Mechanisms By Which Glucose Prevents Aging Of Mouse Oocytes And In V:。o Preservation Of Mouse Oocytes

PART Ⅰ: Mammalian oocytes can undergo aging when ovulated oocytes are retained inthe oviduct for a relatively long period of time. Oocyte aging after ovulation significantlyaffects embryo development.In vivo fertilization of aged oocytes gives rise to mice sufferingfrom nervous and emotional abnormalities and decreased reproductive fitness and longevity.Like detention in the oviduct, in vitro culture of matured oocytes also led to oocyte aging.Humans and some animals potentially undertake sexual activity on any day of the estrouscycle, which may cause fertilization of aged oocytes. Many experimental designs in bothresearch and clinical applications involve culture of matured oocytes prior tomicromanipulation or insemination. For example, the cell cycle stage of the recipientcytoplasts must be finely controlled for the success of nuclear transfer for cloning; rates ofcell fusion and embryonic development of nuclear transfer embryos decreased significantlywhen aged oocytes were used for recipient cytoplasts.Therefore, studies on the mechanismsand control of oocyte aging are important for the healthy reproduction of both humans andnonhuman mammals.Studies have shown that subtle changes in the energy substrate composition of theculture medium can have profound effects on meiotic maturation of mammalian oocytes. Forexample, the meiosis-inducing action of FSH on cumulus-oocyte complexes (COCs) waseliminated when glucose was removed from the culture medium. Millimolar concentrationsof pyruvate reversed the meiotic arrest mediated by hypoxanthine or dibutyryl cAMP in theabsence of glucose. In addition, FSH treatment increased hexokinase activity of the COCsand greatly augmented glucose metabolism through the glycolytic pathway. Although thissuggests that the positive effect of glucose is mediated by glycolytic production of pyruvatethat can then be oxidized to generate the energy necessary for nuclear maturation, other dataindicate that the glucose requirement for meiotic induction does not depend on its glycolysisto pyruvate. For example,2-deoxyglucose block to glycolysis was not necessarily associatedwith suppression of FSH-induced meiotic maturation in hypoxanthine-arrested oocytes.Iodoacetate inhibition of glycolysis did not impair LH-induced resumption of oocyte meiosis.Furthermore, pyruvate alone was unable to increase ATP production in COCs, and COCs didnot show increased oxygen consumption when exposed to pyruvate or gonadotropinstimulation.Based on their finding that purine nucleotide-generating pathways participated ingonadotropin stimulation of meiotic maturation, Downs et al. proposed that the pentose phosphate pathway (PPP) was the alternative pathway to glycolysis that mediated the positiveaction of glucose, because the PPP can provide ribose-5-phosphate for synthesis ofphosphoribosyl pyrophosphate (PRPP), an important substrate for the purinenucleotide-generating pathways. They tested this hypothesis and concluded that glucosemetabolism through the PPP was involved in the meiotic induction through the generation ofPRPP.Our recent studies indicated that while cumulus cells accelerated, pyruvate preventedaging of mouse oocytes,However, the mechanisms by which cumulus cells and particularlypyruvate regulate oocyte aging are unknown. Furthermore, since oxidative stress has beenshown to cause an imbalance of the intracellular redox potential, leading to apoptosis of theearly embryo and postovulatory aging of the egg, it is important to understand howintermediary metabolism regulates the oocyte redox potential. In this paper, we have studiedthe glucose metabolism using the aging mouse oocyte model, The results indicated that1) While the oocyte itself can utilize pyruvate or lactate to prevent aging, it cannot useglucose unless in the presence of cumulus cells, suggesting that cumulus cells inhibitoocyte aging by producing pyruvate and/or lactate.2) Freshly ovulated COCs were cultured for6h in CZB containing glucose and differentconcentrations of DHEA (the PPP inhibitor) and/or iodoacetate (the glycolysis inhibitor)The results suggest that oocyte activation rates increased with concentrations of bothdrugs. when glycolysis was inhibited by iodoacetate, only60%of the oocytes becameaged, When PPP was inhibited, however, all the oocytes became aged. Then wemeasured intra-oocyte contents of GSX and the GSH/GSSG ratio.The results suggestedthat PPP prevented oocyte aging mainly by maintaining GSX in a reduced state.3) Lactate