The Protective Effect Of Metal Chelator And Antioxidants On The “prior Matuartion Aging” Of Immature Oocyte And Subsequent In Vitro Maturation

Part 1 The chromosome errors and the changes of mitochondrial distribution on the “prior matuartion aging” of immature mouse oocyteIntroduction In recent years,the renewed of assisted reproductive technology(ART)has developed rapidly,which help the infertility couples to solved the problem and pregnant a baby.But due to various factors such as the pressure of work and life,more and more women were with advanced maternal age that hard to have a baby.Before the arrival of menopause,however,the female fertility showed age-related decline significantly.As the growth of the age,especially over 35 years old,quantity and quality of aged oocytes gradually decline,leading to the problem of infertility.Therefore,oocyte aging problem is the more to more attention.Embryonic cytoplasm almost directly come from the oocytes,so the quality of the oocytes directly affect the follow-up developmental ability of embryo.In ART,because of individualization,patients always with many different types of controlling stimulate ovulation(COH)protocol.However,when stimulating development of different oocytes in different follicle was not all synchronized,there are some eggs in the follicle grown faster,and some of the eggs grown slower,these eggs are not synchronized development before HCG stimulating at the same time,eggs in GV stage that grow faster is aging before HCG stimulating,and some eggs are still young.We called these GV stage aging oocytes “ prior matuartion aging” of immature mouse oocyte.The phenomenon of prior matuartion aging” of immature oocyte often occurin patients with advanced maternal aged or PCOS,that may be one of the important reasons for the low quality of embryo in these patients.Once the immature eggs aging occurs,will change the fertilization outcome of oocytes.Chromosome aneuploidy or errors are often considered to be to one of the reasons that leading to the fertilized failure or embryonic chromosome errors that affecting the pregnancy outcome after transplantation.Mitochondria is an important ATP producer in oocytes,its changes of distribution will also greatly affects the normal operation of a variety of physiological activities in the cell.So far,many studies are focus on the aging after oocytes maturation,rare researches are focus on the prior matuartion aging of immature oocyte and its chromosomes structure errors and the change of mitochondrial distribution,this area is still unknown.Understand the chromosome structure errors and the change of mitochondrial distribution is helpful to clarify mechanism of high loss rate and low growth potential in immature aging oocytes,and it is helpful to improve the quantity and quality of aging eggs in clinic to help the patients get more healthy embryos.Objective In this study we use mice immature oocytes in vitro pre-maturation culture with 8-Methoxymethyl-3-isobutyl-1-methylxanthine(IBMX)for 12hrs-36 hrs aging to prevent GV oocytes developing to MII stage respectively,the oocytes of different immature aging period then further in vitro maturation,and we further check the chromosome abnormalities and mitochondrial distribution of different GV stage aging oocytes,which is very helpful to prevent oocyte aging and also provide the assisted reproductive strategy for the women in ART therapy.Methods 1.Experiment design and in vitro culture of immature mice oocytes Collected mouse GV oocytes were divided into four groups: 1)Oocytes were pre-IVM cultured in 200?mol/ml IBMX for 0hrs(recorded as the group 0hrs);2)Oocytes were pre-IVM cultured in 200?mol/ml IBMX for 12hrs(recorded as the group 12hrs);3)Oocytes were pre-IVM cultured in 200?mol/ml IBMX for 24hrs(recorded as the group 24hrs);4)Oocytes were pre-IVM cultured in 200?mol/ml IBMX for 36hrs(recorded as the group 36hrs);5)In vivo maturation oocytes were collected(recorded as Fresh group).After pre-IVM culture,Oocytes were further cultured for maturation in vitro for 14(12-16)hrs in M2 media supplemented with 10% FBS,recombinant FSH/h CG(75m IU/m L and 0.5 IU/m L),and recombinant epidermal growth factor(5ng/m L).2.Examination of chromosome errors and assessment of mitochondrial distribution on fresh oocytes and pre-maturation aging oocytes.The oocytes with nuclear membrane breakdown was considered as GVBD,and the extrusion of the first polar body(PB1)was considered as MII stage.The number of GVBD oocytes and MII oocytes was counted,MII oocytes were used for the CNV examination and the GV oocytes were used for the mitochondrial distribution assessment.Result 1.Prior maturation aging oocytes maturation rate decline after long-time pre-maturation aging.2.The number of oocytes with abnormal chromosome increase as the pre-maturation aging culture prolong.3.The abnormal mitochondrial distribution pattern increase on the pre-maturation aging mouse oocytes.Conclusion 1.Most of in vivo/in vitro maturation oocytes showed normal chromosome,oocytes with long-time pre-maturation aging appeared