卵泡刺激素及戊酸雌二醇对小鼠卵母细胞体外成熟的影响
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摘要
背景及目的
未成熟卵体外成熟培养技术(in vitro maturation, IVM)是指不经超促排卵或应用少量促性腺激素(gonadotropin, Gn),从卵巢中获取未成熟卵,经过适宜的条件进行体外成熟培养,使卵子成熟达到减数分裂中期(MetaphaseⅡ,MII)并具备受精能力。在临床上,部分行体外受精患者的卵子取出后未达到MII期,如果不对这些卵子进行体外成熟培养,则其全部不能受精,将给患者造成极大的经济损失和精神创伤。另外,部分卵巢高反应患者(如多囊卵巢综合症患者)因对Gn敏感,促排卵时易出现卵巢过度刺激综合症(ovary hyperstimulation syndrome,OHSS)等严重并发症。如果研究一个优良的体外培养体系,可将体外受精周期取出的生发泡破裂期(germinal vesicle breakdown,GVBD)、减数分裂前期(MetaphaseⅠ,MI)卵子经体外成熟培养后再行体外受精,可减少取消周期,提高取卵周期的妊娠率。对卵巢高反应患者,则可以减少Gn的应用及OHSS等严重并发症的发生。虽然卵母细胞能够在体外培养系统中发育成熟并受精,胚胎移植后获得成功妊娠,但其体外成熟率、受精率及妊娠率仍较低。近年来,为了提高未成熟卵母细胞体外培养的成熟率和质量,大量学者致力于研究在体外培养系统中添加各种物质,希望可以改善培养微环境进而促进未成熟卵母细胞的体外成熟。卵泡刺激素(follicle stimulating hormone, FSH)是卵泡发育必须的激素,在卵泡发育晚期,雌二醇与卵泡刺激素有协同作用,因此许多学者研究在卵母细胞体外培养系统中添加卵泡刺激素及雌二醇来促进卵母细胞的体外成熟,但其添加浓度尚无定论。本实验研究卵泡刺激素、戊酸雌二醇对小鼠未成熟卵母细胞体外成熟、受精及卵裂能力的影响。选择最适的卵泡刺激素、戊酸雌二醇浓度,优化IVM培养体系。
材料与方法
1.研究对象
8～12周龄雌性昆明小白鼠30只,体重25～30克,用于获取未成熟卵母细胞；14～18周龄雄性昆明小鼠15只,体重35～40克,用于获取精子。
2.研究方法
对处于动情前期的雌性小鼠进行超促排卵,48小时后颈椎脱臼法处死小鼠,取出双侧卵巢,在解剖显微镜下用4号半注射器针头刺破半透明的卵泡,收集未成熟卵母细胞。将收集到的形态较好的未成熟卵母细胞随机分为6组,在不添加激素的基础培养液中以及分别添加10OIU/ml FSH、50IU/ml FSH、1μg/ml戊酸雌二醇、10μg/ml戊酸雌二醇、100μg/ml戊酸雌二醇的培养液中培养24小时后,观察各组卵母细胞的成熟情况。对已成熟的卵母细胞进行体外受精,观察卵母细胞受精情况。受精后继续培养48～72小时,观察其卵裂情况。比较各组的成熟率、受精率和卵裂率。
3.统计学方法
应用SPSS 13.0软件包进行统计学分析。结果以百分率表示,率的比较采用X2检验。以α=0.05为检验水准。
结果
1.各浓度FSH组及各浓度戊酸雌二醇组与对照组相比,成熟率(GVBD率、MII率)及卵裂率的差别均无统计学意义(P>0.05)。
2lOIU/ml FSH组、50IU/ml FSH组以及10μg/ml戊酸雌二醇组的受精率分别为41.18%、40%、42.86%,均显著高于对照组(19.44%),差异有统计学意义(P<0.05)；1μg/ml戊酸雌二醇组、100μg/ml戊酸雌二醇组与对照组相比,受精率的差别均无统计学意义(P>0.05)。
3.各浓度FSH组之间,以及各浓度戊酸雌二醇组之间,成熟率(GVBD率、MII率)、受精率及卵裂率的差别均无统计学意义(P>0.05)。
结论
1.在小鼠体外成熟培养液中添加50IU/ml FSH,均能促进小鼠卵母细胞胞浆的成熟。
2.在小鼠体外成熟培养液中添加10μg/ml戊酸雌二醇,可促进小鼠卵母细胞胞浆的成熟。
Background and Objective
In vitro maturation (IVM) is to conduct a proper condition of in vitro culture to immature eggs so that oocytes reach maturity and have fertilization ability as Mll, while without the application of a small amount of induced ovulation or gonadotropin (Gn). IVM can effectively avoid the application of super-ovulation drugs which may lead to breast, ovarian or other hormone-dependent cancers incidences and potential risk of ovarian hyperstimulation syndrome (OHSS) and other serious complications. It also has obvious advantages such as simplifying treatment, reducing treatment time, decreasing the mental burden of patients and lowering treatment costs. In addition, IVM can be combined with freezing, IVF-ET, ICSI and other technologies in order to pertain reproduction insurance and solve the source of oocytes for premature ovarian failure (POF) patients, and young patients who were operated ovariectomy because of surgery, radiotherapy, chemotherapy and need to preserve the reproductive capacity, as well as other situations of childbearing age women. Moreover, IVM technology can effectively supply oocytes used for basic medical research, and can avoid the ethical disputes. Therefore, IVM could be studied and applied more by scientists all over the world. Although the oocytes can mature and fertilized under in vitro maturation and fertilization system, and can attain the success of pregnancy after fertilized, the maturation, fertilization and pregnancy rates are still low. In recent years, in order to improve the immature oocytes in vitro maturation rate and quality, a large number of scholars have been working on in vitro culture system by adding