人类卵母细胞和早期胚胎发育异常的遗传学研究进展

卵母细胞和早期胚胎的正常发育是获得健康后代的关键,遗传因素在卵母细胞成熟和早期胚胎发育过程中发挥重要作用。调控卵母细胞和早期胚胎发育的关键基因突变可导致卵母细胞成熟障碍、受精失败或早期胚胎发育停滞,进而导致不孕或早期流产等。随着全基因组测序和全外显子组测序技术的广泛应用,越来越多的致病基因突变被发现,为不孕患者提供了可靠的诊疗靶点。该文系统回顾了导致人类卵母细胞成熟、受精和早期胚胎发育异常的致病基因,以期促进其在辅助生殖遗传咨询中的应用。     

关于小鼠裸卵体外成熟培养系统的研究

获取高质量的体外成熟卵母细胞是成功进行人类体外受精、动物胚胎生产和克隆的关键。尽管大多数哺乳动物卵母细胞能够在体外自发完成细胞核成熟,但卵母细胞成熟质量远不如体内成熟卵母细胞,其受精后胚胎发育能力较差。目前认为,这可能是由于体外成熟的卵母细胞的胞质成熟不充分造成的。研究表明,卵母细胞体外成熟培养系统与受精率和囊胚发育率有强相关性。     

哺乳动物核移植中线粒体命运

线粒体是哺乳动物细胞中一种重要的产能、供能细胞器,与生长、发育、衰老和凋亡等多种细胞事件以及多种疾病有关.哺乳动物核移植中,供体细胞和受体卵胞质两种来源的线粒体在重构胚胎发育进程中的变化一直是科学家们研究的热点.对哺乳动物同种胚胎细胞核移植、同种体细胞核移植、异种核移植研究中线粒体的变化进行了综述.     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

核移植与线粒体

线粒体是哺乳动物的产能、供能细胞器,与生长、发育、衰老和凋亡等多种细胞事件及疾病有关。哺乳动物核移植可能导致克隆胚胎及后代中线粒体的杂合性,从而影响到个体的表型甚至导致线粒体疾病。文章阐明了哺乳动物中线粒体的生物学功能及遗传特性,并分析了核移植中供体细胞和受体卵胞质两种来源的线粒体在同种胚胎细胞核移植、同种及异种体细胞核移植重构胚发育进程中的变化以及可能影响线粒体杂合性的一些因素,对其可能导致的线粒体疾病及解决方法进行了简单的阐述。

     

线粒体与卵母细胞发育

卵子发育、成熟是一个复杂的过程, 细胞核成熟和细胞质成熟过程必须同步化, 才能保证卵子的正常受精和进一步的发育。作为细胞质内最重要的细胞器, 线粒体在卵子成熟过程中的分布的变化、氧化磷酸化产生ATP的能力以及线粒体ＤＮＡ的含量和拷贝数或转录水平对卵母细胞发育成熟有着重要的影响。因此, 对卵子成熟过程中线粒体的分布和功能状况及线粒体DNA的研究, 有利于进一步了解生殖生理, 并为解决辅助生殖技术中及克隆胚胎技术所面临的困难提供新的思路。     

表观遗传药物对体细胞核移植胚胎发育的促进作用

体细胞核移植(somatic cell nuclear transfer,SCNT)是利用卵母细胞胞质中的重编程物质对高度分化体细胞核进行重编程作用使其恢复全能性并发育为新个体的技术。在SCNT过程中,表观遗传修饰参与卵母细胞的重编程,如DNA甲基化修饰和组蛋白的翻译后修饰。这些重编程的异常修饰会对SCNT胚胎的发育产生不良影响。表观遗传药物,如DNA甲基转移酶抑制剂和组蛋白去乙酰基酶抑制剂,可改善表观遗传修饰的异常现象,促进体细胞核移植重构胚的重编程。该文对SCNT胚胎重编程过程中的异常表观遗传修饰以及近年来报道的表观遗传相关药物进行综述,并进一步探讨了这些药物对SCNT胚胎发育的促进作用。     

哺乳动物线粒体遗传和在体细胞克隆胚胎中的命运

线粒体是哺乳动物重要的细胞器之一,为细胞的生命活动提供能量.线粒体是除细胞核外唯一含有功能性基因组DNA的细胞器.由于线粒体在哺乳动物早期胚胎的发育中有多方面重要的作用,因此线粒体对体细胞克隆胚胎发育的影响成为体细胞克隆动物研究的热点.就线粒体的结构特点和遗传特性及其在同种、异种动物克隆早期胚胎发育过程中的命运以及可能的遗传机制进行综述.同时,也将比较注射异源线粒体后,线粒体在注射胚胎中的发育命运.     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

