Embryo Aggregation Research Articles

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.

Non-diabetogenic insulitis in NODB10.GD allophenic mice in spite of permissive class I MHC antigens.

Allophenic mice (embryo aggregation mouse chimeras) enable us to dissect the process of spontaneous autoimmunity under physiological conditions. Our previous experiments showed that the autoimmune process in allophenic mice of the NOD<-->C57B1/6 strain combination does not progress from insulitis to diabetes. One possible explanation for this protection is that H-2 Kd-restricted CD8+ T cells kill only NOD beta cells (Kd,Db) in the chimeric islets, while the B6 beta cells (Kb,Db) are spared from destruction. To test this hypothesis we analysed 22 NOD<-->B10.GD chimeras in which the class I MHC are shared by both parental strains. Therefore all the beta cells in these chimeras express H-2 Kd molecules. Ten allophenic mice were killed at 7 weeks and studied for early pathology. No evidence for intra-islet infiltration was obtained at this age, suggesting that the autoimmune process in NOD<-->B10.GD chimeras is slower than in NOD mice. Twelve chimeras were followed up for 1 year for disease development and all failed to progress to full-blown diabetes, despite the occurrence of intra-insulitis in six out of 12 mice. The lack of disease in NOD<-->B10.GD chimeras demonstrates that class I MHC chimerism does not account for diabetes resistance in NOD-allophenic mice.

Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene.

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.

Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.

Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell <==> tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.

A new method for explanting early postimplantation rat embryos for culture

Rat embryos were explanted either on day 8, 3 PM (primitive streak stage) or on day 9, 8 AM (presomite stage) and divided randomly into two groups (plug day = day 0). In one group, Reichert's membrane was removed starting from the side opposite to the ectoplacental cone which was left intact (standard method). In the second group, Reichert's membrane was removed starting from the same side as the ectoplacental cone after the latter structure was excised and the proamniotic cavity (day 8) or the ectoplacental cavity (day 9) destroyed (new method). Embryos were then cultured and examined at 10 AM on day 11. All embryos developed normally in size and general morphology. However, there was a high incidence of inverted heart, allantoic placenta and tail in all groups, except for day 9, new method group; this was the only group in which the ectoplacental cavity did not expand abnormally during culture. The new method, which is quick and easy to perform even when Reichert's membrane does not protrude well, lessens the chances of damaging embryonic ectoderm during the removal of Reichert's membrane. Furthermore, the absence of ectoplacental cone decreases embryo aggregation during culture. We hypothesize that the abnormally expanded ectoplacental cavity generates abnormal physical forces within the egg cylinder that disrupt the control of body asymmetry.

A multivariate morphometric analysis of hippocampal anatomical variation in C57BL/6 ↔ BALB/c chimeric mice

We investigated hippocampal anatomy in artificially-produced chimeras derived by the aggregation of embryos from two widely-studied inbred mouse strains, C57BL/6J and BALB/cJ. Contrary to expectations, the chimeras were not always intermediate between the parental strains. For a number of characters, the chimeras exceeded qualitatively as well as quantitatively the phenotypical range displayed by both inbred parental strains. These findings imply that if only one parent is available for comparison, for instance, in studies involving a normally inviable genotype, separating effects of this genotype from idiosyncratic effects inherent to the chimeric model will be very difficult, if not impossible.

Intraspecific Blastocyst Reconstitution in Mice Using Giant Trophectodermal Vesicles Produced by Multiple Embryo Aggregation