various substances to improving the microenvironment and thus promote the possibilities of immature oocytes in vitro maturation. Follicle stimulating hormone (FSH) is necessary for follicular growing. In the later phase of the growth of follicular, estradiol and FSH are synergized. Therefore, lots of scholars researched adding follicle stimulating hormone and estradiol to the IVM system to promote the mature of oocytes, but its mechanism and the concentrations are still inconclusive. We researched the effect of Follicle Stimulation Hormone (FSH) and pentanoic acid estradiol on mouse immature oocytes maturation and to examine the capacity of in vitro-matured oocytes underwent in vitro fertilization and merogenesis. Choose the most proper concentration of FSH and pentanoic acid estradiol, and promote the IVM system.
Materials and methods
1 Research Object:
30 female Kuming mice of 8～12 week old and 15 male Kunming mice of 14～18 week old. We use female mice to obtain immature oocytes and use male mice to obtain sperm.
2 Research methods:
Conducted superovulation on the 8-12 week old female Kuming mice, and killed them after 48 hours by cervical dislocation. Acquired their ovarian and pierced the follicles with 41/2 translucent syringe needle to collect immature oocytes. Immature oocytes were classified as six groups:matured for 24h in the basal culture fluid without hormone and the culture fluid added with 1μg/ml pentanoic acid estradiol, 10μg/ml,100μg/ml pentanoic acid estradiol, and 10IU/mL and 50IU/mL FSH, respectively. The maturation of oocytes in each groups were observed. The matured oocytes were then inseminated in-vitro;and observe the capability of fertilization. Cultivated the inseminated oocytes for another 48～72 hours, and then observed their cleavage. The maturation, fertilization and cleavage percentages were compared byχ2 test.
3 Statistic analysis:
Statistical analysis used SPSS 13.0 software package. Percentage was used to express results.χ2 test was used to compare rates. Tookα= 0.05 as test standard.
Results
1. Compared with the groups without hormone, the groups which added with FSH and pentanoic acid estradiol of different concentration had similar percentage of undergoing GVBD, similar percentage of MⅡand similar percentage of cleavage. The differences between them had no statistical significance (P>0.05).
2. Fertilization rate in groups which added with 10IU/ml FSH,50IU/ml FSH and 10μl/ml pentanoic acid estradiol, were 42.86%,41.18% and 40%, respectively. These rates were all higher than that in control group(19.44%). The differences between them had statistics significance (P<0.05). Compared with the groups without hormone, the fertilization rate in groups which added with 1μl/ml and 100μl/ml pentanoic acid estradiol had similar percentage of cleavage. The differences between them had no statistical significance (P>0.05).
3. The percentage of GVBD, MⅡand cleavage between groups which treated by FSH and pentanoic acid estradiol of different concentrations were similar. The differences between them had no statistical significance (P>0.05).
Conclusion
1. The addition of 10IU/ml and 50IU/ml FSH in the mouse IVM culture solution could promote the mature of mouse oocyte cytoplasm.
2. The addition of 10μg/ml pentanoic acid estradiol in the mouse IVM culture solution could promote the mature of mouse oocyte cytoplasm.
