Objetivo
The project will contribute to an evaluation of the radiosensitivity of guinea-pig oocytes. Several parameters can be used to assess the radiosensitivity of the germ cells: the project will concentrate on long term reproductive effects and on cytogenetic effects.
The influence of various factors (strain, methodological factors) on the response of preimplantation mouse embryos to X-irradiation was investigated. The BALB/c zygote was used to study the mechanisms of radiation induced 1 cell block (a particular and very pronounced form of G2 arrest). Radiation induced G2 arrest is considered by some to be a passive consequence of damamge to a proteinaceous target whose integrity is a prerequisite for progression to mitosis. Recent evidence suggests that repair of this lesion is not essential for G2 cell progression, and that G2 arrest could be an active reponse to irradiatial, triggered by defects not directly concerned with progression. Results obtained in this study support the active response hypothesis; the 1 cell block could not be induced before 17 h after giving human chorionic gonadotrophin, a time corresponding to the formation of the pronuclei in the zygotes.
However some of the results also lent support to the passive response hypothesis: Synthesis of critical proteins was found to occur after the cycloheximido transition point in irradiated embryos. Such protein synthesis is insufficient alone to induce the division of blocked embryos, but is necessary to allow the recovery from G2 arrest in the presence of caffeine. Studies with doses of 1 or 2 Gy or X-rays showed that zygotes cleaving after G2 arrest carry multiple chromosome aberrations and die after a few more divisions, and with much lower doses zygotes undergoing even a slight division delay are also unable to reach the blastocyst stage.
The absence of division in the mouse zygote could be indirectly linked to an absence of modifications of critical polypeptides, occurring at a posttranslational level during mitosis. This is supported by the absence of specific phosphorylations observed in the blocked zygotes at the normal time of mitosis and which, with the absence of the second round of replication, constituted the only visible qualitative difference with control embryos, up to the end of the second cycle.
The study showed that the use of superovulating hormones did not increase the radiosensitivity of the mouse zygote and that zygotes displayed similar radiosensitivities in vitro and in vivo.
The radiosensitivity of resting oocytes is being investigated in the guinea pig. The guinea pig possesses 2 populations of oocytes, 1 with a large nucleus and comparable to that of other mammals including man, and another with a contracted nucleus which appears a few days after birth and predominates as the animal ages. The study concentrates on the long term reproductive effects and cytogenetic effects of X-ray exposure.
Normal 26 day old embryo ovaries were found to contain only oogonia, either in interphase or in mitotic prophase or metaphase. Ovaries of 32 day embryos showed the additional presence of oocytes in the first stages of meistic prophase (leptotenes and a few zygotenes), while those of 41 days showed oogonia and all stages of meiotic prophase, with great majority of zygotenes and pachytenes and a few diplotenes. Newborn animals possessed only large type diplotene oocytes plus low numbers of maturing oocytes. In adult animals (from 4 months) the large diplotenes had mostly been replaced by those of the contracted type (85-95%).
For cytogenetic studies guinea pig oocytes were matured in vitro. Meiotically competent oocytes were incubated in culture medium for 6 hours (for first meiotic metaphane) and for 20 hours (for second meiotic metaphase). Surroudning cumulus cells and zona pellucida were removed, followed by fixation and chromosome preparation. Cytogenetic analysis could only successfully be carried out on first meiotic metaphase preparations. Female guinea pigs were irradiated at various times of pregnancy with either 2 or 6 Gy of X-rays, and the influence of this treatment on delivery and subsequent fertility (4 months after treatment) examined. Fetuses irradiated in utero were also tested for fertility, as well as 1 animal irradiated with 2 Gy on day 0.5 postpartum at the end of the experiment ovaries from each group were fixed and examined. The conclusions of the experiment are as follows:
Irradiation with 6 or 2 Gy before day 32 of pregnancy is lethal to the embryos.
After that time, irradiation of adult (pregnant) animals and their fetuses with 2 Gy does not appear to effect the oocyte populations and still allows pregnancy 4 months after treatment.
Irradiation of adult (pregnant) animals and their fetuses with 6 Gy strongly reduces the number of resting and maturing oocytes. However, this effect still allows pregnancy 4 months after treatment.
Irradiation of the newborn animal with 2 Gy strongly reduced the number of resting oocytes and could increase the proportion of large oocytes. However this did not affect subsequent fertility.
On the basis of these results doses of 2 and 4 Gy X-rays were chosen for the long term reproductive study. These doses were administered to adult pregnant and not pregnant animals, fetuses (32 day) in utero, and newborn (0 to 1 day old) animals.Preliminary histological analysis show the target cells to be, respectively, the contracted resting oocytes (adults), the oogonia and oocytest at the beginning of meiotic prophase (fetuses in utero) and the large resiting oocytes (newborn).
Guinea pigs were irradiated at day 0.5 postpartum and mated at 6 months. No difference could be seen between experimental groups (control, 2 Gy and 4 Gy) for the parameters tested (percentage of females giving young; number of young/females; weight of young). In addition all young were normal. Ovaries from each group were examined. Irradiation with 2 Gy did not affect the transformation of large oocytes into contracted oocytes, and the number of oocytes at the different stages was not diminished 7 to 8 months after treatment. In animal given 4 Gy the proportion of large oocytes to contracted oocytes was not affected, however there was a reduction in the number of resting oocytes in 2 of the 3 animals examined.
In these experiments, irradiation will be performed during intrauterine life, early postnatal life or adult life, in order to compare the radiosensitivities of oocytes at different stages. Two doses of XArays will be delivered, based on results obtained in a preliminary study. 6 and 12 months after treatment, exposed females will be mated with untreated males and the fertility of the animals will be followed. 3 to 4 weeks after the second mating, females will be killed and, in pregnant animals, the number of corpora lutea, live and dead embryos will be counted and the percentage yield of dominant lethal mutations will be calculated. In each experimental group some ovaries will be fixed for histological examination in order to determine the effects of treatment on oocyte killing.
Simultaneously with the long term reproductive study, a method will be developed for culturing the guinea-pig oocytes and for obtaining preparations of their metaphase I and metaphase II chromosomes. This will be followed by investigations on the induction by radiation of translocations in the female germ cells of this species. The guineaApig differs from other rodents in that the ovary of the postnatal animal contains two different populations of oocytes at diploten. A large oocyte comparable to that of other mammals and a contracted type, which predominates as the animal ages. The sensitivities of these two cell types of resting oocytes to the induction of translocations by radiation will be evaluated and compared.
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2400 MOL
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