Normal Control Embryos Research Articles

The effects of caffeine and bisphenol A singularly or in combination on cultured mouse embryos and yolk sac placenta

Pregnant women drink caffeinated beverages using bisphenol A (BPA)-coated cans without knowing the potential risks. In this study, mouse embryos (embryonic day 8.5) surrounded by yolk sac placenta were cultured with caffeine (30, 60, and 120 μg/ml) and/or BPA (35 μg/ml) for 48 h. In response to a single administration of BPA or caffeine dose, embryonic development was similar to normal control embryos. However, the combined exposure to caffeine and BPA dose-dependently increased embryonic anomalies, and thinner ventricular wall and trabeculae disorders of heart were observed. The mRNA levels of various anti-oxidative, apoptotic, and hypoxic genes were significantly altered in the treated embryos. Furthermore, abnormal vasculogenesis, reduced vasculogenic growth factor expressions, and apoptotic cell death were detected in yolk sac placentas. These findings indicate that the combined exposure to caffeine and BPA induces embryonic anomalies and injuries of the yolk sac placentas through oxidative stress, apoptosis, hypoxia, and vasculogenic defects.

Understanding the etiology of ventral body wall defects

Ventral body wall (VBW) defects are observed in 1 in 3000 births. The main types of defects include body wall dysplasia, gastroschisis, omphalocele and primary thoracoabdominoschisis. There is little evidence suggesting that genetic changes are associated with these defects. An important number of these occur as a result of exposure to chemical pollutants or radiation. In this project chick embryos exposed to cadmium were used as a model of VBW defect. The project aimed to identify when the ventral wall in chicks is fully closed and to investigate the migration of different cells and tissues in normal control embryos and compare it to embryos exposed to cadmium.

Effect of Cytoplasmic Volume on Developmental Competence of Buffalo (Bubalus bubalis) Embryos Produced Through Hand-Made Cloning

This study examined the effects of cytoplasmic volume on the developmental competence of hand-made cloned buffalo embryos. Two different cell types, that is, buffalo fetal fibroblast (BFF) and buffalo embryonic stem (ES) cell-like cells were taken as donor cell and fused with one, two, or three demicytoplasts to generate embryos with decreased, normal (control), and increased cytoplasmic volume. Using BFF as a nuclear donor, the cleavage rate was similar in all the groups (p > 0.05), but the blastocysts rate was significantly lower (p < 0.05) for embryos generated with decreased cytoplasmic volume. Using ES cell-like cells, the cleavage and blastocyst rate with increased cytoplasmic volume was significantly higher (p < 0.05) compared that with reduced cytoplasmic volume. Blastocysts produced from embryos having increased cytoplasmic volume had significantly higher (p < 0.05) cell number than normal (control) embryos in both BFF and ES cell-like cells groups. Pregnancies were established in all the groups except for the embryos reconstructed with decreased cytoplasmic volume. The pregnancy rate was almost double for embryos reconstructed using increased cytoplasmic volume compared to that with the controls. Most of the pregnancies aborted in the first trimester and one live calf was delivered through Caesarean, which died 4 h after birth.

Novel highly efficient generation of disease-specific human embryonic stem cells from genetically abnormal embryos

