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PubMed1994

Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle.

Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embryos after release from the drug was determined. The minimum effective concentration (> 95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5 microM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P < 0.001) and the length of exposure to nocodazole (P < 0.001). Exposure to any concentration of nocodazole within the range 2.5-10 microM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5 microM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embryos developing to the blastocyst stage and to term (P < 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60-90 min and started DNA synthesis at 120-150 min after release from nocodazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Analysis of thymidine kinase gene expression in preimplantation mouse embryos.

Thymidine kinase (TK) activity was examined during the development of preimplantation mouse embryos. TK activity was increased approximately 20-fold from day 2 embryos (2-cell) to day 5 embryos (late blastocyst). TK activity did not change along with the progression into S-phase of the first and the second cell cycles but increased sharply at S-phase of the third cell cycle. Analysis of TK mRNA with a reverse transcription-polymerase chain reaction (RT-PCR) method showed that the level of TK mRNA was low in ovulated eggs and 1-cell embryos and was hardly detectable in day 2 embryos (2-cell), but sharply increased in day 3 embryos (mixture of 5- to 8-cell and morula). The functional role of 5'-flanking sequence of TK gene was also investigated in preimplantation embryos after microinjection with the DNA construct of 5'-flanking sequence of TK (2.4 kb) linked to bacterial lacZ gene (TK2.5lacZ) into the pronucleus of 1-cell and subsequently by histochemical staining with X-gal. beta-Galactosidase activity was first detected in day 3 embryos (8-cell), and 30% of embryos were stained with X-gal in day 4 and day 5 embryos, respectively. These results show that an increase in TK activity occurred after 2-cell stage, and this increase was primarily due to the embryonic activation of TK gene expression. Also, it appears that the 5'-flanking sequence of TK may directly regulate the TK gene expression at the transcriptional level during preimplantation murine development.

Animals↗
PubMed2004

Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro.

Recent data have demonstrated that fast-cleaving embryos produced in vitro are more likely to develop to blastocyst stage, and that the postfertilization culture system used impacts considerably on the mRNA expression and quality of blastocysts produced. The present study is the first to investigate the relationship between the developmental speed of embryos produced in vivo or in vitro and the temporal transcription pattern. Genes related to important preimplantation events are monitored during the first 4 days of embryo development in embryos with fast or slow development. The set of genes analyzed in the present study characterizes several important physiological processes including: transport and metabolism of fructose (Glut-5), stress (SOX), mitochondrial activity and detoxification of reactive oxygen species (MnSOD), cell communication (Cx43), maternal recognition of pregnancy (IFN-tau), imprinting (IGF-II), apoptosis (Bax), growth factor binding and metabolism (IGF-IR), and oxidative stress (G6PD). Using real time PCR, we have found that for all the genes analyzed there are differences in mRNA expression between embryos with fast and slow developmental speed produced both in vitro and in vivo. Frequently, genes that may be stress induced such as SOX, MnSOD, BAX, IFtau, and G6PD were highly transcribed in in vitro produced embryos and in embryos with slow developmental speed. On the other side, transcripts from genes related with metabolism, growth, and differentiation (Glut-5, Cx 43, IGF-II, and IGF-IR) were detected in higher amounts in in vivo produced embryos and in embryos with fast developmental speed. Moreover, it is interesting to stand out that for some genetic markers (such as SOX and G6PD) there are in vivo and in vitro differences that can be observed even before materno-zygotic transition, which probably reflects a differential mRNA degradation. These transcription patterns reflects the embryonic response to the adverse in vitro culture conditions, and connect the low quality of embryos which slow developmental speed produced in vivo and in vitro, with the mRNA expression pattern of some embryonic genes.

Animals↗
PubMed2006

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals.

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.

Adenine↗
PubMed2002

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos.

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.

Animals↗
PubMed1986

Neurites show pathway specificity but lack directional specificity or predetermined lengths in Xenopus embryos.

The earliest outgrowth of nerve fibers from identified spinal neurons labeled with horseradish peroxidase (HRP) was traced along surgically rearranged pathways in the central nervous system (CNS) of Xenopus embryos. Parts of the CNS were misaligned or inverted rostrocaudally by grafting a segment of labeled spinal cord in place of the same or different spinal cord segment of an unlabeled embryo or by joining two rostral half embryos (head-to-head) or two caudal half embryos (tail-to-tail), one half of which was derived from a labeled embryo in each combination. Donor embryos were labeled by injection of HRP into a selected blastomere at the 16- or 32-cell stage. Host embryos were unlabeled. Grafts from labeled donors to unlabeled host embryos were made at early neural tube stages before outgrowth of any nerve fibers had started (Jacobson and Huang, 1985). Routes taken by labeled nerve fibers growing into unlabeled CNS were observed at later stages, and the rates of nerve fiber elongation were calculated. Labeled nerve fibers were normal in appearance, and elongated without branching, at normal rates (22-71 micron/h). In head-to-head and tail-to-tail embryos and in embryos with inverted spinal cord grafts, nerve fibers continued elongating without branching in the direction opposite to normal in the CNS. Many fibers reached lengths that were far greater than normal. No reorientation of such maldirected nerve fibers was seen. These results indicate that nerve fiber elongation is not guided by axially polarized pathway cues or markers and that nerve fibers do not grow to predetermined lengths. However, neurites preferred to grow along stereotyped nerve fiber pathways even when forced to grow in the wrong direction or when confronted with nonneural tissue.

