蛋白激酶Cα在小鼠孤雌及四倍体胚胎早期发育过程中的分布和作用

为了观察蛋白激酶Cα(protein kinase Cα,PKCα在昆明白小鼠受精卵、孤雌激活和四倍体胚胎早期发育阶段的亚细胞定位和致密化进程中的表达变化,本实验利用免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,对受精卵、孤雌激活和四倍体胚胎早期发育阶段PKCα的表达进行了定位观察,并利用Western blot对三组胚胎致密化进程中PKCα的表达进行定量分析.结果显示,PKCα在上述三组胚胎发育的2-细胞期至囊胚期均有表达,虽然不同胚胎PKCα的分布在同一发育阶段存在差异,却表现出在各胚胎期主要分布于卵裂球核染色质内,以及在胚胎致密化开始,PKCα在卵裂球连接处发生重新分布的共同特点.此外,三组胚胎PKCα在致密化进程中的表达呈升高趋势,即致密化后的表达高于敛密化前.结果表明,PKCct对胚胎致密化的调节具有重要作用,其在8-细胞/4-细胞期的重新分布是胚胎进入桑椹胚期的必然事件,是胚胎致密化的前提,同时伴随蛋白表达增多.此外,PKCα在囊胚期发生了植入前的第二次重新分布.PKCα在三组胚胎各发育阶段表达情况各不相同,它对小鼠胚胎发育的影响体现在整个早期发育阶段.PKCα在小鼠受精卵早期发育阶段的两次重新分布可能与在致密化开始时启动的细胞黏附事件存在某种必然联系.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.     

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.     

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.     

p62/p56 are cortical granule proteins that contribute to formation of the cortical granule envelope and play a role in mammalian preimplantation development

The purpose of this study was to identify specific cortical granule protein(s) that form the cortical granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the cortical granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster cortical granules at fertilization and contribute to formation of the cortical granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the cortical granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as cortical granule proteins that contribute to the formation of the cortical granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.     

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.     

Embryonic cytoplasmic extracts rescue murine androgenones to the blastocyst stage.

Androgenones (paternally derived genome) show a significant inability to form a blastocoele cavity. Eighty percent of these embryos die or arrest at earlier stages. Factor(s) from both normal and parthenogenetic late preimplantation embryos injected into each blastomere of androgenetic 4-cell stage can rescue more than twice as many to the blastocyst stage (47.2% versus 19.2% for non-injected androgenones). This factor(s) becomes available beginning at the 4-cell stage and is titratable. Injected total cytoplasmic mRNA will also cause a rescue response. Isolating this specific factor message(s) will permit the eventual cloning of possibly the earliest parentally imprinted gene(s) expressed during development.     

Early patterning of cloned mouse embryos contributes to post-implantation development

Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.