increasing aneuploidy and/or chromosomal errors.2.As the pre-maturation aging culture prolonging,the impaired mitochondria function,abnormal chromosome and inhibited nuclear maturation maybe themain reasons on aging oocytes that caused a low rate of embryonic formation and poor embryonic developmental ability.Part 2 Protective effect of metal chelator and antioxidants on the “prior matuartion aging” of immature mouse oocyte and subsequent in vitro maturationIntroduction Assisted reproductive technology,especially in vitro fertilization(IVF)– embryo transfer,has been widely used for treatment of infertility in human.However,transferrable embryos and embryo implantation rates are still low.As oocyte growth in different follicles and/or ovaries does not occur simultaneously,some oocytes in fast growing follicles may be aging at the germinal vesicle(GV)stage,through a process we designated as “pre-maturation aging”,before triggering for oocyte maturation by gonadotropins.Pre-maturation aging could adversely affect the outcome of maturation.Oxidative stress(OS)is considered to be a prominent mediator associated with oocyte aging and causes poor embryonic development.Quantity and quality of oocytes are greatly decreased because of apoptosis induced by OS.However,during ovarian stimulation for multiple oocyte growth and collection,10-14 days are necessary for oocytes to become fully grown at GV stage,and some follicles grow faster than others.In order to allow slowly growing follicles to reach the appropriate size so that a fully grown oocyte can be obtained,fast growing oocytes have to wait until most follicles reach a size suitable for triggering to induce meiosis resumption.During this waiting period(up to 36-72 hours),the oocytes in the fast growing follicles may undergo aging.This is frequently observed in patients with advanced maternal age.Although the exact mechanism(s)for poor embryo development from these patients are unknown,it is possible that oocytes in fast growing follicles become aged before maturation triggering due to unsynchronized follicle growth.If excessive ROS are produced by mitochondria during oocyte aging and there are insufficient antioxidants to remove them,the equilibrium of ROS and antioxidants will be disrupted,and apoptosis is accelerated.3-isobutyl-1-methylxanthine(IBMX)can be used as a nonspecific phosphodiesterase inhibitor for modulating cyclic adenosine monophosphate(c AMP)levels to prevent GV-stage oocytes from meiosis resumption during pre-maturation.Supplementation of IBMX in a culture medium can maintain the oocytes at GV and can be used to mimic in vivo human ovarian stimulation before triggering for maturation.Metal chelator and antioxidants supplementation has been proven to protect oocytes against ROS and OS.Metal ions can be accumulated through food,water or air during a lifetime.Sodium citrate,?-lipoic acid(ALA)and acetyl-L-carnitine(ALCAR)are antioxidants that protect the oocyts attaching by oxidative stress.EDTA is a metal chelator that play important roles in protecting mammalian cells against oxidative stress by scavenging free radicals.Mouse oocytes at germinal vesicle stage were prevented from meiosis resumption in a culture medium supplemented with 3-isobutyl-1-methylxanthine and with or without metal celator(EDTA)and/or antioxidants(sodium citrate,?-lipoic acid and acetyl-L-carnitine)for 12,24 and 36 h to allow oocytes to undergo aging.After aging,oocytes were cultured in the same medium without 3-isobutyl-1-methylxanthine for in vitro maturation.Nuclear maturation,mitochondria activity,spindle integrity and DNA integrity were examined after in vitro maturation.As of our knowledge,no study so far has reported such effects on immature oocyte aging,i.e.pre-maturation aging.The pre-maturation aging of oocytes at GV stage is a common phenomenon in human IVF when oocytes are not triggered for maturation at an appropriate time.Therefore,in the present study,mouse oocytes were maintained at GV stage in a medium supplemented with IBMX and antioxidants for 12-36 h,and then processed to IVM to investigate the effects of metal chelator(EDTA)and/or antioxidants(sodium citrate,ALA and ALCAR)on nuclear maturation and other cellular functions,especially mitochondria activity,meiotic spindle formation,chromosome configuration and DNA integrity.Objective Mouse oocytes at germinal vesicle stage were prevented from meiosis resumption in a culture medium supplemented with 3-isobutyl-1-methylxanthine and with or without metal celator(EDTA)and/or antioxidants(sodium citrate,?