Giant trophectodermal (TE) vesicles, produced by aggregating multiple embryos, were evaluated as a means of enhancing the viability of reconstituted murine blastocysts. Previous studies have indicated that the development of chimeric conceptuses produced by multiple embryo aggregation is impaired, thus the initial objective here was to examine how this is affected by 1) differences in developmental rates of individual embryos comprising the aggregates, 2) lengths of in vitro culture, and 3) incompatibilities between maternal hosts and chimeric fetal allografts. The results indicate that the viability of aggregates was influenced by embryo genotype and length of in vitro culture. The average number of B6D2-F1 x BL/6 offspring resulting from aggregates cultured for 0.7 day was not significantly different (p greater than 0.05) from that obtained from either embryo singletons cultured for 0.7 and 1.7 days (2.9 +/- 0.3 and 3.9 +/- 0.4, respectively) or from nonmanipulated controls (3.2 +/- 0.2 and 4.3 +/- 0.3, respectively). Blastocyst reconstitution studies were then conducted using giant TE vesicles from B6D2-F1 x BL/6 mice and inner cell masses from C3H mice. The average number of C3H offspring produced (2.7 +/- 0.1) was not significantly different (p greater than 0.05) from that of B6D2-F1 x BL/6 embryo aggregates (3.2 +/- 0.6) or of nonmanipulated controls (4.3 +/- 0.3). The results demonstrate that giant TE vesicles can be effectively used for intraspecific blastocyst reconstitution in mice.

Intraspecific blastocyst reconstitution in mice using giant trophectodermal vesicles produced by multiple embryo aggregation

Giant trophectodermal (TE) vesicles, produced by aggregating multiple embryos, were evaluated as a means of enhancing the viability of reconstituted murine blastocysts. Previous studies have indicated that the development of chimeric conceptuses produced by multiple embryo aggregation is impaired, thus the initial objective here was to examine how this is affected by 1) differences in developmental rates of individual embryos comprising the aggregates, 2) lengths of in vitro culture, and 3) incompatibilities between maternal hosts and chimeric fetal allografts. The results indicate that the viability of aggregates was influenced by embryo genotype and length of in vitro culture. The average number of B6D2-F1 x BL/6 offspring resulting from aggregates cultured for 0.7 day was not significantly different (p greater than 0.05) from that obtained from either embryo singletons cultured for 0.7 and 1.7 days (2.9 +/- 0.3 and 3.9 +/- 0.4, respectively) or from nonmanipulated controls (3.2 +/- 0.2 and 4.3 +/- 0.3, respectively). Blastocyst reconstitution studies were then conducted using giant TE vesicles from B6D2-F1 x BL/6 mice and inner cell masses from C3H mice. The average number of C3H offspring produced (2.7 +/- 0.1) was not significantly different (p greater than 0.05) from that of B6D2-F1 x BL/6 embryo aggregates (3.2 +/- 0.6) or of nonmanipulated controls (4.3 +/- 0.3). The results demonstrate that giant TE vesicles can be effectively used for intraspecific blastocyst reconstitution in mice.

Factors influencing the developmental competence of intraspecific murine chimeras produced by multiple embryo aggregation

Fourteen mature anestrous bitches were used to determine the effectiveness of pretreatment with an orally active progestogen to prevent premature luteolysis of induced corpora lutea (CL) in the anestrous bitch. In Group 1, seven bitches were treated orally with megestrol acetate (Ovaban®) at the rate of 2.2 mg/kg body weight for eight days. Three days later, the bitches were treated daily with pregnant mare's serum gonadotropin (PMSG) (44 IU/kg body weight) administered intramuscularly for nine consecutive days, and each bitch was given 500 IU human chorionic gonadotropin (HCG) on day 10, or on the first day of induced estrus if the bitches exhibited estrus while being treated with PMSG. A control group (Group 2) of seven bitches was not treated with Ovaban® but was similarly given PMSG and HCG. Estrus was detected twice daily using a vasectomized male dog and verified by vaginal cytology. Blood samples were obtained on the first day of induced estrus (day 0) and every other day until day 90 post-estrus. Plasma progesterone (P4) concentrations were determined by a non-extraction solid phase radioimmunoassay (RIA), and data were analyzed by Student's t-test. There was no significant difference between the progesterone profiles of both groups of bitches. In addition, P4 values were less than 1 ng/ml by day 50 post-estrus. Results of this study suggested that pretreatment with an orally active progestogen was not effective in preventing premature luteolysis of induced CL in the anestrous bitch.