Animals↗
PubMed2007

FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos: the added value of extended aneuploidy detection.

OBJECTIVE: Screening for an increased number of chromosomes may improve the detection of abnormal embryos and thus contribute to the capability of preimplantation genetic screening (PGS) to detect the embryo(s) for transfer in IVF with the best chance for a healthy child. Good-quality day 4 and 5 embryos were analyzed after cryopreservation for the nine chromosomes mostly recommended for screening (13, 14, 15, 16, 18, 21, 22, X and Y), next to six additional chromosomes which are less well studied in this context (1, 2, 7, 6, 10 and 17). METHOD: The copy numbers of 15 chromosomes were investigated by fluorescence in situ hybridization (FISH) in three consecutive rounds. The proportion of aneuploid and mosaic embryos was determined and compared in retrospect to results in case only the recommended probe set had been analyzed. RESULTS: A total of 52 embryos from 29 infertile women were analyzed. Screening the embryos for six additional chromosomes increased the proportion of abnormal embryos from 67 to 81% (P = 0.03), owing to an increase in mosaic embryos. CONCLUSION: All but one of the meiotic aneuploidies found in this study would have been detected by the probe set most frequently used in PGS clinics. However, aneuploid cell lines originating from mitotic errors could be detected for almost all chromosomes, so screening of six additional chromosomes mainly increased the proportion of mosaic embryos. The added value of screening for six additional chromosomes in PGS for clinical practice will remain undetermined as long as the fate of mosaic embryos after transfer is unclear.

Aneuploidy↗
PubMed1994

In utero exposure to caffeine causes delayed neural tube closure in rat embryos.

We have investigated the effect of caffeine on embryo growth and development. Caffeine (25 mg/kg) was administered on gestation day (g.d.) 8-9 and the embryos examined histologically 24 h after the final dose. The crown-rump length of caffeine treated embryos (1.92 +/- 0.08 mm) was significantly smaller (P < 0.001) than the controls (2.91 +/- 0.26 mm) as was the circumferential length (caffeine vs. controls, 3.79 +/- 0.16 mm vs. 6.03 +/- 0.61 mm; P < 0.001). Additional measures, such as development of the heart, eye, and limb buds, were also reduced in the caffeine treated embryos. The most striking difference between the control and caffeine treated embryos was the larger proportion of treated embryos with regions of open neural tube. This was most marked in the caudal region of the embryos where 91% of treated embryos had regions of open neural tube compared with 14% of controls. The amount of open neural tube in any individual caffeine treated embryo did not relate to the crown-rump or circumferential length of that embryo nor was the effect restricted to particular litters. These results indicate that caffeine had a significant effects on embryonic growth and development.

Animals↗
PubMed1995

Synthesis of glutathione in the preimplantation mouse embryo.

Depletion and repletion of glutathione in two-cell to blastocyst stage mouse embryos was examined. Reduced (GSH) and oxidized glutathione (GSSG) were measured by fluorimetric HPLC after derivatization of extracted embryo samples with dansyl chloride. Addition of buthionine sulfoximine (BSO) to culture medium for 16 h decreased GSH levels in both two-cell and blastocyst stage embryos; however, GSH decreased more drastically in blastocysts. Addition of diethyl maleate (DEM) to culture medium depleted GSH in both two-cell and blastocyst stage embryos. After removal of DEM, GSH levels returned to normal in blastocysts, and addition of BSO or removal of cystine from medium blocked GSH repletion. Two-cell stage embryos were unable to recover GSH levels after depletion and exhibited decreased in vitro development. Addition of methionine to culture medium was unable to substitute for cystine as a source of cysteine in glutathione synthesis, indicating that the embryos do not use the cystathionine pathway. Embryos collected early on Day 3 of development were unable to recover GSH levels within 5 h, whereas embryos collected late on Day 3 recovered GSH within 5 h. Addition of cycloheximide to culture medium at noon on Day 3 of development decreased the ability of blastocysts to recover their GSH levels late on Day 3. These data indicate that GSH turnover and synthesis increases between the two-cell and blastocyst stages. The increase in the ability of embryos to synthesize GSH on Day 3 is dependent on protein synthesis. Cleavage stage embryos have limited capacity to synthesize GSH and appear susceptible to adverse effects of toxicants or conditions that deplete glutathione.

