生长因子提高牦牛IVF、SCNT胚胎发育和卵母细胞冷冻适应性的机制研究

发布时间：2018-04-23 21:47

  本文选题：牦牛 + 生长因子　； 参考：《甘肃农业大学》2016年博士论文

【摘要】：牦牛作为青藏高原地区重要物种资源,通过辅助繁殖技术提高其繁殖性能对我国“丝绸之路经济带”畜牧业的发展至关重要。为了提高体外生产牦牛胚胎的质量,阐明生长因子对牦牛体外胚胎发育的调控作用,及其对细胞凋亡的影响,并试图通过生长因子改善牦牛卵母细胞和胚胎冷冻技术,本研究进行了以下实验并取得了一定成果。(1)采用Real-time PCR和间接免疫荧光方法检测COCs成熟及体外受精胚胎发育过程中EGF及EGFR表达动态,分析EGF对牦牛卵母细胞成熟率、胚胎发育能力、囊胚质量的影响,并检测细胞凋亡相关基因的表达变化。研究发现卵母细胞成熟和胚胎发育各个时期均可表达EGF和EGFR,且成熟卵母细胞的表达水平高于未成熟卵母细胞,囊胚的表达水平高于其他发育时期的胚胎,EGFR的表达水平高于EGF。加入EGF可以显著提高卵母细胞的成熟率、胚胎的发育能力和囊胚质量,最佳的作用浓度为100 ng·mL-1。EGF作用后成熟卵母细胞中促凋亡基因Bax表达显著降低(p?0.05),而抗凋亡基因Baxi的表达水平显著增加(p?0.05),囊胚中HSP70和Survivin的表达也可在100 ng·mL-1 EGF处理组中显著提高。结果表明牦牛卵母细胞成熟和胚胎发育过程中EGF和EGFR作为重要的自分泌或旁分泌因子,可通过调控Bax、Baxi、HSP70和Survivin等凋亡相关基因的表达提高牦牛卵母细胞和体外受精胚胎的发育能力。(2)运用Real-time PCR、WB和间接免疫荧光方法从基因和蛋白水平检测COCs成熟及体外受精胚胎发育过程中IGF-1及IGF-1R表达动态,同时在牦牛卵丘细胞体外培养、体外胚胎发育中加入不同浓度的IGF-1,分析卵丘细胞的凋亡率及凋亡相关基因的表达变化,及其对胚胎发育能力的影响。研究发现卵母细胞成熟和胚胎发育各个时期均可表达IGF-1和IGF-1R,成熟卵母细胞的表达水平高于未成熟卵母细胞,囊胚的表达水平高于其他发育时期的胚胎,IGF-1R的表达水平高于IGF-1、外源的IGF-1可以显著提高卵母细胞的成熟率、胚胎的发育能力和囊胚质量,浓度为100 ng·mL-1时,作用效果最佳。体细胞培养过程中IGF-1可以显著降低细胞凋亡率(p?0.05),在IGF-1浓度为100 ng·mL-1处理组中卵丘细胞的凋亡率最低,且Bax的表达水平最低,HSP70和Bcl-2的表达水平最高。研究结果表明,igf-1可调节卵丘细胞的hsp70、bax和bcl-2表达,降低细胞凋亡率,且igf-1在牦牛移植前胚胎发育过程中作为重要的生长因子,可以提高囊胚的形成和细胞增殖。(3)采集6月龄和24月龄牦牛睾丸组织,real-timepcr、wb和ihc方法从基因和蛋白水平分析牦牛睾丸发育过程中egf和egfr的表达变化;在牦牛冻精体外获能过程中加入不同浓度的igf-1,分析获能后精子的活性、评估不同处理组受精后胚胎的卵裂率,并采用real-timepcr、wb和间接免疫荧光方法从基因和蛋白水平检测igf-1对牦牛精子bax和bcl-2表达的变化。研究发现24月龄牦牛睾丸组织中egf和egfr的表达水平显著高于6月龄睾丸组织(p?0.05),egfr的水平高于egf,24月龄睾丸组织中表达更广,睾丸间质细胞、曲精小管肌上皮细胞、睾丸支持细胞、生殖细胞和精子均可表达egf和egfr。100ng·ml-1igf-1可以显著提高牦牛的精子活性(p?0.05),该处理组中受精后胚胎卵裂率也显著提高(p?0.05),而精子中bax基因的表达显著降低,bcl-2表达增加。综上表明生长因子可调控牦牛精子的生成和活性,从而促进后续胚胎的发育,且这种调控作用与细胞凋亡有关。(4)牦牛卵母细胞体外成熟过程中加入不同浓度的bmp6,分析卵母细胞成熟率、用成熟的卵母细胞生产克隆胚胎,并评估其发育能力,采用real-timepcr和间接免疫荧光方法分析2细胞胚胎和囊胚中h3k9ac,h3k18ac,和h3k9me3表达的变化,同时分析囊胚bax和bcl-2的表达。研究发现100ng·ml-1bmp6可以显著提高卵母细胞成熟率、克隆胚胎卵裂率和囊胚形成率(p?0.05),囊胚质量也有所提高。在bmp6处理组中,h3k9ac、h3k18ac、bcl-2基因表达增加、而bax、h3k9me3基因表达降低,h3k9ac,h3k18ac,和h3k9me3在2细胞克隆胚胎的表达差异更显著(p?0.05)。研究结果表明bmp6可以提高牦牛卵母细胞和后续克隆胚胎的发育潜能,其作用类似于其他osfs,且这种调节作用与其调控组蛋白修饰和细胞凋亡有关。(5)收集牦牛cocs,采用ct和ssv方法对其进行玻璃化冷冻,冷冻-解冻后分析不同处理组卵母细胞的成熟率和胚胎发育能力,采用real-timepcr、wb从基因和蛋白水平检测成熟卵母细胞、卵裂胚胎、和囊胚中hsp90的表达变化;同时在牦牛卵母细胞体外成熟过程中加入不同浓度的igf-1,分析卵母细胞的成熟率和CIRP的表达水平,将不同处理组成熟的卵母细胞采用CT法玻璃化冷冻,冷冻解冻后进行孤雌激活,分析胚胎的发育能力。研究发现CT和SSV冷冻组卵母细胞成熟率、卵裂率和囊胚率比较接近、其中成熟率和卵裂率显著低于对照组(p?0.05),HSP90的表达水平也低于对照组,而三组囊胚率和HSP90的表达水平比较接近。100 ng·mL-1 IGF-1可以提高成熟卵母细胞CIRP表达和卵母细胞的成熟率,冷冻-解冻后孤雌激活胚胎的卵裂率和囊胚率有所提高,但囊胚质量差异不显著。结果表明CT和SSV玻璃化冷冻方法对牦牛未成熟卵母细胞具有相同的冷冻效果,且其对发育能力的影响主要集中在成熟期到早期卵裂期,并与HSP90的表达水平有一定关联性,而卵母细胞成熟过程中IGF-1可以调控CIRP的表达,提高成熟卵母细胞对低温的适应能力,降低低温对卵母细胞损伤,从而提高后续发育能力。
[Abstract]:As an important species resource in the Qinghai Tibet Plateau, yak is very important to the development of the animal husbandry of the "Silk Road Economic Belt" in China through assisted reproduction technology. In order to improve the quality of the yak embryo in the production of yak in vitro, the regulation effect of growth factor on the development of yak in vitro embryo and its effect on the cell apoptosis are clarified. In order to improve yak oocyte and embryo cryopreservation through growth factors, the following experiments have been carried out and some results have been achieved. (1) Real-time PCR and indirect immunofluorescence were used to detect the expression of EGF and EGFR during the development of COCs and in vitro fertilization embryos. The maturation rate of yak oocytes and embryos were analyzed by EGF. The effects of developmental ability, blastocyst quality and the expression of apoptosis related genes were detected. The study found that EGF and EGFR could be expressed at all stages of oocyte maturation and embryo development, and the expression level of mature