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PubMedDate not supplied

[Embryo- and genotoxic effects of two endosulfan forms in the culture of rat and mouse pre- and postimplantation embryos].

We studied embryo- and genotoxic effects of two forms (powder and microcapsular) of organochlorine insecticide and acaricide endosulfan in rat and mouse cultures of pre- and postimplantation embryos. The substance caused embryonic death, teratogenesis, and inhibited embryonic growth. Embryo cultivation in the serum obtained from the rats 1-48 h after single peritoneal introduction of highest bearable endosulfan dose allowed us to compare time pattern of the embryotoxic factors in the animal blood for the both substance forms. Microcapsular endosulfan induced much later appearance of the embryotoxic factors in the rat blood, had lower embryolethal and teratogenic effects, but prolonged the presence of pathogenic factors in the animal as compared to the powder form. We propose that embryo- and genotoxic activity of endosulfan increases in the course of its biotransformation.

Animals↗
PubMed1993

The spare embryo--a red herring in the embryo experimentation debate.

Whenever embryo experimentation is discussed, the question of whether it is preferable to use spare or specifically produced ('research') embryos for destructive embryo experimentation always enters the debate at some stage. This question is analysed, and it is suggested that the distinction is morally uninteresting, but rhetorically useful for both sides in the debate. It is further suggested that part of the force of this distinction is caused by the fact that it is parasitic on a real moral distinction based on the degree to which the gametes used in the experiment are obtained by explicit or implicit coercion.

Coercion↗
PubMed2005

Effect of oxygen concentration during the incubation of embryos of women undergoing ICSI and embryo transfer: a prospective randomized study.

The hypothesis that ICSI outcome can be improved by culturing human embryos in an atmosphere of controlled O(2) concentration (5%) compared with 20% was tested in a prospective randomized study of 712 transfer cycles. The cycle characteristics and the embryology parameters were similar between groups. The embryo qualities were similar with day 2 transfers; however, they were better with day 3 transfers incubated in 5% O(2) than in 20% O(2). The clinical outcome parameters did not differ between groups according to the O(2) concentration. The results indicated that culture of embryos under atmospheric conditions of O(2) for the first 2 or 3 days did not alter the clinical outcome in ICSI cycles.

Blastocyst↗
PubMed1989

Early pregnancy wastage following late implantation of embryos after in-vitro fertilization and embryo transfer.

Serial plasma concentrations of human chorionic gonadotrophin (HCG), progesterone and oestradiol were measured in pregnancies after in-vitro fertilization and embryo transfer. The first detection day of HCG after embryo transfer (8.4 +/- 1.1) and the HCG doubling time (DT) of 64 normal singleton pregnancies were compared to those of 14 first-trimester miscarriages. The same parameters were evaluated in nine late-implanted conceptions, seven of which resulted in early pregnancy wastage. The HCG DT of late-implanted pregnancies was consistent with that of singleton term pregnancies in the first 12 days, while first-trimester miscarriages which had implanted at the usual time had a significantly longer DT from implantation onwards. The reduced trophoblastic secretory rate suggests poor embryo quality in these cases. A decreased progesterone/oestradiol ratio was observed in late-implanted pregnancies but because of the small number of individuals, no definite conclusion can be drawn. More patients with delayed implantation should be tested to justify this observation.

Adult↗
PubMed1991

Preimplantation embryo biopsy: detection of trisomy in a single cell biopsied from a four-cell mouse embryo.

This paper describes an efficient technique for the production of metaphase spreads from single blastomeres biopsied from four-cell preimplantation mouse embryos. The karyotype obtained by chromosomal analysis of single biopsied cells is shown to be fully predictive of subsequent fetal karyotype. The data in this study also demonstrate that the entire process of embryo biopsy and karyotypic analysis of biopsied blastomeres does not adversely affect the ability of biopsied embryos to form fetuses after transfer into pseudopregnant recipients. This study has potential clinical relevance in that it demonstrates that chromosomally defective embryos can be accurately identified before implantation. In addition, the techniques developed in this study may facilitate more efficient procedures for the genesis of animal models for human disorders such as Down syndrome and Alzheimers disease.

Animals↗
PubMed1990

Preimplantation murine embryos are more resistant than human embryos to bacterial endotoxins.

