Synergistic Effect Between Lh And Estrogen In The Acceleration Of Cumulus Expansion In Mouse And Goat

In the mammal,the oocyte is arrested in meiotic prophase during the embryonic period,and undergoes a long diapause lasting for several months,years or even decades until puberty.Luteinizing hormone?LH?,which is released from the pituitary gland,is a crucial hormone triggering meiotic resumption and ovulation.The preovulatory LH surge is coupled with a rapidly increased estrogen concentration in females in vivo,and ovulation accurs after the estrogen wave and LH peak.However,it is still unclear how the estrogen weve involves in the process of oocyte maturation.Estrogen serves different physiological functions by activating the receptors located on the cell nuclei and cell membranes.The nuclear receptors?ERs?have a high affinity for estrogen and the membrane receptor GPR30 has a low affinity for estrogen.In another word,only a lower level of estrogen is need for activation of ERs,but a higher level of estrogen is required for activation of GPR30.In the previous research of our laboratory,estrogen was found to activate ERs in lower concentration to prolong the duration of CNP-induced meiotic arrest,however,estrogen in higher concentration activated GPR30 to attenuate the effect of CNP and facilitate oocyte meiotic resumption.In this study,we focused on the estrogen wave along with LH peak before ovulation and explored the role and mechanism of estrogen and LH on the reguluation of cumulus cells expantion of cumulus-oocyte-complex?COCs?.The main researches and results were as follows:?1?LH signal increases the GPR30 protein levels in cumulus-oocyte complexsThe GPR30 m RNA and protein levels in mouse and goat COCs were detected after LH/EGF?or EGF-like growth factors?stimulation by using RT-q PCR,western blot and immunofluorescence technologies.The EGFR inhibitor AG1478 was used to verify the pathway that involved in LH/EGF?or EGF-like?signal upregulates GPR30 protein levels.Furthermore,GPR30 protein levels in cumulus and oocyte were analyzed,respectively.The results showed that LH and EGF?or EGF-like growth factors?activated the EGFR signaling pathway on cumulus cells,and upregulated the level of GPR30 protein in cumulus cells through non-genomic regulatory pathways,but played no significant effect on GPR30 protein levels in oocytes.?2?LH inhibits the degradation of GPR30 protein by activating the EGFR signaling pathway in granulosa cellsThe half-life of GPR30 protein was detected in mouse follicle granulosa cells before/after EGF stimulation;the degradation pathway of GPR30 was investigated using the proteasome inhibitor MG132 and the lysosome inhibitor bafilomycin A1;after that,the activity of lysosome was measured by the immunofluorescence staining and lysosome probe.These results showed that GPR30 protein in granulosa cells were degraded by the autophagy-lysosome pathway,activation of EGFR signaling pathway decreased the quantity and inhibited the activity of lysosomes,and prolong the half-life of GPR30 protein,leading to the accumulation of cellular GPR30 protein.?3?Estrogen increases EGFR protein levels in cumulus cells by activating the GPR30signaling pathwayEGFR protein levels in mouse COCs maturated in vivo and goat COCs maturated in follicles were detected and compared with COCs maturated in vitro by using western bolt,then the EGFR protein levels were detected after the COCs were treated with 17?-estradiol?17?-E₂?,G1,G15 and ICI 182780.The results showed that,the EGFR levels of mouse COCs maturated in vivo and goat COCs maturated in follicles in vitro were increased and sustained activating for a longer time during maturation process.However,the EGFR levels of COCs maturated in vitro were decreased and sustained activating for a shorter time.17?-E₂ and G1increase EGFR protein level in COCs,G15,but not ICI 182780 could reverse the increase of EGFR induced by 17?-E₂.These results indicated that estrogen increase EGFR protein levels in cumulus cells by activating the GPR30 signaling pathway.?4?The estrogen-GPR30 signaling pathway enhances the response of COCs to EGF and promotes cumulus expansion17?-E_2,G1,EGF,EGF+17?-E₂,EGF+G1,EGF+17?-E₂+G15,and EGF+17?-E₂+ICI182780 were added in the medium to culture mouse and goat COCs in vitro.The cumulus expansion index,the rates of COCs in different expansion grades and the expression of expansion-related genes were detected to explore the effect and mechanism of estrogen promoting cumulus expansion.The results showed that 17?-E₂and G1 significantly promoted cumulus expansion and upregulated the expression of expansion-related genes in the present of EGF;however,neither 17?-E₂ nor G1 could promote cumulus expansion in the absence of EGF.These results indicated that 17?-E₂ could not promote cumulus expansion directly,instead,17?-E₂ enhanced the responsibility of COCs on the EGF stimulation and promoted cumulus expansion.In conclusion,there is a synergistic effect exist between LH and estrogen to accelerate cumulus expansion during maturation process.Briefly,LH positively stimulates GPR30accumulation in cumulus cells by activating the EGFR pathway and inhibiting lysosomal activity.In parallel,estrogen increases the EGFR levels to accelerate cumulus expansion by promoting the response to EGF signals in COCs via the GPR30 signaling pathway.This study increased the understanding of the maturation mechanism of mammalian oocytes and provided a theoretical basis for improving the oocyte maturation culture system in vitro.