oocytes was higher than that of immature oocytes. The expression of blastocyst was higher than that of other developmental embryos, and EGFR expressed water. The oocyte maturation rate, embryo development ability and blastocyst quality were significantly higher than that of EGF., and the best action concentration was 100 ng. ML-1.EGF, the expression of Bax was significantly reduced (P? 0.05) in mature oocytes (P? 0.05), while the expression level of anti apoptotic gene Baxi increased significantly (P? 0.05), HSP70 and Survivi in blastocysts. The expression of N can also be significantly improved in the 100 ng. ML-1 EGF treatment group. The results show that EGF and EGFR are important autocrine or paracrine factors during the maturation and embryonic development of yak oocytes, which can improve the development of yak oocytes and in vitro fertilization embryos by regulating the expression of apoptosis related genes such as Bax, Baxi, HSP70 and Survivin. (2) Real-time PCR, WB and indirect immunofluorescence were used to detect the expression of IGF-1 and IGF-1R during the development of COCs and IVF embryos from gene and protein levels. At the same time, the culture of yak cumulus cells in vitro, and in vitro embryo development were added to different concentrations of IGF-1, and analyzed the apoptosis rate and the apoptosis related groups of the cumulus cells. The expression of IGF-1 and IGF-1R can be expressed at all stages of oocyte maturation and embryo development. The expression level of mature oocytes is higher than that of immature oocytes. The expression level of blastocyst is higher than that of other developmental embryos. The expression level of IGF-1R is higher than that of IGF-1, and the exogenous I is higher than that of the other developmental stages. GF-1 can significantly increase the maturation rate of oocytes, the embryonic development ability and the blastocyst quality, when the concentration is 100 ng. ML-1, the effect is the best. IGF-1 can significantly reduce the apoptosis rate (P? 0.05) in the process of somatic cell culture. In the IGF-1 concentration 100 ng. ML-1 treatment group, the apoptotic rate of the cumulus cells is the lowest, and the expression of Bax is the lowest. The expression level of HSP70 and Bcl-2 is the highest. The results show that IGF-1 can regulate the expression of HSP70, Bax and Bcl-2 in cumulus cells and reduce the rate of apoptosis. And IGF-1 can be used as an important growth factor in the development of yak embryo development, which can improve the formation and proliferation of blastocysts. (3) collect 6 month old and 24 month old yak testis tissue, real-t Imepcr, WB and IHC methods were used to analyze the expression of EGF and EGFR during the development of yak testis from gene and protein levels. In the process of obtaining energy in Yak frozen sperm, the activity of different concentrations of IGF-1 was added to analyze the sperm activity after the acquired energy, and the cleavage rate of different treatment groups after fertilization was evaluated, and the real-timepcr, WB and indirect immunofluorescence methods were used. The changes in the expression of Bax and Bcl-2 of yak sperm by IGF-1 were detected from gene and protein level. The expression level of EGF and EGFR in 24 month old yak testis was significantly higher than that of 6 month old testicular tissue (P? 0.05). The level of EGFR was higher than that of EGF, 24 month old in the testicular tissue, and in the Leydig cells, in the seminiferous tubules, and in the testicular branches. The expression of EGF and egfr.100ng / ml-1igf-1 could significantly increase the sperm activity of Yak (P? 