Bacterial endotoxins have been correlated with increased fragmentation of early cleavage-stage human embryos and decreased pregnancy rates in human in vitro fertilization programs. The purpose of the present study was to test the direct effects of increasing concentrations of endotoxins on in vitro fertilization and development of mouse embryos to the blastocyst stage. Sexually mature B6D2F1 female mice were superovulated and oocytecumulus complexes were collected from the oviducts and randomized into control and treatment groups. Oocytes (n = 867) were inseminated with capacitated sperm. Treatments included Ham's F-10 supplemented with 3 mg/ml bovine serum albumin and increasing amounts of endotoxin (0.35, 0.64, 0.92, 1.5, 2.08, 3.21, 6.07, and 11.79 ng/ml). Percentage cleavage, percentage fragmentation at the four-cell stage, and percentage expanded blastocyst formation (of cleaved embryos) were evaluated. Statistically significant decreases in cleavage at 6.07 ng/ml (P less than 0.05) and blastocyst formation at 11.79 ng/ml (P less than 0.05) of endotoxin were observed. Fragmentation at the four-cell stage was significantly increased at 3.21 ng/ml (P less than 0.05) of endotoxin. We conclude that the levels of endotoxin necessary to decrease murine preimplantation development significantly is higher than that reported for human embryos.

Animals↗
PubMed1993

Production and characterization of monoclonal antibodies to embryo-associated immunosuppressor factor (EASF) produced by human pre-implantation embryo.

Balb/c mice were immunized with pre-implantation embryo-associated immunosuppressor factor (EASF) (purified from embryo growth media of in vitro fertilized human ova). Hybridoma clones were produced by fusing their spleen cells with NS1 and P3X653 myeloma cell lines. The presence of specific anti-EASF monoclonal antibodies (mAb) in the hybridoma culture supernatants were tested by enzyme-linked immunosorbent assay. A total of 15 hybridoma clones were selected, and their products were purified and characterized. Each mAb bound specifically to its antigen in a dose-dependent manner. The affinity-purified EASF from embryo growth media demonstrated immunosuppressive activity on Concanavalin A-induced lymphocytes and the presence of 14 kDa, 24 kDa and 37 kDa factors. No such activity or similar molecules were identified when control growth media were analyzed. This clearly demonstrates that these mAb are indeed EASF-specific and are able to recognize biologically active immunosuppressive components in embryo growth media. These mAbs are presently being tested for the development of EASF-specific assay system.

Animals↗
PubMed1997

Developmental, stress and ABA modulation of mRNA levels for bZip transcription factors and Vp1 in barley embryos and embryo-derived suspension cultures.

The transcription factors VP1 (Viviparous-1), EmBP-1 (Em-binding protein 1) and OSBZ8, originally cloned and analysed in various monocot species, have been implicated in the regulation of the Lea (late embryogenesis-abundant) group 1 genes. We have investigated the modulation of the levels of these mRNAs in barley during embryogenesis, and in developing embryos subjected to various kinds of osmotic stress. The accumulation of mRNA for VP1 and EmBP-1 transcription factors, using cDNAs cloned from barley, starts at 10 and 15 days after anthesis, respectively, whereas Lea B19 mRNA levels are very low or undetectable until 25 days after anthesis during normal development. The EmBP-1 mRNA is predominantly induced in mannitol-stressed immature embryos. Vp1 mRNA was not significantly modulated by ABA, salt or mannitol. Inhibition of ABA biosynthesis by norflurazon showed that the induction of both Vp1 and EmBP-1 mRNAs was ABA-independent. In embryo-derived suspension-cultured cells, neither of the two transcripts would be induced by ABA or osmotic stress, although both OSBZ8 and one member of the Lea B19 family was up-regulated by ABA. Electrophoretic mobility shift assays using a Lea B19.1 probe with an ABRE (abscisic acid-responsive element) similar to that which binds EmBP-1 and OSBZ8 in the wheat and rice Em promoters show that the binding activity is increased by ABA and osmotic stress. Taken together, these data show that both VP1 and EmBP-1 are involved in embryo-specific signal transduction pathways, that they are differentially regulated at the mRNA level, and that EmBP-1 can be induced by osmotic stress independently of any increase in endogenous ABA. The difference in mRNA regulation patterns of OSBZ8 and EmBP-1 may suggest that they are involved in different signal transduction pathways in connection with osmotic stress/ABA regulation of Lea genes.

Abscisic Acid↗
PubMed2002

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos.

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.

Bioreactors↗
PubMed2004

Nuclear chromosomal localization in human preimplantation embryos: correlation with aneuploidy and embryo morphology.