Polyclonal origin of mouse skin papillomas.

We show that, from the earliest morphologically recognisable stages of development, mouse skin papillomas induced by a chemical initiation-promotion regime are polyclonal. We have demonstrated polyclonality directly by immunohistochemical staining of the mosaic cell populations in embryo aggregation chimaeras, a method which removes some of the uncertainties of previous conclusions based on analysis using electrophoretic polymorphisms. The findings may imply that initiation within a single cell and promotion of its clonal descendents is not a sufficient explanation for the origin of these tumours and that interaction between cells of more than one clone is involved.

Co-existence of gametic and agametic seminiferous tubules in a chimeric mouse. A light- and electron-microscopic study.

Six chimeras, including 4 phenotypic males and 2 females, were produced by aggregation of F1 (C57BL x BALB/c) and Swiss white embryos. All were fertile, except 1 male, whose deviation in testicular structure prompted this light- and electron-microscopic study. This chimera had a well-developed sperm-conducting system, sperm in the epididymis and active accessory sex glands. The testes displayed typical parenchymal and stromal components with the important exception of co-existence of gametic and agametic seminiferous tubules. These tubules were organized in territories of quasi-lobular configurations which appeared to open separately into rete testis. The former corresponded to normally developed and active seminiferous tubules, while the latter were solid testicular cords devoid of any germ cells and embedded in solid masses of interstitial (Leydig) cells. Special mitochondrial transformations were identified in sustentacular (Sertoli) cells of both types of tubules, in maturing spermatids and sperm. These and other submicroscopic sperm defects might be the cause of infertility.

Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling.

The effects of zona drilling on mouse embryo development in vitro were evaluated. Following insemination, sperm were immediately concentrated at the area of drilling, and in zona-drilled eggs, pronuclei appeared 30-50 min earlier than in zona-intact controls. Zona-drilled oocytes fertilized at significantly higher rates than undrilled controls and, consequently, a greater percentage of eggs inseminated after zona drilling reached the blastocyst stage. The attrition rates of zona-drilled embryos at each cleavage stage did not differ significantly from controls. Manipulated embryos exhibited unique cleavage patterns. Some embryos lost their zonae entirely, whereas others became partially extruded at early cleavage stages. These anomalies led to separation of blastomeres from the zygote proper, aggregation of embryos to form giant composite morulae and blastocysts, and occasionally to formation of miniature twin blastocysts. These characteristics of cleavage indicate that although zona drilling of a cohort of oocytes is likely to lead to an increased number of live births relative to controls, some developmental abnormalities can be encountered, and these may be associated with embryo loss, spontaneous chimerism, or possibly with conception of monozygotic twins.

An Assay Using Embryo Aggregation Chimeras for the Detection of Nonlethal Changes in X-Irradiated Mouse Preimplantation Embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Production of somatic embryos from carrot tissues in hormone-free medium

Carrot “seed” after the germination of their zygotic embryos, contain cells which can develop in aseptic culture into somatic embros in a reliable and predictable manner without the use of exogenously added growth regulators. In the course of further development beyond the cotyledonary stage, somatic embryos form secondary embryos along their axes. If continually subcultured, they do so in a cyclical manner yielding loosely attached aggregates of embryos and plantlets. Subcellular embryogenesis stops and normal plantlets are recoverable if the medium is allowed to become stale. Excised zygotic embryos of carrot in vitro are also able to form somatic embryos in the same way, but only when broken in half, and at a much lower response level. Component(s) of the basal medium responsible for inducing somatic embryogenesis remain to be elucidated, but a lowered sucrose concentration prevents secondary somatic embryogenesis. Cytological and histological examinations show that the somatic embryos are diploid and are most likely derived both from cells of the fruit wall and the endosperm.

Epileptic Convulsion in Chimera Mice

SummaryUsing a method of aggregation of embryos from the Fl progenies of ddY X El and ddY X C57BL/6J, seven chimera mice were produced. These seven chimera mice manifested various distributions of the white coat color derived from the Fl progeny of ddY X El. It was found that the chimera mouse, which had a larger percent of the white coat color, had a higher susceptibility to epileptic convulsion, the susceptibility having been proportional to the degree of the tissue mosaicism.

