哺乳动物卵母细胞生发泡移植

生发泡(GV)移植是指将GV期卵母细胞的GV移入到去核的受体细胞(GV期卵母细胞、MII期卵母细胞或受精卵)透明带下,经融合形成一个重组卵的过程。GV移植对研究卵母细胞的细胞周期调控、成熟及受精时细胞核与细胞质之间的相互作用非常重要,可用于研究卵母细胞减数分裂异常和与年龄相关变化之间的关系及细胞质衰老与卵母细胞非整倍性之间的关系。现简要介绍了GV移植的基本程序,GV核体与胞质体的融合,重组卵的培养条件,重组卵成熟后的受精、人工激活和胚胎发育能力以及GV移植的意义。     

绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响

本研究旨在探讨次黄嘌呤 +dbcAMP或IBMX +dbcAMP对绵羊卵母细胞体外成熟的可逆性抑制作用 ,以及这种抑制作用对卵母细胞胚胎发育潜力的影响。自绵羊卵巢分离卵丘 -卵母细胞复合物进行体外培养 ,培养基中分别加入或不加入上述抑制剂。培养 6h后各组取部分卵母细胞固定染色检查卵母细胞核成熟情况 ;将其余的卵母细胞分别移入无抑制剂的成熟培养基中继续培养 18h后 ,再次检查各组卵母细胞核成熟情况 ,并进行体外受精和胚胎培养。结果表明 :次黄嘌呤 +dbcAMP或IBMX +dbcAM都分别能使 6 0 %以上的绵羊卵母细胞抑制在GV期。这种抑制是可逆性的 ,去除抑制剂后卵母细胞能恢复减数分裂 ,并加快由GVBD到MⅡ的成熟过程。各处理组受精率、卵裂率和囊胚发育率与对照组相比无显著性差异 ,表明卵母细胞的胚胎发育潜力没有受损。上述物质对卵母细胞成熟的可逆性抑制可用于研究卵母细胞成熟及其胚胎发育潜力的调节机制。     

绵羊卵母细胞体外核成熟和抑制及其对胚胎发育潜力的影响

本研究旨在探讨次黄嘌呤＋dbcAMP或IBMX＋dbcAMP对绵羊卵母细胞体外成熟的可逆性抑制作用,以及这种抑制作用对卵母细胞胚胎发育潜力的影响。自绵羊卵巢分离卵丘－卵母细胞复合物进行体外培养,培养基中分别加入或不加入上述抑制剂。培养6h后各组取部分卵母细胞固定染色检查卵母细胞核成熟情况;将其余的卵母细胞分别移入无抑制剂的成熟培养基中继续培养18h后,再次检查各组卵母细胞核成熟情况,并进行体外受精和胚胎培养。结果表明,次黄嘌呤＋dbcAMP或IBMS＋dbcAM都分别能使60％以上的绵羊卵母细胞抑制在GV期。这种抑制是可行性的,去除抑制剂后卵母细胞能恢复减数分裂,并加快由GVBD到MⅡ的成熟过程。各处理组肥精率,卵裂率和囊胚发育率与对照组相比无显著性差异,表明卵母细胞的胚胎发育潜力没有受损。上述物质对卵母细胞成熟的可逆性抑制可用于研究卵母细胞成熟及其胚胎发育潜力的调节机制。     

卵丘在卵母细胞成熟中的作用

卵丘是指在卵母细胞外周并与之进行代谢联系的颗粒细胞群;卵丘对于卵母细胞成熟有极其重要的作用。主要表现在卵丘参与维持卵母细胞减数分裂阻滞,诱导卵母细胞减数分裂恢复、支持卵母细胞细胞质的成熟。卵丘形态和卵丘扩展影响卵母细胞成熟。了解卵丘在卵母细胞成熟中的作用有助于帮助人们进一步揭示哺乳动物卵母细胞成熟的机制。     

卵母细胞透明带异常及其对助孕结局影响的研究进展

人类透明带(zona pellucida,ZP)是在卵泡发生过程中由卵母细胞和颗粒细胞共同分泌的由ZP1-ZP4四种糖蛋白分子组成的高度有序结构,它与卵母细胞的成熟、受精、胚胎发育及妊娠结局等预后紧密关联。许多生殖中心实验室发现有些患者的卵出现全部或部分的透明带异常,而且不同实验室发现的透明带异常类型各异。研究发现这些透明带异常与卵母细胞受精、胚胎发育及临床结局有一定的相关性。本文综述了关于透明带异常及其对卵母细胞受精、胚胎发育潜能和临床结局的影响的研究进展。     

THE MECHANISM OF SPERM HEAD TRANSFORMATION DURING MOUSE OOCYTE FERTILIZATION IN VITRO

用Ｈｏｅｃｈｓｔ３３３４２标记及氨银反应的方法在光镜和电镜水平上研究不同发育阶段的小鼠卵母细胞转化精核的能力。生发泡期卵母细胞质不能诱发精子组蛋白替代鱼精蛋白及精核解聚。生发泡破裂后,卵母细胞获得使精核解聚的能力,但直到卵母细胞完成成熟前,雄原核均不能形成。进一步研究表明,卵母细胞成熟过程中持续的蛋白质合成是雄原核形成所必需的,鱼精蛋白质磷酸化是精核解聚中的关键步骤。     

非人灵长类的体外受精和胚胎移植

非人灵长类的体外受精和胚胎移植是了解人类生殖机制,如卵的成熟调控,受精卵的成熟与分化,胚胎着床,控制某些遗传疾病以及保护珍稀灵长类和提高实验灵长类质量的有效途径。本文从非人灵长类卵的获取(包括超数排卵,非激素刺激动物取卵),精子处理(精液采集,冻存和精子获能),体外受精和胚胎移植、胚胎的冻存等方面介绍了有关研究概况和发展动态。     

