Fetal abnormalities produced after preimplantation exposure of mouse embryos to ammonium chloride

BACKGROUND: The aims of this study were to determine whether preimplantation exposure of mouse embryos to ammonium resulted in abnormal fetal development and to evaluate similar risks to the outcome of human assisted conception. METHODS: Mouse embryos cultured from the 1-cell stage were exposed to 0.3 mmol/l ammonium chloride for 3 days. Embryos cultured from the 2-cell stage were exposed to 0.3 or 0.6 mmol/l ammonium for 2 days. After transfer to the uteri of pseudopregnant recipient females, post-implantation development was evaluated on embryonic day 15.5 (E15.5) or E18.5. RESULTS: There was no consistent effect of preimplantation exposure to ammonium chloride on fetal or placental weights. All 101 E18.5 fetuses were normal but 5/217 E15.5 fetuses were abnormal (three exencephalic and two polydactylous), which was significantly higher than the 0/363 for the pooled groups of E15.5 control fetuses (P = 0.007). The combined E15.5 and E18.5 frequency was also significantly higher than the controls (5/318 versus 0/433; P = 0.013). CONCLUSIONS: These results support the conclusion that abnormal preimplantation culture conditions can cause fetal abnormalities in mice, but the risks may be lower than previously suggested. Further work is needed to evaluate the risk more fully but this risk should be considered when designing new strategies for human assisted conception.     

Clastogenic effects of transplacental exposure of mouse embryos to nitrogen mustard or cyclophosphamide

Embryos from day 12 pregnant Swiss mice given intraperitoneal injections of nitrogen mustard (HN2) or cyclophosphamide (CP) were evaluated for chromosomal aberrations. Both agents induced dose-dependent increases in the frequency of cells with aberrations observed in embryos from females treated 6 hr before sacrifice. The highest frequencies of cells with aberrations were observed when females were injected 15 or 18 hr before sacrifice on day 12. A teratogenic dose of HN2 (1.0 mg/kg) induced significantly higher frequencies of damaged cells than a teratogenic dose of CP (20 mg/kg). Cytogenetic analysis of rodent embryos from pregnant females exposed to xenobiotic agents may be an effective screening test for evaluation of genetic effects induced by transplacental exposure.     

In vivo staining of mouse preimplantation embryos with hoechst 33342

Female mice bearing secondary oocytes or preimplantation embryos of various stages were injected with Hoechst 33342 (H 33342), either intravenously or intraperitoneally and sacrificed at various intervals afterward. The dye penetrated through the wall of oviduct and stained the nuclei of its cells as well as mitotic chromosomes of secondary oocytes, the pronuclei of one-cell embryos, and nuclei of blastomeres and polar bodies. The nuclear fluorescene was observed in living cells flushed out of the oviduct as well as in those fixed in oviduct, embedded in paraffin, and sectioned. H 33342 injected in a dose of 10 μg/g of body weight failed to interfere with preimplantation development of stained embryos as indicated by the number of blastocysts per mouse and number of cells per blastocyst.     

In vivo staining of mouse preimplantation embryos with Hoechst 33342.

Female mice bearing secondary oocytes or preimplantation embryos of various stages were injected with Hoechst 33342 (H 33342), either intravenously or intraperitoneally and sacrificed at various intervals afterward. The dye penetrated through the wall of oviduct and stained the nuclei of its cells as well as mitotic chromosomes of secondary oocytes, the pronuclei of one-cell embryos, and nuclei of blastomeres and polar bodies. The nuclear fluorescence was observed in living cells flushed out of the oviduct as well as in those fixed in oviduct, embedded in paraffin, and sectioned. H 33342 injected in a dose of 10 micrograms/g of body weight failed to interfere with preimplantation development of stained embryos as indicated by the number of blastocysts per mouse and number of cells per blastocyst.     

