线粒体功能对辅助生殖技术的影响

卵母细胞质量与不孕、发育异常、囊胚数目的减少以及胚胎丢弃有关,而线粒体的功能是影响卵母细胞质量的重要因素,同时也是受精和获得健康后代的重要决定因素。越来越多的女性不孕症患者伴随着诸如糖尿病和肥胖等代谢疾病以及卵母细胞老化等问题,这类人群因为线粒体功能异常,在进行辅助生殖技术助孕的过程中往往也较难取得令人满意的妊娠结局。为了修复卵母细胞质量提高辅助生殖技术的成功率,核置换、极体移植等新技术引起了人们的重视。     

Electrical activation of rabbit oocytes increases fertilization and embryo development by intracytoplasmic sperm injection using sperm from deceased male

Purpose

We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm using ICSI.

Methods

Sperm were collected from a heterozygote GFP male rabbit 5 h after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B₂ medium for 4 days or transferred into the oviducts of recipient rabbits at the 2- or 4-cell stage.

Results

The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group, 23 % of the blastocysts expressed GFP, which was significantly greater than all other groups. However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally, embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered.

Conclusions

One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ISCI that are capable of normal fetal and kit development.     

肥胖引起的线粒体功能及抗氧化异常促进小鼠卵母细胞排卵后老化

目的: 探讨肥胖对小鼠卵母细胞体内老化、体外受精和胚胎发育的影响及其机制。方法: 分别获取人绒毛膜促性腺激素(HCG)注射后14 h、18 h和22 h的卵母细胞-卵丘颗粒细胞复合体,进行体外受精胚胎培养,采用Hoechst 33342和碘化丙啶双染检测卵丘颗粒细胞的凋亡率,JC-1和DCFH-DA分别检测卵母细胞中线粒体膜电位(MMP)和活性氧类(ROS)水平,并检测卵母细胞中ATP和谷胱甘肽(GSH)水平。结果: 刚排卵时肥胖小鼠卵母细胞GSH低于对照组(P<0.05),囊胚形成率、ATP、ROS和卵丘颗粒细胞凋亡率两组无显著差别(P>0.05);HCG注射后18 h,肥胖小鼠卵母细胞中ATP含量及囊胚细胞数目开始明显减少并低于对照组(P<0.05),而卵母细胞中ROS水平和卵丘颗粒细胞凋亡率则开始升高并高于对照组(P<0.05);HCG注射后22 h,肥胖小鼠卵母细胞MMP和体外受精囊胚形成率显著下降(P<0.05),并且低于对照组(P<0.05)。结论: 肥胖促进排卵后卵母细胞的老化,氧自由基对卵母细胞的损伤可能是引起母源老化不孕的重要原因。     

尾吊模型模拟微重力抑制小鼠卵母细胞的成熟

本文研究模拟微重力对小鼠卵母细胞成熟的影响,探讨尾吊模型模拟微重力研究雌性小鼠生殖功能的可行性。采用转壁式生物反应器模拟微重力体外培养小鼠卵母细胞,以及对小鼠进行旋转和尾吊处理,观察小鼠卵母细胞的成熟情况。结果表明:体外旋转培养和尾吊模型产生的模拟微重力条件下,小鼠卵母细胞的成熟受到抑制,异常率增加。表现为体外旋转培养小鼠卵母细胞的成熟率为8.93%,显著低于1g重力条件下的72.33%;尾吊模型小鼠卵母细胞的成熟率为14.54%,显著低于对照组的73.29%。与对照组比较,小鼠旋转模型对小鼠卵母细胞的成熟无明显影响,提示该旋转模型不适用于模拟微重力的研究。尾吊模型可适用于地面研究模拟微重力对母鼠繁殖机能的影响。     

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Objective::There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the fol...     

干细胞条件培养液对小鼠体外成熟卵母细胞发育潜能的影响

目的:探讨骨髓间充质干细胞(MSCs)条件培养液对小鼠体外成熟卵母细胞发育潜能的影响。方法:分离、培养小鼠MSCs,获得MSCs条件培养液;收集4~6周小鼠生发泡期(GV)卵母细胞,在MSCs条件培养液中体外成熟(IVM);同时部分小鼠促排处理,收集体内成熟卵母细胞;体外受精(IVF)后,比较受精率、4-细胞形成率及囊胚形成率差异;Hoechst33342染色后观察内细胞团(ICM)和滋养层细胞形成情况,计数内细胞团数。结果:IVM卵母细胞IVF后的受精率、4-细胞形成率和囊胚形成率及囊胚内细胞总数与体内成熟组比较,无显著差异(P0.05);IVM组各时期的胚胎形态正常,囊胚内细胞染色后的荧光图像与体内成熟组基本一致。结论:MSCs条件培养液能保持小鼠IVM卵母细胞良好的质量和发育潜能,是一种较理想的IVM培养体系。     

颗粒细胞与卵母细胞发育

颗粒细胞在卵母细胞发育、成熟中起着重要的营养及信号传导作用,同时卵母细胞对颗粒细胞的分化及发育也起着中心调节作用。研究颗粒细胞的基因表达改变可以对卵母细胞发育的微环境进行评估。颗粒细胞基因差异表达可能与卵母细胞质量有关,有可能通过对颗粒细胞基因表达谱的分析,寻找卵母细胞及胚胎选择的生物标记,以提高体外受精的受精率、胚胎种植率及临床妊娠率,降低流产率。     

两种冻融方法对小鼠成熟卵母细胞冷冻效果的研究

目的 探讨不同冷冻方法对成熟期(MⅡ)卵母细胞体外受精及胚胎发育能力的影响.方法 收集昆明小鼠MⅡ期卵母细胞,分别行玻璃化冷冻与程序化冷冻.解冻后比较两组复活率、受精率、卵裂率及囊胚形成率.结果 玻璃化冷冻法冷冻MⅡ期卵母细胞解冻后存活率显著高于程序化冷冻组的(80.1% vs.55.5%,P＜0.01),并且两组的囊胚形成率比较,差异有统计学意义(28.6% vs.19.4%,P＜0.05).结论 在进行卵母细胞冷冻保存时,玻璃化冷冻法冷冻效果好于程序冷冻法.     

ATP8基因在小鼠单个GV期卵母细胞中的表达和生物信息学分析

目的:探讨小鼠GV期卵母细胞线粒体中ATP合酶亚基8(ATP8)基因的表达情况及其生物信息学的分析。方法:应用挤压法从卵巢中分离获得生发泡期(germinal vesicle,GV)卵母细胞,用RT-PCR检测GV期单个卵母细胞中ATP8基因的表达;将GV期单个卵母细胞直接进行RT合成cDNA,然后再对回收产物构建克隆质粒并测序,最后用VECTOR NIT9.0、RNAdraw和Treeview等工具对测序结果进行生物信息学分析。结果:琼脂糖电泳结果表明ATP8基因在GV期卵母细胞中有表达;测序结果显示所测昆明小鼠ATP8序列与Genbank序列完全一致;mRNA二级结构图显示12种动物间的ATP8氨基酸序列和ATP8核酸序列有较大差异,其中不同品种小鼠间也有差异;不同物种ATP8核苷酸序列构建出的分子进化树中,小鼠与棕熊进化距离较远,与田鼠、家猫进化距离相对较近。结论:小鼠GV期卵母细胞特异表达的ATP8基因与卵母细胞的正常发育成熟相关。