Animals↗
PubMed1994

DNA-protein interactions in the Caenorhabditis elegans embryo: oocyte and embryonic factors that bind to the promoter of the gut-specific ges-1 gene.

We describe an experimental system in which to investigate DNA-protein interactions in the early Caenorhabditis elegans embryo. A homogeneous population of developmentally blocked mid-proliferation stage embryos can be produced by exposure to the deoxynucleotide analog fluorodeoxyuridine. These blocked embryos remain viable for days and express a number of biochemical markers of early differentiation, for example, gut granules, the gut esterase ges-1, and two regulatory genes, mab-5 and hlh-1. Using the techniques of gel mobility shift and DNase I footprinting, we show that nuclear extracts prepared from these embryos contain factors that bind to the 5'-promoter sequences of the C. elegans gut-specific ges-1 gene. In particular, we examine a putative gut "activator" region, which was previously identified by deletion-transformation analysis and which contains two copies of a consensus GATA-factor binding sequence. Factors that bind to double-stranded oligonucleotides containing the ges-1 GATA sequences are present predominantly in nuclear extracts of embryos but are found neither in cytoplasmic nor in nuclear extracts of unfertilized oocytes. Two proteins, of 43 and 60 kDa, can be uv-crosslinked to double-stranded oligonucleotides containing the ges-1 GATA sequences. The sizes of these proteins correspond to the sizes expected for the elt-1 protein and for the skn-1 protein, two regulatory factors present in early C. elegans embryos and possible candidates for ges-1 control. However, we show that homozygous deficiency embryos (mDf7/mDf7 embryos and eDf19/eDf19 embryos, both of which lack the elt-1 gene, and nDf41/nDf41 embryos, which have no skn-1 gene), still express the ges-1 esterase. We conclude that neither the elt-1 gene nor the skn-1 gene is necessary zygotically for ges-1 expression. We suggest that neither the elt-1 protein nor the skn-1 protein interacts directly with the ges-1 gene and that the observed binding proteins must correspond to products of other genes. More generally, the present experimental system should allow the biochemical study of any gene expressed during early C. elegans embryogenesis.

Animals↗
PubMed2002

Anteroposterior patterning in Xenopus embryos: egg fragment assay system reveals a synergy of dorsalizing and posteriorizing embryonic domains.

Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ).

Animals↗
PubMed2000

Regulation of gamma-glutamate-cysteine ligase expression by oxidative stress in the mouse preimplantation embryo.

The present study examined expression of gamma-glutamate-cysteine ligase (GLCL; also known as gamma-glutamylcysteine synthetase), the rate-limiting enzyme for de novo synthesis of glutathione, in the preimplantation mouse embryo. Previous studies indicated that the cleavage stage embryo is unable to synthesize glutathione de novo. It is hypothesized that GLCL mRNA and protein are not normally expressed in the cleavage stage embryo, but either glutathione depletion or oxidation may induce their expression. In untreated embryos, RT-PCR and Western blotting revealed GLCL heavy subunit (GLCL-H) mRNA and protein only at the blastocyst stage of development. Furthermore, while diethyl maleate (DEM) exposure to deplete cellular glutathione did not induce expression of GLCL-H, exposure to tertiary-butyl hydroperoxide (tBH), an oxidizing agent, resulted in significant upregulation of GLCL-H expression in two-cell embryos. Neither treatment affected expression in blastocysts. Finally, HPLC analysis confirmed that tBH-treated embryos experienced oxidative stress, as indicated by an increase in the ratio of oxidized to reduced glutathione. This oxidative stress induced de novo glutathione synthesis in the cleavage stage embryo, as demonstrated by the subsequent recovery of reduced glutathione levels following DEM-induced depletion. In the absence of tBH treatment, however, cleavage stage embryos could not recover GSH after DEM-mediated depletion. This study demonstrates that the preimplantation embryo has the capacity to upregulate glutathione synthesis in response to oxidative stress but not GSH depletion. These results suggest that, while the preimplantation embryo is well adapted to dealing with oxidative stress, it may be poorly protected from GSH-depleting toxicants.

Animals↗
PubMed1996

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos.

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27-32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5 degrees C by comparison with control embryos cultured at 38 degrees C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.

Animals↗
PubMed1992

The regulative potential of the limb region in 11.5-day rat embryos following the amputation of the fore-limb bud.

