Effects Of Different Treatments On The [Ca²⁺]i Oscillations In Parthenogenetic Activation Of Mouse Oocyte

Oocyte activation failure is one of the most serious problems for poor embryo development.In mouse,the transiently elevated Ca2+ plays an important role in egg activation and fertilization.[Ca2+]i oscillations in mouse eggs last up to several hours after parthenogenetic activation.They switch the oocytes from meiosis to mitosis,and thus,play an important role for PN formation,embryonic development and implantation outcome.In recent years,many studies have shown that[Ca2+]i oscillations regulate the egg activation efficiency and the subsequent embryo development.However,there are few researchs on how the time post-hCG,the medium pH,in vivo/in-vitro maturation and ER stress affect[Ca2+]i oscillations in parthenogenetic activation.The aim of this study was to compare the[Ca2+]i oscillations pattern in parthenogenetic activation of mouse oocyte with different treatments.Briefly,we pre-treated the oocyte with different methods,and then,used strontium chloride to activate it and a time-lapse confocal laser microscope to monitor the[Ca2+]i oscillations pattern.Moreover,the endoplasmic reticulum stress(ER stress)markers were also measured by qRT-PCR.We collected oocyte released from the oviduct at 13 to 21 h post hCG to study how the time post-hCG to affect the[Ca2+]i oscillations pattern in mouse oocyte parthenogenetic activation.In order to investigate the effects of the pH during the activation of the mouse oocyte,[Ca2+]i oscillation patterns were recorded for 3.5 hours under different pH condition.To compare the difference of the[Ca2+]i oscillations of parthenogenetic activation between oocyte matured in vivo and in vitro,we collected oocyte released from the oviduct at 17 h post-hCG as control group,while GV denuded oocytes(DO)collected after 48 h PMSG were cultured in M2 media for 17 h as treatment group.To explore whether there was difference between cumulus oocyte complexes(COC)and DO matured in vitro,COC and DO collected after 48 h PMSG were cultured in M2 media for 17 h respectively.To observe the effect of ER stress induced by tunicamycin(TM)on[Ca2+]i oscillations,we adopted the 0 pM TM group as control group,and concentration of 2.5 μg/ml,5 μg/ml,10μg/ml,20 μg/ml,30 μg/ml,50 μg/ml TM were added to M2 medium,respectively,to treat oocytes for 1.5 hours before parthenogenetic activation.The results showed that[Ca2+]i oscillations of oocyte of 17 h post-hCG exhibited the fastest frequency and shortest cycle.Then we found that pH 6.8 to pH 7.6 didn’t affect the[Ca2+]i oscillations pattern,while the group of pH 6.6 and pH 6.4 showed significantly lower frequency and longer cycle than that of pH 7.2.Furthermore,the[Ca2+]i oscillations frequency and peak value decreased after parthenogenetic activation in oocyte matured in vitro.In addition,COC matured in vitro showed significantly higher frequency,shorter cycle and higher peak than that of DO.However,the frequency and peak of the[Ca2+]i oscillations of COC matured in vitro were not as high as that of the oocytes matured in vivo,so did the PN rate.Finally,ER stress that induced by 2.5-50 μg/ml TM largely affected parameters of[Ca2+]i oscillations,including elongated cycles and lower frequency.Together,these data indicated that the time post-hCG,the pH in the culture medium and in-vitro maturation were the key factors affecting[Ca2+]i oscillations in parthenogenetic activation.Furthermore,ER stress also affects the[Ca2+]i oscillations in mouse oocyte parthenogenetic activation.