0.05). The cleavage rate of the embryos after fertilization was significantly increased (P? 0.05) in the treatment group (P? 0.05), and the expression of Bax gene in the sperm was significantly reduced and the expression of Bcl-2 increased. The results showed that growth factor could regulate the production of yak sperm and the growth factor. Activity, thus promoting the development of subsequent embryos, and this regulatory role is related to cell apoptosis. (4) yak oocyte in vitro maturation in the process of adding different concentrations of BMP6, analysis of oocyte maturation rate, the use of mature oocytes to produce cloned embryos, and evaluate their development ability, using real-timepcr and indirect immunofluorescence method analysis. The changes in the expression of H3K9Ac, H3K18ac, and h3k9me3 in the 2 cell embryos and blastocysts, and the analysis of the expression of Bax and Bcl-2 in the blastocyst. The study found that 100ng ml-1bmp6 can significantly increase the oocyte maturation rate, clone embryo cleavage rate and blastocyst formation rate (P? 0.05), and the blastocyst quality also increased. In the BMP6 treatment group, H3K9Ac, H3K18ac, bcl-2 gene table The expression of Bax, h3k9me3, H3K9Ac, H3K18ac, and h3k9me3 in the 2 cell cloned embryos was more significant (P? 0.05). The results showed that BMP6 could improve the developmental potential of yak oocytes and subsequent cloned embryos. The effect was similar to that of other OSFs, and this regulation was related to the regulation of histone modification and cells. Apoptosis related. (5) collection of yak COCs, using CT and SSV methods to vitrification, freezing and thawing to analyze the maturation rate and embryonic development ability of different treatment group oocytes, real-timepcr, WB from gene and protein level detection of mature oocyte, cleavage embryo, and blastocyst of Hsp90 expression changes; at the same time in Yak eggs The mature rate of oocyte and the expression level of CIRP were analyzed by adding different concentrations of IGF-1 in the process of maternal cell maturation in vitro. The mature oocytes of different treatment groups were vitrified by CT method, parthenogenetic activation was carried out after freezing thawing, and the developmental ability of embryos was analyzed. The maturation rate and cleavage rate of the oocyte in CT and SSV cryopreservation group were studied. Compared with the blastocyst rate, the maturation rate and cleavage rate were significantly lower than that of the control group (P? 0.05), and the expression level of HSP90 was also lower than that of the control group. The three blastocyst rate and the expression level of HSP90 were close to.100 ng. ML-1 IGF-1, which could improve the CIRP expression and oocyte maturation rate of the mature oocyte, and the parthenogenetic activation of the embryos after freezing thawing. The cleavage rate and blastocyst rate increased, but the difference of blastocyst quality was not significant. The results showed that the CT and SSV vitrification methods had the same freezing effect on Yak immature oocytes, and the effects on the developmental ability mainly concentrated in the mature stage to the early cleavage stage, and had some correlation with the expression level of HSP90, and the oocyte was oocyte. In the process of maturation, IGF-1 can regulate the expression of CIRP, improve the adaptability of mature oocyte to low temperature, reduce the damage to oocyte at low temperature, and thus improve the following developmental ability.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823.85
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本文编号：1793764