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Reproduction (2009) 137 35-43
DOI: 10.1530/REP-08-0073
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RESEARCH

Cytogenetic damage in preimplantation mouse embryos generated after paternal and parental {gamma}-irradiation and the influence of vitamin C

Hossein Mozdarani and Elmina Nazari1

Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran 14117 13116, Iran1 Department of Science and Research, Azad University and Royan Institute, Tehran 19877 73354, Iran

Correspondence should be addressed to H Mozdarani; Email: mozdarah{at}modares.ac.ir


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Acknowledgements
 References
 
Cytogenetic damage expressed as micronuclei (MN) in 4–8-cell embryos generated after irradiation of male or male and female mice in the absence and presence of vitamin C was investigated. Male NMRI mice were whole body exposed to 4 Gy {gamma}-rays and mated with non-irradiated superovulated female mice in 6 successive weeks after irradiation in a weekly interval. In experiments involving irradiation of both male and female mice, irradiated male mice for 6 weeks post irradiation were mated with female mice irradiated after induction of superovulation. Effect of 100 mg/kg vitamin C (ascorbic acid) on the frequency of MN was also studied. Pregnant animals were euthanized and embryos flushed from the oviducts and fixed on slides. The rate of MN observed in embryos generated from irradiated male compared with control group dramatically increased (P<0.01). Frequency of MN in this group decreased dramatically after vitamin C treatment (P<0.01). Frequency of MN in embryos generated by mating both male and female irradiated mice was higher than that observed for those embryos generated by irradiated male mice alone. However, a considerable modifying effect of vitamin C was observed for this group too (P<0.05). Results indicate that irradiation of gonads during spermatogenesis and preovulatory stage oocytes may lead to unstable chromosomal aberrations and probably stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos expressed as MN. The way vitamin C reduces clastogenic effects of radiation on germ cells leading to reduced frequency of MN in pre-embryos might be due to its antioxidation and radical scavenging properties.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Declaration of interest
 Acknowledgements
 References
 
Ionizing radiation may lead to the induction of an elevated germ line mutation rate in the directly exposed parents. These mutational events may be an indirect effect on genome stability that is transmitted through the germ line of irradiated parents to their offspring (Dubrova 2003, Dasenbrock et al. 2005). Teratogenic effects of various types of ionizing radiation at different stages of pregnancy, death, and morphological changes in embryos and fetus of experimental animals have been shown (Hall 2001). Zygotes, embryos, and fetuses are known to be highly sensitive to the ionizing radiations (Muller & Streffer 1994, Gu et al. 2002). Experiments performed in laboratory animals suggest that ionizing radiation can induce DNA damage in the germ cells of exposed individuals and lead to various deleterious effects in their progeny, inducing miscarriage, low birth weight, congenital abnormalities, and perhaps cancer, depending on the period of gestation at which irradiation occurs (Jacquet 2004).

There is evidence indicating that in irradiated male mice, post irradiation spermatozoa exhibit increase in the incidence of abnormality shaped spermatozoa, preimplantation loss (Bateman 1958, Searl & Beechey 1974), and transmission of tumors to the F1 progeny (Nomura 1986). However, spermatozoa can retain a high fertilizing ability even after receiving moderate doses of {gamma}-irradiation. This suggests that radiation-induced DNA damages in spermatozoa may be transmitted to the next generation without being selected out at fertilization (Tateno et al. 1996). It has been shown that DNA-damaged sperms have the ability to fertilize the oocyte but, that embryonic development is very much related to the degree of DNA damage (Ahmadi & Ng 1999). The majority of de novo structural chromosome aberrations in fetuses and newborns are considered being of paternal origin, that is of sperm origin (Olson & Magenis 1988). Cytogenetic investigations on oocytes irradiated with various doses of X-rays at the immature stage or at different stages of follicular growth on mouse have shown that the sensitivity of the oocyte to radiation induced chromosome aberrations can vary greatly according to the stage of oogenesis reached at the time of irradiation (Jacquet et al. 2005). Investigation of chromosome anomalies in mouse oocytes after irradiation showed that during the preovulatory phase a high number of chromosome anomalies are induced and increased linearly with increasing dose (Reichert et al. 1975). However, the result of numerous publications suggests that radiation may also have an indirect effect on genome stability that is transmitted through the germ line of irradiated parents to their offspring (Dubrova 2003, Dasenbrock et al. 2005). Due to strong selection against several defects at the earliest stage of pregnancy, this genetic effect of radiation is likely to be difficult to detect in people.

