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1.
This study reports establishment of an in vitro culture system for E5.5 mouse embryos that supports development to the gastrulation stage and allows the use of experimental approaches to study gastrulation during mouse embryogenesis. Recent experiments suggest that the extraembryonic tissues may play a critical role for gastrulation from as early as E5.5. To apply whole embryo culture to E5.5 embryos and analyze gastrulation, it is essential to optimize the conditions so that most of the embryos develop to the gastrulation stage in culture. For this purpose, we established a protocol in which embryos were isolated using micromanipulator and cultured with 50-75% rat serum. Although cultured embryos tended to grow a larger extraembryonic portion, more than 80% of them developed the primitive streak and induce mesoderm, which corresponds to the mid-streak stage.  相似文献   

2.
To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.  相似文献   

3.
Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.  相似文献   

4.
The properties of androgenetic cells and their ability to proliferate and differentiate were examined in post-midgestation chimeras. In several tissues, namely the brain, cardiac muscle, skeletal muscle and intestinal epithelium, the rate of proliferation of androgenetic cells was higher than that of normal cells in day 13 embryos. This higher rate of proliferation was however less pronounced by day 17–18 of development. It is possible that IGF2, a major growth factor regulating fetal growth, could play a role in the increased proliferation of androgenetic cells. Igf2 is also an imprinted gene that is expressed only when inherited paternally. Indeed, in the smooth muscle, cartilage and intestinal epithelium, patches of androgenetic (ag) cells exhibited higher levels of IGF2 mRNA than neighbouring wild-type cells. Surprisingly, we also detected expression of Igf2 in ag cells of ectodermal origin; this gene is not normally expressed in this lineage. This expression was observed in the brain, epidermis and in the epithelium of the tongue. We attempted to confirm the identity and differentiation status of ag cells with the help of cell-type specific antibodies and lectins. Evidence for only one of the cell types analysed, i.e. the goblet cells of the gut, suggests a delay or aberrant differentiation of ag cells.  相似文献   

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6.
Several recent studies have proposed a model that the organization of the mouse blastocyst is determined by the pattern of early cleavages: the plane of first cleavage divides the two-cell embryo into embryonic (Em) and abembryonic (Ab) halves, while the timing of the second cleavages specifies which blastomere becomes the Em half. This model is still controversial because of conflicting observations in various studies. Here, we investigated the possibility that the difference between mouse strains contributed to the discrepancy of the findings of different experiments regarding the relationship between the first two cleavages and the blastocyst axial pattern. First, we showed by using a lipophilic, fluorescent tracer that the plane of the first cleavage bears no consistent spatial relationship to the Em-Ab axis of the blastocyst regardless of the genotypic background. Secondly, the order of the second cleavage does not correlate with the Em-Ab polarity of the blastocyst. This was demonstrated by tracing the lineage of the early- and later-dividing two-cell stage blastomeres in the whole embryo as well as by comparing the developmental potential of isolated early- and later-dividing blastomeres and chimeras made entirely of early- or later-dividing blastomeres. These results suggest that contrary to recent studies, the differences between the early- and later-dividing blastomeres of the two-cell embryo are not functionally evident and do not define the Em-Ab polarity of the blastocyst. The significance of these findings is discussed in relation to human assisted reproduction and preimplantation genetic diagnosis.  相似文献   

7.
8.
The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2-10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two-pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr(2+)-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr(2+)-induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.  相似文献   

9.
《遗传学报》2020,47(5):249-261
Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs) into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS) of Flk-1~(EGFP/ECFP) rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.  相似文献   

10.
Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight-cell embryo placed under the zona pellucida with a two-cell embryo, results in a difference in incorporation of 3H-uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight-cell embryo in a separate zona pellucida and a two-cell embryo. The incorporation of 3H-uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P < .02). A stage-specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P < .01) and another stage-specific protein band (48k) was found to be quantitatively different (more protein; P equals; .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.  相似文献   

11.
Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.  相似文献   

12.
BACKGROUND: Interleukin (IL)‐12 is a cytokine that can exert regulatory effects on T and NK cells. This study was designed to identify potential developmental and reproductive hazards associated with IL‐12p40 knockout in mice. METHODS: In the combined fertility and teratology study, female F0 C57/BL6 wild‐type control mice and female F0 C57/BL6 IL‐12p40 homozgyous knockout mice were assessed for estrous cyclicity, sperm, and mating parameters. Pregnant females were euthanized on gestation day (GD) 18 and their fetuses were assessed for external, visceral, and skeletal development. In the peri and postnatal development study, the F1 wild‐type control and IL‐12p40 knockout mice were assessed for developmental landmarks, sexual development, passive avoidance, motor activity, and morris water maze. RESULTS: The IL‐12p40 knockout male mice exhibited decreased testis weights when compared to the wild‐type control group; however, this finding was not considered adverse, as it had no apparent functional effects on mating, fertility, and pregnancy rates or sperm motility. The IL‐12p40 knockout group exhibited effects on estrous cycle length, passive avoidance, morris water maze, and motor activity when compared to the wild‐type control group. However, since these findings were small in magnitude, transient and/or had no apparent effects on subsequent growth and development, they were not considered adverse. CONCLUSIONS: These results demonstrate that although IL‐12p40 homozygous knockout in mice exhibited effects on developmental and reproductive parameters, these effects were relatively minor and were not considered adverse. Birth Defects Res (Part B) 92:102–110, 2011. © 2011 Wiley‐Liss, Inc.  相似文献   

