The Role Of Calcium Signaling In Mouse Oocyte Meiotic Resumption

The activation of natriuretic peptide receptor 2(NPR2)in response to its agonist,natriuretic peptide type C(NPPC),is essential for maintaining oocyte meiotic arrest.NPR2 is expressed throughout the granulosa cells of the follicle,and is most concentrated in the cumulus cells directly surrounding the oocyte.NPPC is produced only in the mural granulosa cells(MGCs).The LH(Luteinizing hormone)receptor signaling is amplified by the transactivation of epidermal growth factor(EGF)receptor in cumulus cells,resulting in the meiotic resumption.As previously reported,LH-dependent EGF receptor signaling elevated calcium levels of cumulus cells to inactivate NPR2,resulting in the decrease of cGMP and oocyte meiotic resumption.This study focuses on the regulatory mechanism of activating EGF receptor elevates calcium in cumulus cells,and the regulatory mechanism of calcium on NPR2 inactivation is also investigated.Sphingosine-1-phosphate(SIP)could mobilize calcium from internal stores.Sphingosine kinases(SphK)catalyze the ATP-dependent phosphorylation of the sphingosine to generate SIP.There are two isoforms of SphK,named SphK1 and SphK2.In the present study,we found that SKI-?,the inhibitor of SphK,could inhibit EGF-induced oocyte meiotic resumption and cumulus cells expansion in mouse cumulus-oocyte complexes(COCs).We used UPLC-HRMS system to measure the SIP levels of COCs,both hCG and EGF elevated the SIP levels of COCs,whereas SKI-? significantly inhibited EGF-induced increase of SIP levels.These results indicated that EGF activates SphK.Further study found that U0126 could inhibit the increase of S1P which induced by EGF,suggesting that EGF activates SphK through the activation of MAPK3/1.SKI-? reversed the EGF-induced elevated-calcium levels of cumulus cells.In addition,EGF couldn't decrease cGMP levels when treated COCs with SKI-?,suggesting that the elevated-calcium levels of cumulus cells inactivate NPR2.After that,we found that in cultured follicles,SKI-? inhibited LH-induced elevated-calcium of cumulus cells,oocyte meiotic resumption and cumulus cells expansion.Next,we used the SphK1-/-;SphK2f/-;Cyp19-Cre mouse to investigate the effect of SphK1 and SphK2 on meiotic resumption.SphK1-/-;SphK2f/-;Cyp19-Cre mouse which was injected with hCG for 4 h,the percent of GVB was 53.7%±2.4%,whereas the control group was 96.3%±0.9%.In addition,the calcium levels of cumulus cells couldn't increase when SphKl-/-;SphK2f-/-;Cyp19-Cre mouse was treated with hCG for 2 h.In super-ovulation experiment,we found that the average number of eggs ovulated by SphKl-/-;SphK2f-/-,Cyp19-Cre mouse was 29.3 ± 1.2,but the control group was 56.0 ±3.2.The breeding assay for 5 months indicated that the reproductive performance significantly decrease in SphKl-/-;SphKf/-;Cyp19-Cre mouse,the average number of pups for one mouse in 5 months was only 17.8± 1.9,the control group was 43.0±6.9.Next,we investigated the regulatory mechanism of calcium on NPR2 inactivation.In mouse ovarian follicles,LH,through the activation of EGF receptor,significantly elevated calcium levels in cumulus cells,but decreased the binding affinity of NPR2 for NPPC.In cultured COCs,the activation of EGF receptor by EGF mobilized intracellular calcium of cumulus cells to decrease NPR2 affinity and cGMP levels,resulting in meiotic resumption.However,hormone treatments had not changed NPR2 protein levels.Furthermore,SKI-? reversed the decrease of the binding affinity of NPR2 for NPPC which meditated by LH or EGF.These results indicated that LH-dependent EGF receptor signaling increases calcium levels of cumulus cells,but decreases the binding affinity of NPR2 for NPPC,resulting in the inactivation of NPR2 and oocyte meiotic resumption.In addition,we found that magnesium ions are required to maintain functional NPR2.In summary,LH-EGF receptor signaling activates SphK through MAPK3/1 to produce SIP,after then,SIP increases calcium levels of cumulus cells,decreases the binding affinity of NPR2 for NPPC,resulting in NPR2 inactivation,cGMP levels decrease and meiotic resumption.On one hand,these findings contribute to a more comprehensive understanding of calcium on meiotic resumption,on the other hand,they propose a new idea for the research and application about human oocyte meiotic resumption in clinical.