It has frequently been suggested that the death of embryos post implantation is caused by chromosomal aberrations after exposure to ionizing radiation. Structural as well as numerical aberrations may be responsible for this effect. Both types of the aberrations can be expressed as micronuclei (MN) measured with the MN test in interphase cells (Muller & Streffer 1994). The MN test is a reliable in vivo test for evaluation of the clastogenic effects of mutagens and radiation. MN arise from acentric chromosome fragments or chromosomes that are not incorporated into daughter nuclei during mitosis (Fenech & Morely 1985). MN scoring in interphase cells has been proposed and used as the quick and easy substitute for the more difficult and time consuming metaphase aberration analysis (Fenech et al. 2003, Norpa & Falck 2003).

Studies using the MN assay have shown higher radiosensitivity of human spermatozoa in comparison with golden hamster, Chinese hamster, and mouse spermatozoa (Kamiguchi & Tateno 2002). In some experiments a dose and dose-rate-related increase in MN was seen in early spermatids with no difference between the different stages of differentiation were observed (Kunugita et al. 2002). Spermatids exposed to X-rays (400 cGy) during meiotic prophase had ~10-fold increases in MN compared with controls (Collins et al. 1992). Induction of MN by X-rays or neutrons in spermatogonia and spermatocytes showed no effect in spermatogonia but showed significantly different effect for all spermatocyte stages (Tates et al. 1989).

Preimplantation mouse embryos generated with X- or {gamma}-irradiated sperms showed abnormal segregation of chromosomes at the first cleavage with a high frequency of chromosomal aberrations and MN in these embryos (Pampfer & Streffer 1989, Shimura et al. 2002, Mozdarani & Salimi 2006).

It is known that ionizing radiation induces various types of DNA damages mainly through water radiolysis and formation of reactive oxygen species (ROS) such as free radicals. Normally, cells contain enzymatic antioxidant defense mechanisms, which serve to scavenge free radicals. However, ionizing radiation is such a potent free radical former that intracellular enzyme systems fall short of ridding the cell of excessive amounts of free radicals (Weiss et al. 1990, Athar et al. 1993). Various kinds of chemical agents (synthetics or naturally occurring) have been used so far as radioprotective agents in which their main mechanism of action is to scavenge free radicals (Bump & Malaker 1998). Unfortunately, most of these compounds at radioprotective doses were found to be toxic to humans (Foye 1998).

The modifying effect of treatment with several antioxidant agents such as vitamins C, E and β-carotene on the clastogenic activity of {gamma}-rays had shown radioprotective effect in mice (Konopacka & Rzeszowska-Wolny 2001). Vitamin C (ascorbic acid), the major water-soluble antioxidant in blood, tissue, and intracellular fluid, is a very potent free radical scavenger and has attracted tremendous attention. It has been shown that vitamin C and E reduced radiation-induced mutations and chromosomal damage in mammalian cells and radiation-induced lethality (Harapanhalli et al. 1996, Konopacka & Rzeszowska-Wolny 1998, 2001). Also vitamin C added at low concentration before exposure of the cells to radiation prevented induction of MN (Konopacka & Rzeszowska-Wolny 2001) and apoptosis in human peripheral leukocytes (Mozdarani & Ghoraeian 2008). In addition, administration of ascorbic acid has protected mice against radiation-induced sickness, mortality, and improves healing of wounds after exposure to whole-body {gamma}-radiation (Jagetia et al. 2004).

In a recent report, we have clearly shown that maternal irradiation at preovulatory stage prior to mating with unirradiated male mouse lead to a high frequency of MN formation in subsequent preimplantation embryos. The presence of vitamin C at the time of irradiation could effectively reduce the frequency of MN by a dose-reduction factor (DRF) of 2.9 (Mozdarani & Nazari 2007). Despite of numerous published studies on radiation, relatively little information is available on the paternal (only male) and parental (both male and female) irradiation and the incidence of specific chromosomal abnormalities expressed as MN in their preimplantation embryos. Study of parental irradiation and its genetic consequences is very important issue in case of radiological and nuclear accidents, where both males and females are affected. This led us to carry out a systematic study on this relationship and the influence of vitamin C as an antioxidant agent to decrease genetic transmittable abnormalities.

The aim of this study was therefore, to investigate the effects of paternal as well as parental {gamma}-irradiation of mice at various time intervals on the frequency of MN in preimplantation embryos in the absence and presence of 100 mg/kg of vitamin C.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Declaration of interest
 Acknowledgements
 References
 