BACKGROUND: Spatial organization of chromosomes is hypothesized to reflect transcriptional activity and regulatory protein function. Preimplantation genetic diagnosis allows assessment of the spatial relationship of chromosomes in human blastomeres. We thus examined the localization of chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres from 6-8-cell stage embryos, correlating localization to aneuploidy and embryo morphology. METHODS: Following fluorescence in situ hybridization to enumerate chromosomes 13, 16, 18, 21, 22, X and Y, signal positions were localized within one of four concentric shells. Statistical analysis compared chromosome localization between euploid and aneuploid blastomeres as well as morphologically normal and abnormal embryos. RESULTS: Of 98 embryos, 109 blastomeres were evaluated. Within chromosomally normal blastomeres, no difference in the location of all seven chromosomes (P</=0.10) was observed. However, a significant difference was observed between the organization of chromosomes in euploid versus aneuploid blastomeres (P</=0.001). Localization of chromosomes 13, 18, 21 and 22 was significantly different when an abnormality involving that chromosome existed (P</=0.001, P</=0.01, P</=0.025 and P</=0.01 respectively). CONCLUSIONS: We report for the first time that localization of chromosomes is altered in chromosomally aneuploid but not in chromosomally normal nor morphologically abnormal euploid blastomeres.

Adult↗
PubMed1996

Development of a culture medium (BECM-3) for porcine embryos: effects of bovine serum albumin and fetal bovine serum on embryo development.

Media are available that can deliver modest porcine embryonic development from a single-cell zygote to the blastocyst stage. However, few embryos develop to hatched blastocysts by Day 7 in vitro, indicating deficiencies in media that inhibit early embryonic development. A defined culture medium, Beltsville Embryo Culture Medium (BECM-3), was developed to support porcine zygote development to the blastocyst stage. When fetal bovine serum was added by late Day 5 (late morula/early blastocyst stage), 80% of total embryos cultured from Day 2 developed into hatched blastocysts by Day 8. There was also a significant increase in the mean cell number of blastocysts and hatched blastocysts when culture was performed in BECM-3-based media in the absence of BSA fraction V. These studies provide a chemically defined foundation for elucidating key developmental components of preimplantation pig embryos.

Animals↗
PubMed1994

The maize (Zea mays L.) Cat1 catalase promoter displays differential binding of nuclear proteins isolated from germinated and developing embryos and from embryos grown in the presence and absence of abscisic acid.

We previously demonstrated that amounts of Cat1 RNA in developing immature maize (Zea mays L.) embryos change in parallel with endogenous abscisic acid (ABA) content. In excised immature embryos, addition of ABA leads to a large increase in Cat1 RNA accumulation. The Cat1 transcript, however, also accumulates to high amounts in scutella of germinating embryos, where ABA content is low and decreasing. Here we show that application of ABA to germinated embryos no longer results in the up-regulation of the Cat1 transcript accumulation that is seen during embryogenesis. This suggests that factors other than ABA control Cat1 expression at this developmental stage. Using band-shift and southwestern analyses, we show that the change in sensitivity to ABA is paralleled by changes in nuclear proteins binding to a 28-bp region of the Cat1 promoter in vitro. One protein (CAT1BP-20) shows increased accumulation in the absence of ABA, suggesting that a repressor-mediated mechanism accounts for at least a portion of the ABA regulation of Cat1.

Abscisic Acid↗
PubMed1988

ABA-Regulation of Two Classes of Embryo-Specific Sequences in Mature Wheat Embryos.

We have previously described the isolation and characterization of ABA-enhanced sequences from developing wheat embryos. Here we use in vivo RNA labeling and the inhibitors alpha-amanitin and cycloheximide to determine the level at which ABA acts to modulate these sequences in cultured mature embryos. Sequences fell into two classes: one, represented by the 7S globulin clone, p511, appears to be regulated at the level of transcription, while the other, represented by the early methionine-labeled polypeptide (E(m))-protein clone, p1015, has an additional posttranscriptional component. In mature embryos cultured in the absence of ABA, mRNA levels of p511 and p1015 declined rapidly until neither was detected at 3 days postimbibition. Levels of p511 increased in mature embryos cultured in the presence of ABA, but remained low in the presence of ABA + alpha-amanitin, suggesting p511 RNA is regulated at the level of transcription. Levels of p1015, in contrast, remained high not only in the presence of ABA, but also in the presence of ABA + alpha-amanitin or alpha-amanitin alone. This suggests p1015 regulation might be at the level of selective RNA stability. Cycloheximide had no detectable effect on ABA-mediated stabilization of p1015, suggesting that newly synthesized proteins are not involved. E(m)-protein synthesis rates closely paralleled E(m) RNA levels, suggesting E(m) expression is not controlled at the level of translation.

Journal Article↗
PubMed1989

Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development.