Osmotrophy in fossil protoctists and early animals

Summary

The role of Ca²⁺ in activation and early development of locust eggs was examined through measurement of ooplasmic Ca²⁺ levels before and after fertilization, and through experimental activation of unfertilized eggs. Ooplasmic pCa (i.e. the negative logarithm of Ca²⁺ activity) measured in intact eggs decreased from 5.35 before fertilization, to 4.77 and 3.00 by 1 day and 3 days after fertilization, respectively. pCa was also determined for samples of ooplasm collected by rupturing eggs under paraffin oil. The pCa was 5.10 in ooplasm isolated from unfertilized eggs, and 3.84 in ooplasm collected from eggs within 4 h of fertilization. Ooplasmic pCa remained between 3.97 and 3.12 from 1–6 days after fertilization. Since a decline in pCa indicates an increase in ooplasmic Ca²⁺ activity, the data suggest that regulation of ooplasmic Ca²⁺ during post-fertilization development involves release of Ca²⁺ from internal stores. Experimental egg activation was examined in eggs dissected from the oviducts before fertilization and incubated on moist filter paper. Some eggs were first immersed in experimental solutions for 30–60 minutes before incubation. The presence of an embryo 2 or 4 days after fertilization or experimental treatment was used as an indicator of egg activation. Activation occurred in 92% and 12% of fertilized and untreated eggs, respectively. The percentage of unfertilized eggs which activated increased to 47% if eggs were soaked 30–60 minutes in physiological saline, and to as much as 65%-68% if eggs were injected with Ca²⁺ buffers or if a Ca²⁺ action potential was evoked. Up to 36% and 42% of unfertilized eggs activated after incubation in Ca²⁺-free salines or in the presence of the Ca²⁺-channel blocker Cd²⁺, respectively. Taken together, the results suggest that entry of external Ca²⁺ through voltage dependent channels increases the proportion of eggs which activate, but is not an absolute requirement for activation.     

Mitochondrial dysfunction in reproduction

The mitochondrial genome passes from one generation to the next by way of the egg's cytoplasm, so ordinarily an individual's mitochondrial DNA (mtDNA) is entirely derived from his or her mother. A potential mother has a finite number of eggs, or oocytes, all of which were formed when she herself was still a fetus, many years before she can conceive. The eggs are progressively depleted through childhood and her reproductive years at a much faster rate than is accounted for by ovulation. Up to a decade before the ultimate depletion of ovarian follicles (and hence oocytes) at or soon after menopause, cytoplasmic senility of the remaining eggs leads to physiological sterility; a phenomenon that is suspected of being mitochondrially based and has been termed the oopause. When ovulation and conception occur, oxidative phosphorylation and other mitochondrial functions of the fertilized oocyte are thought to be essential to the early embryo well before it implants in the uterus. The competition between follicles to deliver the oocyte that will be fertilized and which will found a new generation could also be mitochondrially based, but the mechanism remains to be elucidated. Increasing experience with the culture of human embryos in vitro is highlighting the importance of mitochondrial metabolism generally, and the avoidance of excessive generation of reactive oxygen species in particular. Paradoxes abound in the experimental data, however. Although natural selection operates on mitochondria only in females (and in extreme cases through the survival of their offspring), reproductive disturbance from mitochondrial mutations is most obvious in males, who typically have reduced sperm motility. mtDNA point mutations such as T8993G, which is serious enough to cause the death of infants from Leigh disease in the first few years of life, can carry through the female germ line apparently unhindered; yet mtDNA deletions that cause a less severe phenotype, and which typically manifest at a later age, are effectively blocked from transmission to offspring--a phenomenon in accord with early experimental observations that deleted mtDNA species are less common in cleaving embryos than in unselected preovulatory oocytes. A mitochondrial basis for ooplasmic aging has not been convincingly established, but the novel IVF-based practice of micro-aspiration and transfer of ooplasm from younger eggs to older eggs, which includes the transfer of mitochondria, appears in preliminary studies to have some clinical efficacy in rejuvenating fertility in older women.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43-48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.     

Mitochondria and reproduction

Mitochondria play a primary role in cellular energetic metabolism. They possess their own DNA, which is exclusively maternally transmitted. The relatively recent idea that mitochondria may be directly involved in human reproduction is arousing increasing interest in the scientific and medical community. It has been shown that the functional status of mitochondria contributes to the quality of oocytes and spermatozoa, and plays a part in the process of fertilisation and embryo development. Moreover, new techniques, such as ooplasm transfer, compromise the uniquely maternal inheritance of mitochondrial DNA, raising important ethical questions. This review discusses recent information about mitochondria in the field of human fertility and reproduction.     

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.