卵母细胞和早期胚胎特异的皮质下母源效应复合体

从卵母细胞成熟后到胚胎合子基因组激活之前,基因转录处于沉默状态.在此期间,胚胎的生命活动主要受母源效应基因调控.受到研究材料和技术的限制,哺乳动物母源效应基因研究长期进展缓慢.近期研究发现,在小鼠(Mus musculus)卵母细胞和早期胚胎中特异存在皮质下母源效应复合体(SCMC).SCMC是由多种母源效应蛋白组成的巨型蛋白复合体,它是小鼠早期胚胎发育必需的.后续研究证明,SCMC不仅可以用于鉴定小鼠中新的母源效应基因,同时有助于其他母源效应基因作用机制的阐明,还可用于其他哺乳动物早期胚胎发育调控机制研究.此外,SCMC可能存在于人类中,其组分基因的突变与临床上一些未明原因的不孕症或出生后疾病相关.以上研究表明,SCMC可能是哺乳动物母源调控过程中的重要调节枢纽.目前SCMC已经成为哺乳动物母源调控研究的重要内容,相关研究明显推动了哺乳动物母源调控领域的发展.     

垂体浸出液诱发蟾蜍卵球成熟的实验分析

冬眠蟾蜍的长足卵母细胞,只需要与垂体促性腺激素作短暂的接触,在其细胞核尚未破裂以前,转入生理水培养,即可恢复成熟分裂,由第一次成熟分裂前期,发育到第二次成熟分裂中期。在垂体浸出液作用下,有核或去核的卵母细胞的细胞质中,都会出现促成熟的活性物质(MPF)。含有这种促成熟因子的微量卵质,一旦注入未经激素处理的卵球,即能诱发后者正常成熟。可是,在同一激素的作用下,卵母细胞细胞核(胚泡)的内含物,却不能诱发未经激素处理的卵球胚泡破裂,继续进行成熟分裂。卵母细胞的细胞质,既然可以不依赖于其细胞核的存在而形成促成熟的活性物质,而蛋白质合成的抑制剂又能妨障此活性物质的产生,这说明,激素导致卵母细胞胚泡破裂和继续减数分裂,所涉及的主要是翻译水平上的蛋白质合成问题。激素对卵母细胞细胞核的作用是间接的,是通过细胞质的活动予以调控的。     

哺乳动物卵泡卵母细胞发生的分子调控

哺乳动物卵泡卵母细胞发生的研究一直是发育生物学研究的重点之一。简要叙述了哺乳动物卵泡卵母细胞发生的一般过程,重点分析了原始生殖细胞向卵母细胞分化过程中gdf9、c-kti、BMP4及TGF家族关键基因的表达调控对卵母细胞发生的影响,以及卵母细胞与颗粒细胞间的相互调节作用,介绍了卵母细胞体外发生的最新研究进展及面临的难题等,为进一步研究原始生殖细胞向卵母细胞分化以及卵泡生长发育的机制提供了理论基础。     

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.     

Effects of in vitro maturation of monkey oocytes on their developmental capacity

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes.     

Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog

The oocyte of the domestic dog is unique from that of other mammalian species studied to date. Ovulation occurs either once or twice per year, with the oocyte released at the germinal vesicle stage, and then completing nuclear and cytoplasmic maturation within the oviduct under the influence of rising circulating progesterone. In vivo meiotic maturation of the bitch oocyte is completed within 48-72 h after ovulation, which is longer than 12-36 h required for oocytes from most other mammalian species. Due to these inherently novel traits, in vitro culture systems developed for maturing oocytes of other species have been found inadequate for maturation of dog oocytes. On average, only 15-20% of ovarian oocytes achieve the metaphase II stage after 48-72 h of in vitro culture. Thus far, no offspring have been produced in the dog (or other canids) by transferring embryos derived from in vitro matured oocytes. This review addresses current knowledge about dog reproductive physiology, specifically those factors influencing in vitro developmental competence of the oocyte. This summary lays a foundation for identifying the next steps to understanding the mechanisms regulating meiotic maturation and developmental competence of the dog oocyte.     

The equine oocyte: Factors affecting meiotic and developmental competence

There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. Mol. Reprod. Dev. 77: 651–661, 2010. © 2010 Wiley‐Liss, Inc.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.     

Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation. Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus expansion suggested progression of oocyte maturation. No differences were found in the expression patterns of analyzed genes, except for heat-shock-protein (Hsp) that was lower in in vivo matured oocytes collected shortly before ovulation. Oocytes collected at this time also had higher developmental ability measured as blastocyst rates (57.6%) after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5%). We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre-capacitation and the capacitation period, probably due to the former having matured in vivo. However, a precisely defined time for aspirating immature oocytes for subsequent in vitro development seems not to be crucial.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

In vitro-produced bovine embryos: dealing with the warts

Bovine in vitro embryo production is an inefficient process; while maturation and fertilization proceed apparently normally, the proportion of embryos reaching the transferable (blastocyst) stage is rarely over 40% and those that do reach this stage are often compromised in quality and competence. There is considerable evidence of a significant influence of follicular origin on oocyte developmental potential and it appears that once the oocyte is removed from the follicle its developmental capacity is capped. Evidence suggests that while culture conditions during bovine in vitro embryo production can impact somewhat the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. This paper highlights some of the problems associated with in vitro production of embryos and discusses some of the ways of overcoming these problems.