未成熟卵体外成熟培养技术(in vitro maturation, IVM)是指不经超促排卵或应用少量促性腺激素(gonadotropin, Gn),从卵巢中获取未成熟卵,经过适宜的条件进行体外成熟培养,使卵子成熟达到减数分裂中期(MetaphaseⅡ,MII)并具备受精能力。在临床上,部分行体外受精患者的卵子取出后未达到MII期,如果不对这些卵子进行体外成熟培养,则其全部不能受精,将给患者造成极大的经济损失和精神创伤。另外,部分卵巢高反应患者(如多囊卵巢综合症患者)因对Gn敏感,促排卵时易出现卵巢过度刺激综合症(ovary hyperstimulation syndrome,OHSS)等严重并发症。如果研究一个优良的体外培养体系,可将体外受精周期取出的生发泡破裂期(germinal vesicle breakdown,GVBD)、减数分裂前期(MetaphaseⅠ,MI)卵子经体外成熟培养后再行体外受精,可减少取消周期,提高取卵周期的妊娠率。对卵巢高反应患者,则可以减少Gn的应用及OHSS等严重并发症的发生。虽然卵母细胞能够在体外培养系统中发育成熟并受精,胚胎移植后获得成功妊娠,但其体外成熟率、受精率及妊娠率仍较低。近年来,为了提高未成熟卵母细胞体外培养的成熟率和质量,大量学者致力于研究在体外培养系统中添加各种物质,希望可以改善培养微环境进而促进未成熟卵母细胞的体外成熟。卵泡刺激素(follicle stimulating hormone, FSH)是卵泡发育必须的激素,在卵泡发育晚期,雌二醇与卵泡刺激素有协同作用,因此许多学者研究在卵母细胞体外培养系统中添加卵泡刺激素及雌二醇来促进卵母细胞的体外成熟,但其添加浓度尚无定论。本实验研究卵泡刺激素、戊酸雌二醇对小鼠未成熟卵母细胞体外成熟、受精及卵裂能力的影响。选择最适的卵泡刺激素、戊酸雌二醇浓度,优化IVM培养体系。
材料与方法
1.研究对象
8～12周龄雌性昆明小白鼠30只,体重25～30克,用于获取未成熟卵母细胞；14～18周龄雄性昆明小鼠15只,体重35～40克,用于获取精子。
2.研究方法
对处于动情前期的雌性小鼠进行超促排卵,48小时后颈椎脱臼法处死小鼠,取出双侧卵巢,在解剖显微镜下用4号半注射器针头刺破半透明的卵泡,收集未成熟卵母细胞。将收集到的形态较好的未成熟卵母细胞随机分为6组,在不添加激素的基础培养液中以及分别添加10OIU/ml FSH、50IU/ml FSH、1μg/ml戊酸雌二醇、10μg/ml戊酸雌二醇、100μg/ml戊酸雌二醇的培养液中培养24小时后,观察各组卵母细胞的成熟情况。对已成熟的卵母细胞进行体外受精,观察卵母细胞受精情况。受精后继续培养48～72小时,观察其卵裂情况。比较各组的成熟率、受精率和卵裂率。
3.统计学方法
应用SPSS 13.0软件包进行统计学分析。结果以百分率表示,率的比较采用X2检验。以α=0.05为检验水准。
结果
1.各浓度FSH组及各浓度戊酸雌二醇组与对照组相比,成熟率(GVBD率、MII率)及卵裂率的差别均无统计学意义(P>0.05)。
2lOIU/ml FSH组、50IU/ml FSH组以及10μg/ml戊酸雌二醇组的受精率分别为41.18%、40%、42.86%,均显著高于对照组(19.44%),差异有统计学意义(P<0.05)；1μg/ml戊酸雌二醇组、100μg/ml戊酸雌二醇组与对照组相比,受精率的差别均无统计学意义(P>0.05)。
3.各浓度FSH组之间,以及各浓度戊酸雌二醇组之间,成熟率(GVBD率、MII率)、受精率及卵裂率的差别均无统计学意义(P>0.05)。
结论
1.在小鼠体外成熟培养液中添加50IU/ml FSH,均能促进小鼠卵母细胞胞浆的成熟。
2.在小鼠体外成熟培养液中添加10μg/ml戊酸雌二醇,可促进小鼠卵母细胞胞浆的成熟。
Background and Objective
In vitro maturation (IVM) is to conduct a proper condition of in vitro culture to immature eggs so that oocytes reach maturity and have fertilization ability as Mll, while without the application of a small amount of induced ovulation or gonadotropin (Gn). IVM can effectively avoid the application of super-ovulation drugs which may lead to breast, ovarian or other hormone-dependent cancers incidences and potential risk of ovarian hyperstimulation syndrome (OHSS) and other serious complications. It also has obvious advantages such as simplifying treatment, reducing treatment time, decreasing the mental burden of patients and lowering treatment costs. In addition, IVM can be combined with freezing, IVF-ET, ICSI and other technologies in order to pertain reproduction insurance and solve the source of oocytes for premature ovarian failure (POF) patients, and young patients who were operated ovariectomy because of surgery, radiotherapy, chemotherapy and need to preserve the reproductive capacity, as well as other situations of childbearing age women. Moreover, IVM technology can effectively supply oocytes used for basic medical research, and can avoid the ethical disputes. Therefore, IVM could be studied and applied more by scientists all over the world. Although the oocytes can mature and fertilized under in vitro maturation and fertilization system, and can attain the success of pregnancy after fertilized, the maturation, fertilization and pregnancy rates are still low. In recent years, in order to improve the immature oocytes in vitro maturation rate and quality, a large number of scholars have been working on in vitro culture system by adding various substances to improving the microenvironment and thus promote