低氧对水牛卵母细胞体外成熟及早期胚胎发育的影响

发布时间：2018-03-19 09:13

  本文选题：水牛卵母细胞　切入点：卵丘细胞　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：卵母细胞体外成熟(In Vitro Maturation, IVM)和胚胎体外培养(In Vitro Culture, IVC)是胚胎体外生产的关键技术环节。近年来,虽然二者均已取得了长足进展,但体外生产的胚胎发育潜力仍较体内胚胎低。本研究旨在研究低氧分压对水牛卵母细胞体外成熟与早期胚胎体外发育的影响,同时初步探讨低氧诱导因子(Hypoxia Inducible Factor, HIF)通过糖代谢途径对水牛卵母细胞体外成熟和早期胚胎体外发育影响的分子机制,以期通过模拟体内卵母细胞成熟和胚胎发育的理化环境来优化卵母细胞和早期胚胎体外培养体系中的气相环境,从而提高水牛卵母细胞体外成熟和胚胎体外培养的效率。首先研究了低氧分压对水牛卵母细胞体外成熟的影响,并初步探讨了低氧分压通过HIF调节的低氧通路及糖代谢影响水牛卵母细胞体外成熟的作用机制。结果显示：水牛卵冠丘复合物(Cumulus-Oocyte Complexes, COCs)在不同低氧分压(2%O2、3.5%O2、5%o2、6.5%O2)环境中体外成熟培养时,相对于常氧(20%02)组,5%02和6.5%02组对卵母细胞的成熟率无显著影响(50.73% vs 55.3 1%,49.58% vs 55.3 1%,P0.05),而2%02和3.5%02组卵母细胞的成熟率均显著降低(21.22% vs 55.31%,29.71% vs55.31%,P0.05)；与常氧(20%02)组相比,2%02、3.5%02、5%02组COCs卵丘细胞扩展指数显著增高(2.84 vs 2.70,2.88 vs 2.70,2.89 vs 2.70,P0.05),而6.5%02组对COCs卵丘细胞扩展指数无显著影响(2.74 vs 2.70,P0.05)。水牛COCs在5%02和20%O2条件下体外成熟培养后进行体外受精(In Vitro Fertilization, IVF),获得的胚胎在20%02中体外培养,结果发现：与常氧(20%02)组相比,COCs在低氧(5%02)条件下IVM组能显著提高其IVF早期胚胎的8-ce11分裂率和囊胚发育率(44.88% vs38.47%,26.81%vs 15.60%,P0.05)。对HIF1α和HIF2α进行免疫荧光检测结果发现：在IVM前水牛COCs的卵丘细胞及在不同氧分压(5%O2、20%02)条件下IVM后的卵丘细胞上均有HIF1α和HIF2α的表达；而在IVM后的卵母细胞中HIF1α均有表达,HIF2α几乎不表达。进一步进行实时荧光定量PCR (Real-time Quantitative PCR, Q-PCR)分析,结果显示：在低氧(5%02)环境中体外成熟培养水牛COCs,显著上调了COCs中卵丘细胞HIF1α、HIF2α、FSH的受体(FSHR)、IGF1的受体(IGF1R)、FOXO1、 VEGF及其受体(VEGFR1、VEGFR2)、糖代谢相关基因(GLUT1、LDHA、 G6DP)、热休克因子(HSP70、HSP90)和五聚环蛋白-3(Ptx3)的mRNA水平(P0.05),且显著下调了前列腺素内过氧化物合酶-2(Ptgs2)及凋亡相关基因Bax和Bc12的mRNA水平(P0.05)。TUNEL检测结果亦发现,水牛COCs在低氧(5%02)中体外成熟培养,能显著降低其卵丘细胞的凋亡率(21.84% vs 30.87%,P0.05)。随后,探讨了低氧分压(5%02)体外培养水牛体外早期胚胎对其发育能力的影响,并对低氧通路中HIF的调控路径及其通过无氧糖酵解影响水牛早期胚胎发育的作用机制进行了初步研究。水牛COCs在5%O2中IVM后进行IVF,受精卵分别在不同氧分压(5%02、20%02)条件下进行IVC，并收集囊胚,结果发现：低氧(5%O2)IVC(M5C5)组的胚胎发育到2-cell、 4-cell和8-cell的比率均显著高于常氧(20%O2) IVC (M5C20)组(72.38%vs 68.98%,62.32%vs 57.18%,49.65% vs 45.23%,P0.05),但两组的囊胚发育率差异不显著(27.24% vs 26.11%,P0.05)；免疫荧光分别检测两组囊胚中HIF1α和HIF2α的表达情况,结果表明：HIF1α有表达,而HIF2α几乎不表达；经Q-PCR分析发现：相对于M5C20组,M5C5组囊胚中HIF1α及调控其表达的相关基因(IGF1R、FOXO1)、糖代谢相关基因(GLU1、 LDHA、G6PD)、热休克因子(HSP70/90)和抗凋亡基因(Bcl2)的mRNA水平显著上调(P0.05),而促凋亡基因(Bax)的mRNA水平显著下调(P0.05)。TUNEL检测结果亦发现,M5C5组囊胚细胞的凋亡率显著低于M5C20组(3.89% vs 6.90%,P0.05)。上述结果表明：(1) HIF1α在水牛COCs的卵丘细胞和卵母细胞以及囊胚中均有表达,而HIF2α仅在水牛COCs的卵丘细胞中有表达； (2)低氧分压(5%02)条件有利于水牛COCs的体外成熟及其IVF早期胚胎的体外发育；(3)在低氧分压(5%02)条件下,FSH和IGF1可能通过PI3K/AKT途径调节低氧通路的核转录因子HIF1α稳定表达,稳定表达的HIF1α可能又通过促进无氧糖酵解有利于水牛卵丘细胞和早期胚胎适应低氧环境,促进水牛卵丘细胞的扩展与增殖、上调发育相关基因的表达、抑制细胞的凋亡,进而促进水牛卵母细胞的成熟,并提高其早期胚胎的发育潜力。