-lipoic acid and acetyl-L-carnitine)for 12,24 and 36 h to allow oocytes to undergo aging.After aging,oocytes were cultured in the same medium without 3-isobutyl-1-methylxanthine for in vitro maturation.Nuclear maturation,mitochondria activity,spindle integrity and DNA integrity were examined after in vitro maturation.The study aimed to evaluate the protective effects of metal chelator and antioxidants on the oocytes in terms of maturation rate,functional mitochondria,spindle morphology and DNA integrity under various experimental conditions.Methods 1.Experiment design and in vitro culture of oocytes Collected mouse GV oocytes were divided into five groups: 1)Oocytes were pre-IVM cultured in IBMX and M16/M2 medium containing 10 ?mol/ml EDTA(recorded as the group MC);2)Oocytes were pre-IVM cultured in IBMX and M16/M2 medium containing 10 ?mol/ml sodium citrite,25 ?mol/ml ALA and 20 ?mol/ml ALCAR(recorded as the group AO);3)Oocytes were pre-IVM culturedin IBMX and M16/M2 medium containing 10 ?mol/ml EDTA,10 ?mol/ml sodium citrate,25 ?mol/ml ALA and 20 ?mol/ml ALCAR(recorded as the group MC+AO);4)Oocytes were pre-IVM cultured in IBMX and M16/M2 media(recorded as Control group).5)GV oocytes were cultured in M16/M2 media for IVM(recorded as Fresh group).After pre-IVM culture,Oocytes were further cultured for maturation in vitro for 14(12-16)hrs in M2 media supplemented with 10% FBS,recombinant FSH/h CG(75m IU/m L and 0.5 IU/m L),and recombinant epidermal growth factor(5ng/m L).Fresh GV oocytes were culture in IVM culture medium directly in fresh group.The number of GVBD oocytes and MII oocytes was counted and then used for further experiments,such as mitochondrial distribution assessment and spindle morphological observation.The arrested GV oocytes were used for examining of DNA double-strand breaks(DSBs)to evaluate how the DNA damage affecting the oocyte maturation.2.Immunofluorescence and confocol microscopic examination of spindles and DSBs 3.Assessment of mitochondrial distribution 4.Statistical analysisResult 1.Metal chelator and antioxidants improved prior maturation aging oocytes maturation rate Fresh GV oocytes were processed IVM without adding any of M and A(Group Fresh).When oocytes were pre-IVM cultured for 12 hrs and then for IVM,no statistical differences were observed between the four groups in terms of GVBD rates and MII rates.When the pre-IVM culture time was prolonged to 24 hrs or 36 hrs before IVM,the proportions of oocytes underwent GVBD still did not show statistical differences among the treated groups and control.However,the proportions of oocytes reached MII were significantly higherin MC and/or AO groups than those rates in control.Such differences were observed in both 24 hrsand 36 hrs although the overall rates dropped in all groups in pre-IVM 36 hrs than groups in pre-IVM 24 hrs.2.Effects of metal chelators and antioxidants on the distribution pattern of mitochondria and delay the mitochondrial dysfunction Our result showed that 30.6% to 47.5% oocytes displayed abnormal distribution pattern in 12 hrs pre-IVM culture groups and no statistical differences were found among these groups.When oocytes from 24 hrs and 36 hrs pre-IVM culture were examined,the proportion of oocytes with abnormal mitochondria distribution pattern in MC,AO and MC+AO groups were significantly lower than that in the control group.The proportions of oocytes with abnormal mitochondria distribution were increased as the pre-IVM times were extended from 12 hrs to 36 hrs in all groups.3.Treatment with EDTA,sodium citrate,ALA and ALCAR maintain a normal spindle during oocyte aging The proportion of oocytes with abnormal spindle and/or chromosome misalignment were higher in control group than MC,AO and MC+AO groups,but statistical differences were observed only in M group as compared with control from pre-IVM 12 hrs to Pre-IVM 36 hrs,and AO or AO+MC groups in Pre-IVM 36 hrs.4.Metal chelator and antioxidants may prevent the formation of DNA damage We found that mean intensity of DSBs fluorescence of 12 hrs pre-IVM oocytes in group A was significantly lower than that in control group but other groups did not differ from control group.As the pre-IVM culture time was extended to 24 hrs,fluorescence intensity A group and M+A group still maintained a lower level than that in control group.Significantly lower level of DSBs were observed in group A+M than those in controls after 24 hrs and 36 hrs pre-IVM culture.However,metal chelator alone in the culture medium can reduce the level of DSBs only in extendedculture of 36 hrs.These results indicated that metal chelators and antioxidants played a protective role in ROS-induced DNA damage when process a long-term in vitro culture,but ALA and ALCAR had a stronger protective effect of DNA damage than metal chelators.Conclusion 1.It was found that metal chelator and antioxidants had protective effects on the oocytes in terms of maturation rate,functional mitochondria,spindle morphology and DNA integrity.2.As aging time was prolonged from 12 to 36 h,the protective effect of metal chelator and antioxidants became more obvious.However,as compared with oocytes without aging,it was found that aging significantly inhibited nuclear maturation,impaired mitochondria function,and damaged spindle and DNA.3.These results indicate that pre-maturation oocyte aging is detrimental to oocytes' competence to undergo maturation and other cellular activities.4.Metal chelator(EDTA)and antioxidants(sodium citrate,ALA and ALCAR)in the culture medium play significant roles to prevent oocytes from damages caused by aging.