Raf , a Trans -Acting Locus, Regulates the α-Fetoprotein Gene in a Cell-Autonomous Manner

Genetic analysis provides an approach for identifying regulatory loci that govern the expression of specific genes within the context of the entire organism. Such analyses have defined two unlinked regulatory loci, termed raf and Rif, that modulate the levels of alpha-fetoprotein in liver. Of primary importance for the isolation and characterization of the raf product is to determine whether it is produced by the hepatocyte or whether it is produced by a different cell type. By means of analysis of alpha-fetoprotein expression in livers of embryo aggregation chimeras derived from mice of different raf genotypes it was possible to conclude that the product of the raf locus is expressed as a hepatocyte autonomous function that acts in trans to regulate the level of alpha-fetoprotein messenger RNA.

Susceptibility of chimera mice to epileptic convulsion.

The ddY mouse-derived E1 strain is a mutant susceptible to severe epileptic convulsion. Using a method of aggregation of embryos from the F1 progenies of ddY female X El male and ddY female X C57BL/6J male, seven chimera mice were produced. These seven chimera mice manifested distribution of white coat color derived from F1 progeny of ddY X El, in two 100%, one 50%, two 24%, and two less than 4% and the frequency of the seizure occurrence were 71.4%, 50%, 28.6%, 28.6%, 21.4%, 0.7% and 0%, respectively. It was found that the chimera mouse which had a larger percent of white coat color had higher susceptibility to epileptic convulsion, the susceptibility having been proportional to the degree of the tissue mosaicism.

The effect of retinoic acid pretreatment on the ability of murine embryonal carcinoma and inner cell mass cells to participate in chimaera development

Certain embryonal carcinoma (EC) cell lines can colonize the embryo following blastocyst injection or embryo aggregation, giving rise to EC-embryo chimaeras. However, such chimaeras often develop abnormally. For example, diploid P19 cells colonize the embryo readily but resulting chimaeras are usually abnormal, with persistence of tumour cells. Retinoic acid (RA) induces differentiation of EC cells to a variety of cell types in vitro but, in this study, it was shown that pretreatment of P19 cells with RA did not result in more normal development of P19-embryo chimaeras. The only significant effect of RA was to reduce the ability of P19 cells to participate in embryonic development at all after blastocyst injection. RA did not have a direct toxic or teratogenic effect on preimplantation mouse embryos and did not affect the ability of pluripotent embryo cells to colonize chimaeras. Therefore, RA may not be the normal inducer of differentiation in early embryogenesis.

The effect of addition of polar molecules on the degree of dispersion of silver iodide sol

The objective of the present study was to investigate the changes in the degree of dispersion in a negative AgI sol under the influence of organic components introduced into the medium only after nucleation. It was found that the polar molecules of those components can stimulate the process of formation of individual colloidal particles from unstable metaphase clusters emerging as a result of embryo aggregation.

Cell lineage analysis of Schwann cells in the PNS determined by shiverer-normal mouse chimeras.

The myelin of the peripheral nervous system from the shiverer mutant mice is characterized by the absence of myelin basic protein, while the other myelin protein components are present at normal levels. Myelin lamella formation is normal in the shiverer mutant. Therefore, by using antiserum against myelin basic protein, we can distinguish the shiverer from the wild-type control myelin immunohistochemically. To study the cell lineage of Schwann cells, chimeras produced by the aggregation of eight-cell embryos from wild-type mice and shiverer mice have been used. Using myelin basic protein as a marker, it was observed that Schwann cells in the sciatic nerve existed as patches of cells with like-genotype. The patches occurred in a linear array along the axons with some intermingling of Schwann cells. Complete randomization by intermingling of Schwann cells was not observed and clones of Schwann cells may persist as contiguous groups throughout peripheral nerve development.