the possibilities of immature oocytes in vitro maturation. Follicle stimulating hormone (FSH) is necessary for follicular growing. In the later phase of the growth of follicular, estradiol and FSH are synergized. Therefore, lots of scholars researched adding follicle stimulating hormone and estradiol to the IVM system to promote the mature of oocytes, but its mechanism and the concentrations are still inconclusive. We researched the effect of Follicle Stimulation Hormone (FSH) and pentanoic acid estradiol on mouse immature oocytes maturation and to examine the capacity of in vitro-matured oocytes underwent in vitro fertilization and merogenesis. Choose the most proper concentration of FSH and pentanoic acid estradiol, and promote the IVM system.
Materials and methods
1 Research Object:
30 female Kuming mice of 8～12 week old and 15 male Kunming mice of 14～18 week old. We use female mice to obtain immature oocytes and use male mice to obtain sperm.
2 Research methods:
Conducted superovulation on the 8-12 week old female Kuming mice, and killed them after 48 hours by cervical dislocation. Acquired their ovarian and pierced the follicles with 41/2 translucent syringe needle to collect immature oocytes. Immature oocytes were classified as six groups:matured for 24h in the basal culture fluid without hormone and the culture fluid added with 1μg/ml pentanoic acid estradiol, 10μg/ml,100μg/ml pentanoic acid estradiol, and 10IU/mL and 50IU/mL FSH, respectively. The maturation of oocytes in each groups were observed. The matured oocytes were then inseminated in-vitro;and observe the capability of fertilization. Cultivated the inseminated oocytes for another 48～72 hours, and then observed their cleavage. The maturation, fertilization and cleavage percentages were compared byχ2 test.
3 Statistic analysis:
Statistical analysis used SPSS 13.0 software package. Percentage was used to express results.χ2 test was used to compare rates. Tookα= 0.05 as test standard.
Results
1. Compared with the groups without hormone, the groups which added with FSH and pentanoic acid estradiol of different concentration had similar percentage of undergoing GVBD, similar percentage of MⅡand similar percentage of cleavage. The differences between them had no statistical significance (P>0.05).
2. Fertilization rate in groups which added with 10IU/ml FSH,50IU/ml FSH and 10μl/ml pentanoic acid estradiol, were 42.86%,41.18% and 40%, respectively. These rates were all higher than that in control group(19.44%). The differences between them had statistics significance (P<0.05). Compared with the groups without hormone, the fertilization rate in groups which added with 1μl/ml and 100μl/ml pentanoic acid estradiol had similar percentage of cleavage. The differences between them had no statistical significance (P>0.05).
3. The percentage of GVBD, MⅡand cleavage between groups which treated by FSH and pentanoic acid estradiol of different concentrations were similar. The differences between them had no statistical significance (P>0.05).
Conclusion
1. The addition of 10IU/ml and 50IU/ml FSH in the mouse IVM culture solution could promote the mature of mouse oocyte cytoplasm.
2. The addition of 10μg/ml pentanoic acid estradiol in the mouse IVM culture solution could promote the mature of mouse oocyte cytoplasm.
引文
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