[Abstract]:In vitro maturation of oocytes (In Vitro, Maturation, IVM) and cultured embryos in vitro (In Vitro Culture, IVC) is the key technology of embryo production in vitro. In recent years, although the two have made considerable progress, but the production of in vitro embryo developmental potential is still relatively low body endodermal. This study was aimed to study fetal hypoxia the partial pressure and influence on early embryonic development in vitro maturation of oocytes in vitro and buffalo, preliminary study of hypoxia inducible factor (Hypoxia Inducible, Factor, HIF) through the metabolic pathways of buffalo oocyte maturation and early embryo development in vitro molecular mechanism, in order to simulate in vivo oocyte maturation and embryo development the physicochemical environment to optimize the oocytes and early embryos in vitro culture system in the gas phase, so as to improve the efficiency of buffalo culture in vitro maturation of oocyte and embryo in vitro. The first study The oxygen partial pressure on the impact of buffalo oocyte maturation in vitro, and to explore the low oxygen partial pressure regulated by HIF pathway and effects of glucose metabolism in hypoxia buffalo oocyte in vitro maturation mechanism. Results: Buffalo OCCC (Cumulus-Oocyte Complexes, COCs) at different oxygen partial pressure (2%O2,3.5%O2,5%o2,6.5%O2) in vitro maturation, compared with normoxia (20%02) group, 5%02 group and 6.5%02 on the maturation rate of oocytes had no significant effect (50.73% vs 55.31%, 49.58% vs 55.31%, P0.05), and the maturation rate of oocytes from group 3.5%02 and 2%02 were significantly decreased (21.22% vs 55.31%, vs55.31% 29.71%, P0.05); and normoxia (20%02) group, 2%02,3.5%02,5%02 group, COCs cumulus cell expansion index increased significantly (2.84 vs 2.70,2.88 vs 2.70,2.89 vs 2.70, P0.05), and 6.5%02 group of COCs had no significant effect on cumulus expansion index (2.74 vs 2.70, P0.05). The buffalo COCs in 5%02 and 20%O2 during maturation after in vitro fertilization (In Vitro, Fertilization, IVF) of embryos in 20%02 cultured in vitro. The results showed that: compared with normoxia (20%02) group, COCs (5%02) under the condition of hypoxia in IVM group significantly to improve the IVF 8-ce11 early embryo cleavage rate and blastocyst rate (44.88% vs38.47%, 26.81%vs 15.60%, P0.05). The HIF1 alpha and HIF2 alpha immunofluorescence assay results showed that: in the former IVM of buffalo COCs and cumulus cells at different oxygen partial pressure (5%O2,20%02) expression of IVM after cumulus cells were HIF1 and alpha HIF2 a condition; while in IVM after oocyte expressed HIF1 alpha, alpha HIF2 almost no expression. Further real-time fluorescence quantitative PCR (Real-time Quantitative PCR, Q-PCR) analysis, results showed that: (5% 02) in the hypoxic environment in vitro maturation of water Cattle COCs, increased COCs in cumulus cells HIF1 alpha, alpha HIF2, FSH receptor (FSHR), IGF1 receptor (IGF1R), FOXO1, VEGF and its receptors (VEGFR1, VEGFR2), glucose metabolism related genes (GLUT1, LDHA, G6DP), heat shock factor (HSP70, HSP90) and five poly ring -3 (Ptx3) mRNA protein level (P0.05), and significantly reduced