An assessment of the effects of cyclophosphamide and sodium valproate on the viability of preimplantation mouse embryos using the fluorescein diacetate test

The fluorescein diacetate test is a measure of both esterase enzyme activity and membrane integrity and it has been shown to be useful in assessing the viability of a variety of cells cultured in vitro, mouse embryos after freezing and thawing, and mouse embryos grown under inadequate culture conditions. In this study, the effects were examined of cyclophosphamide and sodium valproate administered respectively to pregnant inbred CBA mice 60 h after fertilization, on the viability of 84-h blastocysts. Cyclophosphamide 20 and 40 mg/kg bodyweight and sodium valproate 90 mg/kg significantly increased the number of nonviable blastocysts. Cyclophosphamide 4 mg/kg and sodium valproate 23 and 45 mg/kg did not adversely affect blastocyst viability. The intensity of embryo fluorescence correlated well with the subsequent development of the embryos in culture (r = 0.863; p less than 0.001). The test had a sensitivity of 83% and a specificity of 100%. Exposure of embryos to fluorescein diacetate under the conditions of this experiment did not adversely influence the subsequent postimplantation development of 84-h blastocysts cultured in vitro for a further 120 h. These findings suggest that the fluorescein diacetate test is a simple, rapid, and nontoxic procedure that may be useful in assessing the viability of preimplantation embryos after exposure to embryotoxic drugs and chemicals.     

Expression of H-Y antigen on preimplantation mouse embryos

The expression of H-Y antigen on preimplantation mouse embryos has been studied by complement dependent cytotoxicity. Embryos at the 8-cell stage were exposed to anti-H-Y and complement and then scored for cell death. Overall, half of the treated embryos were killed, retarded, or contained dead cells. Of those embryos which were not affected by anti-H-Y, 92% were female. In control experiments, neither normal serum plus complement nor complement alone had any specific effect on male embryos. It is concluded, therefore, that the H-Y antigen is present on preimplantation mouse embryos.     

Cryopreservation of in vitro cultured mouse preimplantation embryos

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.     

Effect of opioids on development of preimplantation mouse embryos

All-Union Research Center for Maternal and Child Care, Ministry of Health of the USSR. N. I. Vavilov Institute of General Genetics, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR E. M. Vikhlyaeva.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 622–624, November, 1989.     

Protein synthesis in vivo by preimplantation hamster embryos

The level of protein synthesis by hamster embryos in vivo, as inferred from the incorporation of ³⁵S methionine, was determined for the four days of the preimplantation period by radioautography. The embryos incorporated ³⁵S methionine throughout the preimplantation period. Furthermore, the hamster eggs continued to incorporate ³⁵S methionine following ovariectomy. Therefore, the pattern of protein synthesis in hamster eggs differs from the mouse with its low levels in cleaving eggs and in blastocysts following ovariectomy and high levels in blastocysts influenced by estrogen and progesterone. This finding is of interest because unimplanted mouse embryos remain viable in ovariectomized females for many days, whereas hamster embryos rapidly degenerate. It is suggested that the inability of hamster eggs to reduce the level of protein synthesis and become “dormant” may prevent their survival if implantation is blocked by ovariectomy.     

Fatty acid metabolism in human preimplantation embryos

BACKGROUND: Little is known of fatty acid metabolism in human embryos. This information would be useful in developing metabolic tests of embryo quality and improving embryo culture media. METHODS: The fatty acid composition of human embryos and their ability to accumulate 13C labelled fatty acids was assessed in relation to the stage of development using gas-chromatography and combustion-isotope-ratio-mass spectrometry. RESULTS: Compared with embryos which did not develop beyond the 4-cell stage, those that did had significantly higher concentrations of the unsaturates, linoleic (12% versus 3%; P=0.02) and oleic (14% versus 7%; P=0.02), and a lower concentration of total saturates (62% versus 77%; P=0.04). There was uptake of both 13C linoleic and palmitic, but the developmental pattern was different for each fatty acid. The net accumulation in pmol/embryo/24h for palmitic was 1 at the 2-cell to <8-cell stage, 4 at the 8-cell-morula stage and negligible at the blastocyst stage. For linoleic, there was little net accumulation at the 2-cell to <8-cell stage, 8 (8-cell-morula stage) and 17 pmol/embryo/24 h (blastocyst stage). CONCLUSION: Preimplantation human embryos actively take up individual fatty acids at different rates at different stages of development. The high unsaturated concentration at the later stages of development may be explained by preferential uptake of linoleic acid.     

