Intracellular signal transduction in mouse oocytes and irradiated early embryos.

In order to determine the effect of X-irradiation on intracellular signal transduction in mouse oocytes and embryos, JNK, ERK and p38 kinase activities were measured by the state of phosphorylation of their respective substrates (c-Jun, Elk-1 and ATF-2, respectively) in two mouse strains differing in radiation sensitivity, namely C57BL and BALB/c. In a first step, control oocytes and embryos were compared for their respective kinase activities at various stages of oocyte maturation (germinal vesicle and metaphases of 1st and 2nd meiosis stages) and early embryonic development (1-, 2-, 4-, 8- and 16-cell, morula and blastula stages). Levels of p38, ERK or JNK kinase activities were shown to vary with the stage of oocyte maturation and embryo development. In a second step, 1- and 2-cell embryos were X-irradiated with 2.5 Gy during the S-phase of the 1st or the 2nd cell-cycle, respectively. There were no significant differences in p38, ERK and JNK kinase activities between control and irradiated embryos, whatever the stage or mouse strain was considered. In conclusion, p38, ERK and JNK kinase activities were shown to vary during oocyte maturation and early embryonic development. Apparently, X-irradiation did not affect these kinase activities at the 1- and 2-cell stages in either mouse strains regardless of their difference in radiation sensitivity.

Treatment with 5-aminolevulinic acid and photoactivating light causes destruction of preimplantation mouse embryos.

OBJECTIVE: To evaluate the direct effect of photodynamic treatment with 5-aminolevulinic acid (ALA) on preimplantation mouse embryos in an in vitro setting. DESIGN: Preimplantation mouse embryos were incubated with or without ALA for 5 hours and followed immediately by light exposure for 0, 5, or 15 minutes. Comparison of the viability and blastocyst formation was made among different treatment groups. SETTING: A conventional laboratory setting with embyro culture facilities. INTERVENTIONS: Female CD1 mice were superovulated with pregnant mare serum gonadotropin and hCG before mating. Four-and eight-cell embryos and compacted morulae were flushed from the oviducts and incubated with 0, 0.1, 0.5, 1.0, or 5.0 mM ALA for 5 hours. Embryos subsequently were exposed to photoactivating light for 0, 5, or 15 minutes. MAIN OUTCOME MEASURES: Microscopic assessment of embryos quality at 12 hours and determination of the percentage of embryos progressing to the blastocyst stage at 36 or 60 hours. RESULTS: Incubation of embryos with 0.1, 0.5, 1.0, 5.0 mM ALA without light resulted in 87.3% +/- 1.6%, 84.9% +/- 3.4%, 81.4% +/- 1.8%, and 82.8% +/- 4.7% of the embryos developing to blastocysts, respectively. In the absence of ALA, light exposure for 0, 5, or 15 minutes resulted in 93.8% +/- 2.3%, 92.3% +/- 2.2%, and 85.9% +/- 1.7% blastocyst formation. Combining treatment of ALA at the same concentrations with light resulted in 33.3% +/- 2.1%, 0.7% +/- 0.9%, 0%, 0% (5-minute light), 13.3% +/- 1.0%, 0%, 1.6% +/- 1.3%, 0% (15-minute light) blastocyst formation, respectively. When gross morphology was used to assess embryo viability at 12 hours, similar results were observed. Measurement of the fluorescent spectrum of embryos incubated with ALA indicated that protoporphyrin IX had been formed. CONCLUSION: Photodynamic ablation of mouse embryos was achieved with ALA under in vitro conditions. These results indicate that preimplantation mouse embryos are capable of converting ALA to the photosensitizer, protoporphyrin IX, and are susceptible to subsequent photoablation. A photodynamic effect on the embryo may be important to the successful application of this technique to the treatment of human ectopic pregnancy.

The biology and radiobiology of in utero development in relation to radiological protection.

Aspects of intrauterine development relevant to radiological protection but not considered in the recently revised Recommendations of ICRP (1991a) include (i) recently acquired evidence on the totipotency of cells of the morula with implications for dosimetry and risk in the first week of human development in utero; (ii) much older evidence on the earliest stages of myelo- and lymphopoiesis in the human subject that bears on radiosensitivity to leukaemia induction in utero; (iii) much older evidence on species differences that may affect the application to the human of experimental work on radionuclide transport from mother to embryo and fetus; (iv) recent proposals of alternative mechanisms for localization of functions in the developing mammalian forebrain with implications for risk of mental impairment caused by low level irradiation in utero, and (v) a recent demonstration that UNSCEAR (1977) was mistaken when concluding that a high radiosensitivity for teratogenesis is characteristic of the embryo and of preimplantation irradiation. The 1990 Recommendations of ICRP (1991a) to limit exposure of the fetus to radionuclides by reference to the occupation ALI for adults, i.e. women of reproductive age, seems to be a serious mistake.

Ultrastructure of preimplantation rabbit embryos exposed to visible light and room temperature.

Early cleavage stage embryos (day 1 p.c.) and morulae (day 3 p.c.) of rabbits were exposed to visible (standard) lighting (1600 lx) and room (standard) temperature (23 degrees C) during a 24 h in-vitro culture. Control embryos were cultured in darkness at 37 degrees C. Development was assessed by light and electron microscopy as well as by the cytochemical demonstration of glycogen. In day 1 and day 3 embryos standard temperature induced swelling of the SER and Golgi complex vesicles. Major changes in day 1 embryos consisted of smallish microtubules - like crystalloids, and in day 3 embryos of unusually large SER vesicles. In both embryonic ages cleavage rate and development was more retarded by standard temperature than by standard lighting. Standard lighting, however, led to distinct signs of degeneration and cell death. The mode of cell damage seemed to be different in light exposed early cleavage stages and morulae: In day 1 embryos cytoplasmic degeneration was predominant while the majority of cells in day 3 embryos died by apoptosis. Despite clear indications of cell damage, cleavage rate was not notably impaired compared with non-exposed controls. Glycogen increased during development from cleavage stages to early blastocysts. The distribution was not changed either by exposure to standard temperature nor by standard lighting. The results demonstrate that day 1 embryos were clearly more susceptible to lighting whereas day 3 embryos were more affected by temperature. The mode of damage exerted by both the physical environmental factors was different. Reduction to standard temperature interfered mainly with the organization of the cytoskeleton and intracellular transport of organelles, while exposure to standard lighting led to cell degeneration and death.

Lethality of a tritiated amino acid in early mouse embryos.

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.