Resveratrol Improves Oocyte Maturation During In Vitro Culture

Capture 1 To investigate the organelle orphological changes of oocyte in aged miceObjectives:A growing body of evidence suggests that age-related subfertility in mammals is primarily linked to the compromised quality of oocyte, which is oocyte aging. There are a series of biochemical changes in the cytoplasm from older female, which cause abnormity of oocyte maturation, fertilization and embryo development. In this study, to investigate the reason for the decline of oocyte developmental capacity in aged mice, we have compared several events, such as condensation and movement, formation and migration of spindle, distribution of cortical granule (CG) and mitochondria in young and aged mouse oocytes.Methods:Kunming mice were from the laboratory animal center of Shandong University, they were divided into young group (4-8W) and old group(48-52W). Female mice were injected i.p. with 10IU/ml PMSG followed 48 h later by 10 IU/ml of human chorionic nadotrophin (hCQ Sigma). MⅡ oocytes were collected from the oviducts 14 h after the hCG injection into M199 medium. The cumulus cells were removed by brief aincubation in 80IU/ml hyaluronidase. The denuded MⅡ oocytes were used for mmunofluorescence staining, and organelle structure analyzed with a Zeiss LSM-780 confocal microscopic system.Results:In the normal oocytes, almost spindle microtubules were normal, chromosomes were symmetric and located at metaphase plate. By studying mice oocytes immunofluorescent stained for chromosomes and spindles, the results showed a high proportion of misaligned chromosome(65.16±1.52%)and aberrant spindle (71.78%± 1.27%) in the oocytes obtained from aged mice. The rates of normal chromosome and spindle of oocytes in aged mice were higher than in young mice (P<0.01, P<0.01, respective).In immunofluorescent stained for microfilament,we thought morphologically normal microfilament had a clear, thick microfilament area containing second metaphase chromatin, and in which the second metaphase plate was not observed near the polar body were considered morphologically abnormal. There were higher proportion of morphologically abnormal microfilament in the MⅡ oocytes from aged mice (48W-52W) than from young mice (4W-8W).Mitochondria distribute in the whole ooplasm at MⅡ stage in the oocytes of young and aged mice, but then again mitochondria are enriched close to the spindle.The fluorescence intensity is higer in young mice(44.44±32.97) than in aged mice(18.82±6.42)by analysis of zen 2012(P<0.01).Cortical granules were granulate, densely populated in a line just beneath the oolemma, with a typical normal CG-free domain at MⅡ-stage oocytes in young mice. While,cortical granules clumped together or disappeared from most parts of the cortex in the oocytes of aged mice. The cortical granules of grageⅢ were higher in the oocytes from young mice(94.04%±0.71%) than from aged mice(51.00%±1.00%)(P<0.01).Concluctions:The abnormal rate of the main organelles (spindle, chromosome, microfilament, cortical granules) in the oocytes of aged mice was increased, which is the main reason for the low fertility rate in aged women.Capture 2 Resveratrol improves oocyte maturation during in vitro culture in aged miceObjectives:Resveratrol is found in red wine and most of the plants. It has anti-inflammatory, anti-aging, anti-cancer, antioxidation, protecting mitochondria, improving fertility and other effects.The present study assessed the effect of resveratrol on the maturation of oocytes and embryonic development, then explored the effect of resveratrol on the oocyte organelle of the aged mice.Methods:GV-stage oocytes were obtained by puncturing ovaries with sterile syringe.Four different concentrations of resveratrol (0,0.1,1.0, 10μM/L) were supplemented IVM medium, in which GV-stage oocytes were cultured for checking the maturity of oocytes after 16h. Our aims were to explore the appropriate concentration of resveratrol, then sequentially cultured the mature oocyte to observe the fertilization, cleavage and blastocyst. Mitochondrial, spindle, chromosome were evaluated by confocal laser scanning microscopy. The expression of SIRT1,CAT,SOD1,GPx4 were analyzed by RT-PCR.Results:When resveratrol was used at the concentration 1.0μM/L in the IVM culture medium for 16h, the discharge rate of the first polar body (93.15%±1.46%) was significantly higher in the oocytes derived from aged mice, percentage of fertilization (76.93%±2.40% vs 60.86%±2.13%,P<0.01) and embryos developing to blastocysts at 108 h (58.61%±2.22% vs 41.23%±1.69%,P<0.01) was markedly increased than control group in aged mice.The immunofluorescence intensity of mitochondria was stronger in the concentration of 1.0μM/L resveratrol group (93.01±19.34), compared with control group (1.0μM/L DMSO) (63.06±13.11,P<0.01).The spindle morphology and chromosome alignment have been notably enhanced from oocytes of MⅡ stage in the concentration of 1.0μM/L resveratrol group than control group.Supplementation of culture medium with 1.0μM/L resveratrol increased SRIT1 levels and up-regulated antioxidant gene CAT,SOD1,GPx4 expression in the oocytes from aged mice.Conclusions:(1) Resveratrol treatment improved the ability of fertilization and blastocyst formation of matured oocytes from IVM culture in aged mice.Resveratrol treatment ameliorated oocyte mitochondrial function, and decreased aging-associated oocyte aneuploidy and meiotic spindle defects and enhanced the expression of SIRT1 and antioxidant gene CAT、SOD1、GPx4.(2) 1.0μM/L resveratrol was the appropriate concentration in IVM medium.Capture 3 Resveratrol improves oocyte maturation during in vitro culture in aged womenObjectives:In the course of assisted fertility treatment, the fertility has gradually declined in aged women because of the decrease of the number of eggs and the decline of the quality of eggs. But resveratrol has played an important role in antioxidant,the protection of fertility and inmature oocytes.The present study assessed the effect of resveratrol on the maturation of oocytes, mitochondrial quality and quantity, spindle and chromosome’s morphology in vitro maturation (IVM) of oocytes in aged women.Then we explored the effective culture condition of IVM.Methods:GV-stage oocytes were collected from patients(aged>38 years old) undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility from February 2014 to February 2015 at reproductive hospital affiliate to Shandong University. The maturity of GV-stage oocytes were checked by culturing in IVM medium with or without 1.0μM/L resveratrol for 24h,36h. Mitochondrial, spindle, chromosome were evaluated by confocal laser scanning microscopy in aged women.Results:Formation rate of MⅡ-stage oocytes in the culture medium with 1.0μM/L resveratrol group were apparently higher than in control group when GV-stage oocytes were cultured for 24h,36h in aged women (55.26% vs 37.84%, P<0.05;71.05% vs 51.35%,P<0.05).The immunofluorescence intensity of mitochondria was stronger in the culture medium with 1.0μM/L resveratrol group (53.02±15.10), compared with control group (31.14±6.05)(P<0.05).The percentage of abnormal spindle morphology and chromosome alignment have been notably reduced from oocytes of MⅡ-stage in the concentration of 1.0μM/L resveratrol group than control group(68.75% vs 88.89%,P<0.05; 68.75% vs 88.89%,P<0.05).Conclusions:1.0μM/L resveratrol treatment improved the ability of maturation of GV-stage oocytes in aged women.1.0μM/L resveratrol treatment ameliorated oocyte mitochondrial function, and decrease aging-associated oocyte aneuploidy and meiotic spindle defects.So resveratrol can effectively improve the quality of oocytes in aged women, which will be confirmed by large samples.