The regulative potential of the fore-limb region following the removal of the limb bud was investigated in 11.5-day rat embryos. Fore-limb buds were amputated from a total of 54 embryos. Five embryos were immediately examined via scanning electron microscopy (SEM) to assess the quality of the operation and the reproducibility of the technique. In all cases, the forelimb bud and adjacent tissues extending down to the celomic cavity at the same level were completely removed. The remaining 49 operated embryos were cultured in vitro and examined at different time intervals. Gross inspection of embryos which had been cultured for 24 h, revealed that 24 out of 37 had developed a pair of limb bud-like protrusions at the operation site. Twelve formed only a single protrusion, while nothing was found in the remaining embryo. These protrusions were examined in greater detail under SEM and light microscopy. SEM observations showed that these protrusions were covered with an intact layer of ectoderm. In embryos with a pair of protrusions, these outgrowths developed opposite somites 7 to 13. The failure of either one of these two outgrowths to form, produced our second type of experimental embryo, those which had just a single protrusion. Histological examination revealed that an apical ectodermal ridge (AER) was discernible on the protrusions of 36% of the embryos. Finally, we have established how these protrusions were constructed from SEM observations of operated embryos cultured for 6 h and 10 h.

Amputation Stumps↗
PubMed1996

Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies.

PURPOSE: The purpose of the study was to investigate homeobox gene expression in human oocytes and preembryos and in postimplantation embryos with impaired embryonic development determined by chromosomal abnormalities. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets for Homeobox gene sequences was used. RESULTS: The homeobox genes HoxA4, HoxA7, HoxB4, and HoxB5 were present in human oocytes and cleaving normal and triploid embryos. The expression pattern was different between chromosomally abnormal and normal first-trimester embryos. Of four homeobox transcripts (HoxA7, HoxB4+ ++, HoxB5, and HoxC6) that are expressed in diploid embryos, only HoxA7, HoxB4 and HoxC6 were present in a trisomy 7 embryo, and only HoxB4 and HoxB 5 in triploid embryos and an embryo with trisomy 9. Cloning experiments revealed differences in the number of homeobox clones obtained from trisomy 7 and control embryos. CONCLUSIONS: The transcripts of homeobox genes, HoxA4, HoxA7, HoxB4, and HoxB5, were present in oocytes and cleaving embryos. The pattern of expression of homeobox genes in cultured fibroblasts derived from spontaneously aborted embryos with aneuploidies was different from that in control diploid cells.

Abortion, Spontaneous↗
PubMed1992

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes).

When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105,000 g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 micrograms/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non-toxic congener 1,3,6,8-TCDD at concentrations as high as 50 micrograms/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putative Ah receptor in these fish embryos. At 50 micrograms/l, beta-naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8-TCDD did at toxic concentrations. However, at 50 micrograms/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.

Animals↗
PubMed1998

Developmental abnormalities in horseshoe crab embryos caused by exposure to heavy metals

We examined the effects of heavy metals on the development of horseshoe crabs using bioassays in which embryos at different stages were exposed either continuously or for 24 h. Abnormalities included segment-defective embryos, double embryos, embryos with abnormal eye areas, and no-posterior embryos. In terms of their ability to affect normal development, the pollutants are ranked as follows: Hg >== organotin > Cr falling dots Cd > Cu > Pb > Zn. Organotin treatment was associated with a high frequency of abnormal eye embryos and Hg with segment-defective embryos. The specific cause of segment-defective malformations by Hg was investigated by using SH inhibitors and cytochalasin B, an inhibitor of microfilaments. These inhibitors induced Hg-type segment-defective embryos; thus, Hg may also act by inhibiting SH-SS exchange and blocking morphogenetic movements. Horseshoe crab embryos from Delaware Bay and Sandy Hook Bay, New Jersey, were examined for developmental abnormalities. Malformed embryos were extremely rare (<0.6%) at five beaches along Delaware Bay as well as Sandy Hook Bay, suggesting that these horseshoe crab spawning areas are relatively unpolluted by heavy metals.

Journal Article↗
PubMed2004

Polyethylene glycol and abscisic acid improve maturation and regeneration of Panax ginseng somatic embryos.

Embryogenic culture was initiated from mature zygotic embryos of Panax ginseng. Multiple somatic embryos formed and proliferated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.26 microM) and kinetin (0.046 microM). Mature as well as immature somatic embryos grew into plantlets lacking roots on the same media. Histomorphological analysis of somatic embryos treated with abscisic acid (ABA) and polyethylene glycol (PEG 4000) showed a slight improvement in the root meristem organization of torpedo-stage embryos (embryos were more compact and their cells exhibited a lower degree of vacuolation). Shoot regeneration of non-treated somatic embryos was 31% while that for somatic embryos treated with PEG 4000 and ABA was 70%. Moreover, 75% of plants regenerated from PEG- and ABA-treated embryos formed roots while plants from non-treated embryos did not form roots.

Abscisic Acid↗