prevents oocyte aging through its LDH-catalyzed oxidation to pyruvate, pyruvatedoes not depend on its LDH-catalyzed reduction to lactate. lactate inhibited oocyte agingmainly by producing NADH (through its oxidation to pyruvate), which would then beconverted into ATP through mitochondrial electron transport, pyruvate did not rely solelyon electron transport for its inhibition of oocyte aging.4) When monocarboxylate transfer into the cell or mitochondria was inhibited with4-CIN,an inhibitor of monocarboxylate transporters (MCTs), oocyte activation increased to thehighest level in the presence of both pyruvate and lactate. T the inhibition of oocyteaging by both pyruvate and lactate involves mitochondrial electron transport and MCTsare active on the plasma membrane and/or mitochondria of the aging oocyte.5) The PPP and glycolysis in cumulus cells prevented oocyte aging by producing pyruvate, pyruvate inhibited oocyte aging by its metabolism inside the mitochondrion, a highlevel of pyruvate inhibited oocyte aging by regulating both the intracellular redox statusand energy supply but a low level of pyruvate provided only energy supply.6) In the absence of energy supply, however, supplementation of thiol compounds showedeffect on neither aging nor GSX synthesis of oocytes. oocytes synthesize GSX afterovulation in the presence of energy supply, and that pyruvate inhibits oocyte aging byregulating both the intracellular redox status and energy supply when used at a higherconcentration but it regulates only energy supply when used at a lower concentration.PARTⅡ: Inhibiting oocyte aging is important both for healthy reproduction and for theassisted reproduction techniques. It is known that cells generate reactive oxygen species (ROS)as by-products through normal metabolic activities It is known that hypothermiadown-regulates cell metabolism and thus reduces ROS production. Mild hypothermia wasfound to protect against cerebral ischemia by inhibiting post-ischemia apoptosis and also tohave a specific inhibiting effect on cell apoptosis after diffuse brain injury in rats It wasreported that cooling bovine ovaries to10,15or25°C is beneficial to the developmentalcompetence of oocytes after in vitro fertilization or somatic nuclear transfer. Furthermore,reducing culture temperature has been reported to increase the viability while decreasingapoptosis in various somatic cells. Although our recent studies have demonstrated thatpyruvate supplementation prevented oocyte aging in vitro by maintaining energy supply andredox potential, oocyte blastulation potential after fertilization or parthenogenetic activationwas protected for only6h in those studies with sufficient (10mM) pyruvate supplementation.Our hypothesis was that oocyte aging should be prevented for longer time by reducing culturetemperature in combination with sufficient pyruvate supplementation. The results indicatedthat:1) Oocyte sensitivity to ethanol stimulation increased with culture time but decreased withincreasing pyruvate concentrations.2) Oocyte sensitivity to ethanol also decreased with decreasing preservation temperaturesfrom37°C to15°C, but activation rates were significantly higher after culture at5°Cthan at15°C for the same duration and at the same pyruvate concentration.3) Activation rates after Sr2+treatment were high and did not differ between differentpreservation regimens,Blastocyst rates of over60%were achieved after oocytepreservation at25°C for24h,15°C for36h and5°C for24h.4) Fertilization rates decreased significantly, however, after oocyte preservation at5°C for 48h. Acceptable rates (about80%) of blastulation were observed after oocytepreservation at25°C for24h,15°C for36h, and5°C for24h.5) Two-cell or blastocyst embryos derived from in vitro fertilization of oocytes cultured15°C for6h,and freshly collected oocytes without preservation were transferred tothe oviducts or uteri, respectively, No significant difference was observed betweencontrol and preserved oocytes in either percentage term pregnancy, live young perpregnant recipient, percentage live young/transferre embryos or birth weight of young6)Oocytes with impaired developmental potential after preservation at15°C for96h and at5°C for48h showed unrecoverable decreases in GSH contents,GSH/GSSG ratio andBCL2contents, and in numbers of oocytes with normal spindles and cortisal granuledistribution.In short, in appropriate temperature supplemented with high concentration of pyruvatecan effectively inhibit the aging of mouse oocytes, create a new method for long time in vitropreservation of mature oocytes.