OBJECTIVE: Since many frequent, fatal, and incurable diseases do not have an appropriate disease model and with attainable embryos being scarce, with IRB approval we attempted to generate novel disease-specific human embryonic stem (hES) cells with new protocols for more efficient derivation, maintenance, and differentiation.DESIGN: Experimental.MATERIALS AND METHODS: Zona-free human blastocysts (n=14) previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions were transferred onto feeder cells and cultured in DMEM-based media. Pieces of the colonies (n=4) were frozen by liquid nitrogen vitrification with cryoprotectants propylene glycol, DMSO, and acetamide and subsequently thawed. Differentiation of hES cells was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene and protein analysis for pluripotency and differentiation by reverse transcription PCR and immunofluorescence, respectively.RESULTS: Colonies (n=10; 71.4% of transferred blastocysts) could be established and maintained which showed the typical morphological features of hES cells such as compact colony formation. Colonies were derived from affected embryos (one each of cystic fibrosis, trisomy X, 18, 21, 22, and Tay-Sachs disease), from embryos tested inconclusively (one of cystic fibrosis and three of Tay-Sachs disease), and from three normal control embryos. Marker gene and protein expression as well as growth pattern analysis suggest that the colony cells retain their undifferentiated state in culture as well as after vitrification and thawing and that they can be differentiated into a variety of cell types, including the tissues most affected by the conditions.CONCLUSIONS: These newly established protocols for the derivation, maintenance, and differentiation of novel disease-specific hES cell lines should enable the efficient generation of new disease models. This will provide new tools to study diseases as well as to develop new therapeutic approaches. OBJECTIVE: Since many frequent, fatal, and incurable diseases do not have an appropriate disease model and with attainable embryos being scarce, with IRB approval we attempted to generate novel disease-specific human embryonic stem (hES) cells with new protocols for more efficient derivation, maintenance, and differentiation. DESIGN: Experimental. MATERIALS AND METHODS: Zona-free human blastocysts (n=14) previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions were transferred onto feeder cells and cultured in DMEM-based media. Pieces of the colonies (n=4) were frozen by liquid nitrogen vitrification with cryoprotectants propylene glycol, DMSO, and acetamide and subsequently thawed. Differentiation of hES cells was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene and protein analysis for pluripotency and differentiation by reverse transcription PCR and immunofluorescence, respectively. RESULTS: Colonies (n=10; 71.4% of transferred blastocysts) could be established and maintained which showed the typical morphological features of hES cells such as compact colony formation. Colonies were derived from affected embryos (one each of cystic fibrosis, trisomy X, 18, 21, 22, and Tay-Sachs disease), from embryos tested inconclusively (one of cystic fibrosis and three of Tay-Sachs disease), and from three normal control embryos. Marker gene and protein expression as well as growth pattern analysis suggest that the colony cells retain their undifferentiated state in culture as well as after vitrification and thawing and that they can be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSIONS: These newly established protocols for the derivation, maintenance, and differentiation of novel disease-specific hES cell lines should enable the efficient generation of new disease models. This will provide new tools to study diseases as well as to develop new therapeutic approaches.

Reprogramming of Human Somatic Cells Using Human and Animal Oocytes

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

Infusion of anti-nerve growth factor into the cisternum magnum of chick embryo leads to decrease cell production in the cerebral cortical germinal epithelium

There has been considerable recent progress in understanding the processes involved in cerebral cortical development. Several mitogenic and trophic factors have been implicated in the processes of cortical cell proliferation and differentiation. Anti-nerve growth factor (NGF) antibody was administered to 15 days chick foetuses through the cisternum magnum. Control group received phosphate buffered saline (PBS). To identify cells born in the cerebral cortex at the time of antibody or PBS injection, 5'-bromo-2'- deoxyuridine was administered to the foetuses by intravenous injection into an outlying vein using micromanipulation. After injection, the foetuses were re-incubated for another 3 days. All the foetuses were collected on day 18, the brains fixed in paraformaldehyde, cut with a microtome and stained with methyl green pyronin and anti-NGF antibody. Quantitative measurements showed that the thickness of the germinal epithelium (GE) and cerebral cortex in the anti-NGF antibody injected foetuses was decreased when compared with normal control embryos. The number of cells produced in the GE of antibody injected foetuses was decreased when compared with normal control embryos. The results from this study using neutralizing antibody suggests that NGF is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation.

Systolic and Diastolic Ventricular Function Assessed by Pressure-Volume Loops in the Stage 21 Venous Clipped Chick Embryo

Cardiac pressure-volume relations enable quantification of intrinsic ventricular diastolic and systolic properties independent of loading conditions. The use of pressure-volume loop analysis in early stages of development could contribute to a better understanding of the relationship between hemodynamics and cardiac morphogenesis. The venous clip model is an intervention model for the chick embryo in which permanent obstruction of the right lateral vitelline vein temporarily reduces the mechanical load on the embryonic myocardium and induces a spectrum of outflow tract anomalies. We used pressure-volume loop analysis of the embryonic chick heart at stage 21 (3.5 d of incubation) to investigate whether the development of ventricular function is affected by venous clipping at stage 17, compared with normal control embryos. Steady state hemodynamic parameters demonstrated no significant differences between the venous clipped and control embryos. However, analysis of pressure-volume relations showed a significantly lower end-systolic elastance in the clipped embryos (slope of the end-systolic pressure-volume relation: 5.68 +/- 0.85 versus 11.76 +/- 2.70 mm Hg/microL, p < 0.05), indicating reduced contractility. Diastolic stiffness tended to be increased in the clipped embryos (slope of end-diastolic pressure-volume relation: 2.74 +/- 0.56 versus 1.67 +/- 0.21, p = 0.103), but the difference did not reach statistical significance. The results of the pressure-volume loop analysis show that 1 d after venous obstruction, development of ventricular function is affected, with reduced contractility. Pressure-volume analysis may be applied in the chick embryo and is a sensitive technique to detect subtle alterations in ventricular function.

Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei.

The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4. To explore this hypothesis, we used an in silico approach to identify a set of Oct4-related genes whose developmental expression pattern is similar to that of Oct4. When expression of Oct4 and 10 Oct4-related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4-related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4-related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4-related genes may contribute to embryonic lethality in somatic-cell clones.

Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis.

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep.

Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants.

Effects of low frequency electromagnetic fields on the activity of ornithine decarboxylase in developing chicken embryos

In another paper we have reported the results of a study involving over 2500 chick embryos, in which time-varying magnetic fields (MFs) were shown to induce morphological abnormality. In an attempt to understand the mechanism by which these abnormalities arise, the present work explores the effects of weak magnetic fields (MFs) on the activity of the growth related enzyme ornithine decarboxylase (ODC) in chick embryos. The results presented here demonstrate that a 4 μT, 60 Hz sinusoidal magnetic field significantly enhances ODC activity during gastrulation, and diminishes ODC activity during neurulation. In another set of experiments, slightly older embryos were analyzed for gross morphology. The abnormality rate among MF-exposed embryos was approximately three times that of control embryos — with the vast majority of malformations being neural tube abnormalities. When embryos with morphological abnormalities were assayed separately from those which were morphologically normal, it was found that the ODC activity in the abnormal group was significantly less than that in the normal group. However, comparison of normal control embryos with normal MF exposed embryos showed no differences in ODC activity. Similarly comparison of abnormal control embryos with abnormal MF-exposed embryos showed no differences in the ODC activity. We speculate that MF-induced ODC alterations during neurulation may be related to the occurrence of MF-induced neural tube abnormalities at later stages of development.

Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion.

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3-18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.

C-kit Gene Is Expressed by Skin Mast Cells in Embryos But Not in Puppies of Wsh/Wsh Mice: Age-Dependent Abolishment of c-kit Gene Expression

Abstract The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.

C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c-kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein-containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5-day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.

Developmental capacity of non-nucleated embryos of coho salmon, Oncorhynchus kisutch, induced by γ-irradiation

The effects of γ-rays on chromosomes and nuclei were examined by observing embryos of coho salmon, Oncorhynchus kisutch, produced using either sperm or eggs that had been exposed to γ-irradiation. These embryos had smaller nuclei than normal control embryos, and half of the normal chromosome number, suggesting that the genomic DNA of the gametes had been inactivated by γ-irradiation. Furthermore, non-nucleated embryos were produced using both sperm and eggs that had been γ-irradiated under the same conditions. In spite of the fact that the fertilized eggs lacked Feulgen-positive material, cleavage was observed in the embryos, and some developed to the blastula stage. These results suggest that the early stages of embryogenesis of fertilized fish eggs may be influenced by the cytoplasm rather than the nucleus.

Functional gametes derived from explants of single blastomeres containing the “germ plasm” in Xenopus laevis: A genetic marker study

Single blastomeres containing the “germ plasm” were isolated from 32-cell embryos of Xenopus albino ( a p a p ) or wild type and cultured in vitro until the corresponding normal control embryos reached the neurula stage. The resulting explants from albinos were implanted into wild-type host neurulae and vice versa. The formation of functional gametes, eggs or sperm, of donor type was tested when the operated host embryos had reached sexual maturity. The color of the eggs laid by the experimental females and the presence or absence of melanophores in the epidermis and of pigment granules in the eyes of hatched larvae from matings of the experimental males with albino females made possible the identification of donor-type gametes. Twelve males and 12 females of the wild-type hosts, and 16 males and 14 females of the albino hosts survived. Six animals produced donor-type eggs or sperm, most of them being germ line chimeras. This shows that functional gametes can develop from explants derived from single blastomeres containing the “germ plasm.”