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C(19)-GAs, GA(1), GA(4), GA(5), GA(6), and one C(20) GA, GA(44), were identified. From the embryo, five C(19)-GAs GA(1), GA(4), GA(5), GA(6), GA(60) and two C(20) GAs, GA(19) and GA(44) were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.

Journal Article↗
PubMed1978

Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5'-ends of "capped" RNA from imbibing wheat embryos.

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains[methyl-3H]methionine. (2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of "capped" oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2. (3) By way of contrast with the "capped" RNA of animal cells, 5'-terminal "cap" structures (m7Gppp- and m32,2,7 Gppp-) in the "capped" RNA from the higher plant organism are not bonded to pneultimate O2'-methylnucleoside constituents. (4) In an allied study, it has been found that recovery of poly (A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly (A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris (hydroxymethyl) aminomethane buffered (pH 9.0) phenolic emulsions.

Chemical Phenomena↗
PubMed1977

The mutual relations of structures in the growing bill of chick (Gallus domesticus L.) and duck (Anas platyrhynchos L.) Embryos. IV. The size, shape, and position of the upper and lower bills in unilaterally microphthalmic duck embryos.

Unilaterally microphthalmic duck (DUM) embryos studied in stage 39 were obtained by damaging the right eye in stage 17. The development between stages 19 and 39 was observed in samples. After prolonged incubation, one duck hatched and reached adulthood. In DUM embryos partial crossbeak develops. Both the upper and lower bills deviate lateralwards, the upper bill more than the lower. In stage 39 a small orbit is present on the treated side. In stained and cleared heads most of the elements of the skull (the bones surrounding the orbit, the brain-case, and the upper and lower bill elements) are translated in the direction of the rudimentary orbit on the treated side. The bills are twisted around each other at the tip. Measurements of external dimensions as well as those made in stained and cleared stage-39 DUM heads show elongation of the left part of the lower bill. Except for the crossed bills, the changes found in the embryos persisted in the adult. The findings support a hypothesis concerning the mutual influence of bills during morphogenesis. The coherence of the functional cranial components in the embryo is discussed.

Animals↗
PubMed1988

Dispersal of rat uterine mast cells and their functional response to an embryo-derived histamine releasing factor: a possible model for embryo implantation.

Rat uterine tissue was dissociated by enzymatic digestion with collagenase and viable mast cells were obtained. Their viability was assessed by the ability to exclude trypan blue dye and to respond functionally to different stimuli. Challenge with anti-IgE gave a calcium-dependent histamine release of 49%, whilst the undigested uterine fragments gave 23%. Moreover, they were capable of releasing histamine on challenge with the compound 48/80, suggesting a similarity with connective tissue mast cells. This similarity was further supported by their insensitivity to aldehyde blocking of dye binding. The final dispersed cell preparation contained 3 X 10(5) mast cells/g of uterine tissue, representing about 2% of total nucleated cells. The total histamine content of the undigested uterus was 2.5 micrograms/g of tissue, whilst after digestion the histamine determined was 1.2 pg per mast cell with a yield of 14%. The total histamine content of the uterus changed throughout the reproductive cycle, increasing before ovulation, reaching a maximum during ovulation and then decreasing after embryo implantation. This suggests that the implanting embryo, interacting with the uterus, may be capable of inducing the release of histamine. The embryo-derived histamine releasing factor (EHRF) that we have described previously is capable of inducing 22% histamine-release on uterine mast cells, thus supporting this hypothesis.

Animals↗
PubMed2005

Embryo afterloading: a refinement in embryo transfer technique that may increase clinical pregnancy.

OBJECTIVE: Given the importance of ET technique during assisted reproductive technology cycles, we evaluated the effect of embryo afterloading subsequent to placement of the ET catheter on pregnancy rates vs. a standard direct ET. DESIGN: Retrospective cohort analysis. SETTING: University-based assisted reproductive technology program. PATIENT(S): Patients undergoing a fresh nondonor day 3 ET by a single provider over a 1-year period. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): One hundred twenty-seven patients met inclusion criteria, and the overall pregnancy rate was 46.5%. There was no difference between the two groups with respect to age, basal FSH, or number of embryos transferred. The ET method used was at the discretion of the provider. There was no difference between the two groups in the presence of blood on the transfer catheter. However, there were significantly more transfer catheters with mucus contamination in the direct transfer group (25.58% vs. 5.95%). The clinical pregnancy rate in the group with ET using the afterloading technique was higher than in the direct ET group (52.4% vs. 34.9%). CONCLUSION(S): There was a trend toward an increase in pregnancy rate when an embryo afterloading technique was used. A prospective randomized trial is needed to examine this issue.

Adult↗