the prostaglandin endoperoxide synthase -2 (Ptgs2) and apoptosis related gene Bax and Bc12 mRNA (P0.05).TUNEL test results also found that Buffalo COCs in hypoxia (5%02) maturation in vitro, can significantly reduce the apoptosis of cumulus cells the rate (21.84% vs 30.87%, P0.05). Then, discusses the low oxygen partial pressure (5%02) on the impact of buffalo early embryos in vitro development ability of cultured in vitro, and regulatory pathways to hypoxia and HIF pathway via anaerobic glycolysis mechanism of early embryonic development of buffalo were studied. In the buffalo COCs 5% IVF IVM O2, the fertilized eggs respectively at different oxygen partial pressure (5%02,20%02) of IVC, and collecting blastocysts, results showed that hypoxia (5%O2) IVC (M5C5) group of embryonic development to 2-cell, 4-cell and 8-cell ratio were significantly higher than those in normoxia (20%O2) group (IVC (M5C20) 72.38%vs 68.98%, 62.32%vs 57.18%, 49.65% vs 45.23%, P0.05 two), but the blastocyst development rate had no significant difference (27.24% vs 26.11%, P0.05); the results showed that immunofluorescence was used to detect the expression situation, two groups of blastocysts of HIF1 alpha and HIF2 alpha HIF1 alpha HIF2 alpha expression, while almost no expression by; Q-PCR analysis showed that: compared with M5C20 group, the related gene HIF1 and regulation of M5C5 expression in the blastocyst group (IGF1R, FOXO1), glucose metabolism related genes (GLU1, LDHA, G6PD), heat shock factor (HSP70/90) and anti apoptosis gene (Bcl2) of mRNA was significantly increased (P0.05), and apoptosis (Bax) mRNA gene Levels were significantly reduced (P0.05).TUNEL test results also found that the rate of blastocyst cell apoptosis in M5C5 group was significantly lower than that of M5C20 group (3.89% vs 6.90%, P0.05). The results showed that: (1) HIF1 in Buffalo COCs cumulus cells and oocytes and blastocysts were expressed, and HIF2 alpha only in Buffalo COCs cumulus cells; (2) low oxygen pressure (5%02) growth conditions in favor of buffalo COCs IVF in vitro maturation and early embryo in vitro; (3) in the low oxygen partial pressure (5%02) conditions, the expression of nuclear transcription factor HIF1 alpha stable FSH and IGF1 pathway regulated by PI3K/AKT pathway of hypoxia, stability the expression of HIF1 may be through promoting anaerobic glycolysis in favor of buffalo cumulus cells and early embryos adapt to the hypoxic environment, promote the expansion and proliferation of buffalo cumulus cells, up-regulated expression of genes related to growth, inhibit cell apoptosis, and promote buffalo eggs The maturation of the mother cell and the development potential of its early embryos.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.83
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