Development of the embryonic trisomy 1 mouse

Trisomic and normal control embryos 9.5 through 12 gestational days were examined externally and on serial section with standard light microscopy techniques. Trisomic embryos showed significant retardation in length, weight, optic angle, and somite numbers at most times (p less than .05), and were 1/2 day behind normal animals in overall development. The eye showed the most defects, and these appeared at every period. They included aphakia, large intraretinal space, and dysmorphia of optic cup layers. Brain defects were found at all times. The most common were narrow brain vesicles and folding of the vesicle walls. Some animals showed neural tube defects. The major defect in neural crest derivatives was deficient frontonasal processes in the most defective embryos. The study supports the conclusion of previous investigators that Ts 1 is a syndrome of growth and developmental retardation with a wide range of defects. Definitive evidence, previously lacking, indicates that developmental delay and defective morphogenesis are apparent before 11 days of gestation. In addition, according to our measurements, optic angles are larger in trisomic mice than in normal littermates. This is in contrast to the only previous investigation that included such measurements. Our data do not give definitive support to the suggestion that trisomy 1 belongs to the holoprosencephaly-cyclopia spectrum. However, the hypothesis that murine trisomy 1 shows a parallel in development to human trisomy 13 is not supported by our data. Possible causal mechanisms for the defects and developmental delay are discussed.

Modification of Dorsal-Ventral Polarity in Xenopus laevis Embryos Following Withdrawal of Egg Contents before First Cleavage: (Dorsal-ventral Polarity/Xenopus laevis/Cytoplasmic exudation/Pricking).

When fertilized Xenopus laevis eggs were pricked just beneath the marginal zone with a thick glass needle prior to the first cleavage, a small amount of cytoplasm escaped into the exudate. Those eggs were placed in a poly L-lysine-coated plastic dish filled with 10% Ficoll solution. The location of the sperm entrance site (SES) of each egg was marked by scratching the surface of the plastic dish. The pricked embryos were anchored to the dish through poly L-lysine, and developed, therefore, without changing their original position. Consequently, development of the dorsalventral polarity was conveniently monitored with respect to the location of the SES. Embryos which developed from eggs pricked on the side opposite the SES showed modification of the dorsal-ventral polarity: Semi-quantitative studies showed that an exudation approximately 1.5-12.5% of the whole egg contents from the presumptive dorsal side caused a reversal of the dorsal-ventral polarity. That is, the dorsal lip of the blastopore formed on the same side of the SES, whereas the dorsal lip formed on the side opposite the SES in the normal control and sham-operated embryos. Half of the embryos which had larger cytoplasmic exudates more than 12.5% of the whole egg contents failed to form the dorsal lip by the time all controls and the embryos with smaller exudates showed normal dorsal lip formation. When eggs were pricked on the SES side, the normal topographic relationship between the SES and future dorsal lip side was reinforced.

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It: (Xenopus laevis/germ cell determinant/germ plasm/PGC induction).

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in 3 H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography. Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.

Transplantation studies to investigate mesoderm-ectoderm adhesive cell interactions during gastrulation.

Experiments involving transplantation of the roof of the blastocoel in Rana pipiens embryos reveal that the inner surface of the roof of the blastocoel must be coated with a fibrous extracellular matrix (F-ECM) to serve as a substratum for mesodermal cell migration. When the roof of the blastocoel is inverted the original outer surface, now projecting toward the blastocoel, does not become coated with F-ECM and does not support mesodermal cell migration. When the roof of the blastocoel is removed from a normal embryo and transplanted into an interspecific arrested hybrid embryo known to be deficient in F-ECM synthesis, the grafted ectodermal fragment does not become coated with F-ECM and does not support normal mesodermal cell migration. When a hybrid graft is placed in a normal embryo, the grafted ectodermal fragment becomes coated with F-ECM and supports mesodermal cell migration. In normal control embryos migrating mesodermal cells are polarized due to formation of lamellipodia on their leading but not their trailing edges. These cells are arranged in overlapping layers. The leading cells form lamellipodia on the roof of the blastocoel and trailing cells form lamellipodia on one another.