Role Of Caffeine In Regulation Of Mouse Oocyte Aging

Most mammalian oocytes would be blocked at MII stage after maturation in vivo or in vitro until sperm penetrating. There are some morphological and physiological changes happened in oocytes as fertilization is delayed, which is usually called OOCYTE AGING. Aged oocytes have poor developmental potential accompanied with low fertilization rate and simultaneously higher polyspermous fertilization would be obtained. Organelles in aged oocytes such as spindel and cortical granules(CGs) are wrong assembled and localized. When these oocytes are activated by chemical treatment, cytoplasmic fragmentation ratio would increase with increased activation rate. It is necessary to understand the mechanism and regulating way of oocyte aging happening which would be a favor to the developmetal and reproductive experiments such as in vitro fertilization (IVF) or somitic cell nuclear transfer(SCNT). To study the role of caffeine in mouse oocyte aging inhibition, we compared different concentration, treatment duration and procedure of caffeine on chemical activation(CA) ability and embryos developmental potential of treated oocytes. After that we observed the spindel assembly morphous and CGs location in cytoplasm in oocyte treated with caffeine or not. Experiments results as below:1. Caffeine could inhibit mouse oocyte aging which matured either in vivo or in vitro, but the treatment concentrations were different. Oocyte matured in vivo(IVO group) aging was efficiently inhibited by 6mM caffeine treatment for 6h. When the conceration increased to 10mM, aging was fully inhibited. At the same time the efficient and the full inhibitory caffeine treatment concetration in oocytes matured in vitro(IVM group) were 0.5mM and 8mM respectively.2. The inhibitory effect of caffeine on oocyte aging was reversible. After oocytes in IVO group treated with different conceration caffeine(6mM, 8mM and 10mM respectively) were recoveried in CZB without caffeine for 6h, the ratio of activated oocytes was similar to control group post chemical activation. In IVM group, oocytes also could recover from 0.5mM and 8mM caffeine treatment whose CA ratio was also not different with control group.3. The inhibition of caffeine on oocyte aging could affect embryo development in vitro. When 6mM caffeine was used to inhibit IVO oocyte aging, the oocytes could develop to blastocyst which was similar to the control group(32.9% vs. 35.5%, P>0.05). But the 4 cell developmental ratio were different between two groups(P<0.05). 8mM and 10mM inhibition decreased oocytes blastocyst developmental ability(20.9% and 11.0% respectively) and the last group had the lower blastulation ratio(P<0.05). Then we chose 8mM caffeine as inhibitory conteration and prolong the treatment duration to 12h, the oocytes blastulation decreased more to 3.3%. The results indicated that although caffeine could inhibit oocyte aging and had no effect on CA after recovery, the injury was still happened and the stronger treatment condition increased the harm to oocytes.4. Caffeine treatment could reverse the aged oocytes. When the cumulus-oocyte complexes(COCs) were obtained at 18h post hCG injection(the oocytes were aged for 6h in vivo), 8mM or 10mM caffeine could not reverse the COCs back to the state of control group, and whose CA ratio were still high(85.8%, 91.1%). On the contrary when cumulus cells were removed before caffeine treatment(denuded oocytes were used, DOs), the aged state of oocytes were reversed and CA ratio was decreased to 28.7%. These results indicated that cumulus cells could offset caffeine's reversiable effect on aged oocytes from IVO. The interesting was that in IVM group cumulus cells had no effect on caffeine's treatment. 0.5mM or 8mM caffeine could reverse COCs and DOs obtained from 20h IVM group( the oocytes were aged for 6h in vitro ).5. Inhibition oocyte aging with 8mM caffeine affected spindle morphous and cortical granules(CGs) location in cytoplasm. Spindle morphous in oocytes treated with 8mM caffeine for 6h was not changed obviously. But when the treatment duration was prolonged to 12h, the ratio of oocytes with a tip pole spindle(Which was proved being a normal spindle morphous in control group oocytes) decreased(31.8% vs. 51.2%, P<0.05) obviously. It is interesting that if we recover the treated oocyte in medium without caffeine, the spindle could reassemble and the tip pole spindle oocytes ratio incresed to the control group level(68.3%). On the other hand CGs location in oocytes was also affectd by caffeine treatment. Oocytes with normal CGs location(form a monolayer under plasma membrance and with a CGs free domain) reduced(9.6% vs. 85.5%, P<0.05) when they were treated with 8mM caffeine for 6h. And if the treatment duration was prolonged to 12h, the ratio decreased to zero. What was different from spindle reassembly was that CGs location could not be recovered after removal of caffeine.