13.
At the 16 cell stage, three kinds of borealis–laevis and eight kinds of laevis–laevis chimeric embryos were produced by replacing a particular blastomere of albino embryos of Xenopus laevis with that of wild-type embryos of X. borealis or X. laevis , and then leaving the embryos to develop into frogs.
In the borealis–laevis chimera frogs, we found that all the melanized cells (retinal pigment cells and melanophores) were derived from a transplanted wild-type blastomere with a nuclear marker of X. borealis and that all the albino-mutant cells derived from the host did not become melanized. Thus, retinal pigment cells and melanophores differentiated according to their own genotype. We then examined the origin of these two types of cells, using melanin as a cell-marker in the borealis–laevis and laevis–laevis chimeras.
Retinal pigment cells derive from A1 (dorso-animal) and A2 (latero-animal) blastomeres. A1 of one side contributes to retinal pigment cells in both eyes. Though the blastomeres of one side contribute to the formation of bilateral melanophores, the major contribution is to melanophores of the same side. A1, A2 and V2 (latero-vegetal) form the anterior part of the neural fold, and A2 and V2 contribute to melanophores of the head region. The most anterior part of the neural fold derived from A1 does not make a significant contribution to melanophores. Though V2 is a vegetal blastomere, it forms the anterior part of the neural fold by upward movement against the downward movement for gastrulation. A3 forms the middle and posterior parts of the neural fold and contributes to melanophores of the trunk and hindlimbs. Melanophores of hindlimbs also come from A2, A4 and V2. It is to be noted that A4 contributes to melanophores of hindlimbs, despite no apparent contribution to the neural fold.
Development of the retinal pigment cells and melanophores is discussed from the point of pigmentation patterns of the chimeras.  相似文献   

14.
The spatial distribution of cells in chimaeric tissues, composed of two genotypes, provides insights into the extent of cell mixing during development and growth. However, direct measurement of patch sizes is not usually meaningful because, when the proportion of one genotype is high, a single patch may encompass several adjacent coherent clones of like genotype (clone aggregation). Two previously used methods of comparing patch lengths were evaluated to overcome this problem. The corrected mean patch length (corrected for the predicted effects of random clone aggregation) is a more useful summary statistic than the median patch length of the minor genotype, because its use is not restricted to grossly unbalanced chimaeras, but its validity has been questioned. The two methods gave almost identical numerical summaries of patch sizes in the retinal pigment epithelium of fetal chimaeras, thereby validating the use of the corrected mean patch length for this tissue. The present study also showed that the corrected patch length was unaffected by the presence of cells hemizygous for the TgN(Hbb-b1)83Clo transgene and that the proportion of pigmented cells in a single histological section was representative of the overall composition of the chimaeric fetus.  相似文献   

15.
Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (>16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development. © 1996 Wiley-Liss, Inc.
  • 1 This article is a US Government work and, as such, is in the public domain in the United States of America.
  •   相似文献   

    16.
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    18.
    The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.  相似文献   

    19.
    In sessile modular marine invertebrates, chimeras can originate from fusions of closely settling larvae or of colonies that come into contact through growth or movement. While it has been shown that juveniles of brooding corals fuse under experimental conditions, chimera formation in broadcast spawning corals, the most abundant group of reef corals, has not been examined. This study explores the capacity of the broadcast spawning coral Acropora millepora to form chimeras under experimental conditions and to persist as chimeras in the field. Under experimental conditions, 1.5-fold more larvae settled in aggregations than solitarily, and analyses of nine microsatellite loci revealed that 50 per cent of juveniles tested harboured different genotypes within the same colony. Significantly, some chimeric colonies persisted for 23 months post-settlement, when the study ended. Genotypes within persisting chimeric colonies all showed a high level of relatedness, whereas rejecting colonies displayed variable levels of relatedness. The nearly threefold greater sizes of chimeras compared with solitary juveniles, from settlement through to at least three months, suggest that chimerism is likely to be an important strategy for maximizing survival of vulnerable early life-history stages of corals, although longer-term studies are required to more fully explore the potential benefits of chimerism.  相似文献   

    20.
    Hybrid Mus musculus × Mus caroli embryos were produced by inseminating M. musculus (C57BL/Ola Ws) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3½ days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preim-plantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 31/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 31/2-day samples (12 samples of compacted morulae) but were consistently detected at 4½ days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 elec-trophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele. © 1992 Wiley-Liss, Inc.  相似文献   

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