Biogenic monoamines in mouse oocytes and preimplantation embryos

N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. Institute of Experimental Biology, Academy of Sciences of the Kazakh SSR, Alma-Ata. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 6, pp. 577–578, June, 1990.     

Effects of in vivo and in vitro exposure to rhodamine dyes on mitochondrial function of mouse embryos

Cationic rhodamines (Rh 123 and Rh 6G) can cause developmental toxicity in mice and inhibit embryonic mitochondrial respiration following in vivo or in vitro dye exposure. Rh B, a neutral rhodamine, fails to show such effects at comparable doses. To assess effects of rhodamines on development, F0F1ATPase activity and ADP translocation were measured on gestation day (GD) 12 in embryonic and adult mitochondria. ATP synthesis in embryonic mitochondria transplacentally exposed to Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) given to dams by i.p. injection from GD 7 to 10 were inhibited 39% and 49%, respectively. When isolated mitochondria were treated, dose-dependent inhibition was seen; at 5 micrograms of dye/mg mitochondrial protein, ATP synthesis was inhibited 65% and 81% by Rh 123 and Rh 6G, respectively. When F0F1ATPase activity was assessed, in vitro Rh 123 and Rh 6G exposures at levels up to 8 micrograms/mg mitochondrial protein resulted in enzyme inhibition, but at 10 micrograms/mg, ATPase activity was stimulated. Uncoupler-stimulated ATPase activity was also inhibited. ADP translocation was decreased by 19.1% and 37.7% by Rh 123 and Rh 6G, respectively, at dye concentrations of 20 micrograms/mg. Results of in vitro exposure of maternal liver mitochondria were similar to those for embryonic mitochondria, whereas liver from dams exposed in vivo on GD 7-10 was unaffected on GD 12. In vivo or in vitro treatment with Rh B did not affect any embryonic or maternal parameters. Such results support the hypothesis that inhibition of mitochondrial energy metabolism is a mechanism for the developmental toxicity of cationic rhodamines.     

Localization of green fluorescent protein in mouse preimplantation embryos

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 39–44, January, 2007     

One-step versus two-step culture of mouse preimplantation embryos

Sir, We would like to offer a different perspective on the studyreported by Biggers et al. (2005) whose findings are contradictoryto our own previous studies on these embryo culture media systems. In our previous publication comparing KSOMAA to sequential media(Gardner and Lane, 2002, 2003; Reed et al., 2003), we were carefulto ensure that all conditions used were exactly the same forthe medium being tested, e.g. the same volume of medium. Thiswas done because in our extensive experience, the volume ofmedium has a significant impact on culture outcome if thereare problems with the oil source used. Specifically, when suboptimaloil is used, embryo development increases with increasing volumesof medium. In the     

Quantitative analysis of cellular glutathione in early preimplantation mouse embryos developing in vivo and in vitro.

The relative levels of reduced glutathione (GSH) have been measured fluorimetrically in individual eggs and early embryos from two mouse strains, one of which shows developmental arrest in vitro. GSH levels fell by approximately 20-25% at fertilization and by approximately 45% by the late 2-cell and early 4-cell stages. No differences were observed between strains or between embryos cultured in vitro or in vivo. Addition of exogenous H2O2 or diethylmaleate depleted GSH. GSH levels were not affected significantly after inhibition of GSH-peroxidase by mercaptosuccinate nor of catalase by aminotriazole. Mercaptosuccinate did not inhibit development but catalase inhibition caused arrest at the 2-cell stage. Addition of exogenous GSH or thioredoxin did not promote development of 'blocking' embryos through the 2-cell block. It is concluded that early embryos lack a mercaptosuccinate sensitive peroxidase activity for removing H2O2, which may be removed by catalase or the glutathione-S-transferase system. It is suggested that GSH may have a role in detoxifying peroxidated lipids. The results are consistent with a role for reactive oxygen species in the 2-cell block.