin on cAMP and cGMP (cyclic guanosine monophosphate) in oocytes and cumulus cells

Results

Discussion

Conclusion