The results obtained in three independent experiments are summarized in Tables 1 and 2, also shown in Figs 1 and 2. A total number of 795 embryos with 2970 cells in male irradiate alone group and 870 embryos containing 5358 cells in irradiated male treated with vitamin C group were analyzed for the presence of MN. As seen in Table 1 different numbers of pregnant mice were used to retrieve preimplantation embryos for each experimental group. This difference is because all mated female mice were not vaginal plaque (VP) positive in the morning following mating. Control value for embryos generated from untreated male and female mice was 11% and for male mice treated with 100 mg/kg vitamin C was 5.8%. The data in Table 1 indicate that in all irradiated groups, the yield of MN is dramatically higher than controls statistically significant with P<0.01. In the embryos generated from irradiated males after mating with unirradiated female mice, from the first until the sixth week post irradiation, the frequencies of MN were about 20, 24, 47, 49, 39, and 60% respectively which are significantly higher than control (P<0.01; Fig. 1A). The frequencies of MN in the embryos generated from irradiated males in the presence of 100 mg/kg vitamin C after weekly interval mating with unirradiated female mice for 6 successive weeks were about 9, 9, 16, 28, 23, and 38% respectively which are significantly higher than control (P<0.01) but significantly different from and lower than those only irradiated (P<0.01; Fig. 1A). A similar trend of MN formation following irradiation alone or irradiation in the presence of vitamin C was observed when the frequencies of MN were analyzed per 100 cells instead of 100 embryos; although with lower frequencies of MN and with higher protective effects of vitamin C (Fig. 1B).


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  		Table 1 Mean number of embryos, cells, and micronuclei analyzed after irradiation of male mice with 4 Gy {gamma}-rays alone or in combination with 100 mg/kg vitamin C, before mating at weekly intervals with non-irradiated female mice.

 

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  		Table 2 Mean number of embryos, cells, and micronuclei analyzed after irradiation of both male and female mice with 4 Gy {gamma}-rays alone or in combination with 100 mg/kg vitamin C, before mating at weekly intervals.

 

Figure 1
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  		Figure 1 Frequency of MN (percent) (A) per embryo and (B) per cell following mating of irradiated male in the presence and in the absence of 100 mg/kg vitamin C with an unirradiated female mouse at weekly intervals. Error bars show standard error of mean values obtained from three independent experiments.

 

Figure 2
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  		Figure 2 Frequency of MN (percent) (A) per embryo and (B) per cell following mating of irradiated male with an irradiated female mouse in the presence and in the absence of 100 mg/kg vitamin C at weekly intervals. Error bars show standard error of mean values obtained from three independent experiments.

 
In the other experimental group, where both male and female mice were mated after irradiation, a considerably high number of embryos and embryonic cells were analyzed for the presence of MN (1047 embryos containing 5394 cells for only irradiated mice and 735 embryos containing 4224 cells for irradiated mice after vitamin C injection). Detailed results are shown in Table 2. The control value for the frequency of MN in this group was about 14% and those controls treated with vitamin C exhibited about 9.5% MN in their embryos. The frequencies of MN in embryos generated from irradiated males after mating with irradiated female mice, from the first until the sixth week post irradiation increased dramatically compared with control value (P<0.01). MN frequencies was found to be about 44, 64, 70, 77, 69, and 87% respectively for successive 6-week mating of irradiated male and irradiated superovulated female mice at weekly intervals (Fig. 2A). These values are considerably higher than those obtained with the irradiated male group (Fig. 1A). Treatment of male and female mice with 100 mg/kg vitamin C 1 h before irradiation led to a marked decrease in the frequency of MN in preimplantation embryos generated from these mice (P<0.05; Fig. 2A). Similar frequencies of MN were found for both irradiated parents in the presence of vitamin C when the number of analyzed cells was considered for statistical analysis, although with lower values of MN for both treatments (Fig. 2B).

DRF calculated for the effect of vitamin C in this study for males only irradiated and mated with unirradiated female mice at various time intervals range from 1.6 to 2.98, when embryos are considered; and 2.3 to 3.78 when calculated for reduction of MN per 100 cells (Table 3). DRF for both male and female irradiated group was lower than the male only irradiated group. DRF with a range of 1.3–2.14 for embryos and 1.6–2.6 for cells were calculated for this group (Table 3). These figures are indicative that overall DRF produced by vitamin C for male only irradiated is about 3 and for male and female irradiated mice is about 2 for embryonic cells, which are desirable reducing effects for a radioprotector agent.


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  		Table 3 Dose-reduction factor (DRF) calculated for treatment with {gamma}-rays in the presence of vitamin C.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Declaration of interest
 Acknowledgements
 References
 
Micronuclei are the result of chromosomal aberrations induced during preceding mitotic division of blastomeres. These are from acentric fragment or lagging chromosomes induced by mutagens or clastogens such as ionizing radiation or result of non-disjunction (Fenech & Morely 1985, Norpa & Falck 2003, Mozdarani & Nazari 2007). Irradiation of embryos in the first mitotic division could induce chromosomal abnormalities after several blastomere divisions in embryos. Recently, it has been shown that irradiation of germ cells before mating lead to increased frequencies of chromosomal aberrations in subsequent preimplantation embryos (Mozdarani & Salimi 2006). These chromosome abnormalities can be expressed and visualized as MN.

The time required for spermatogenesis in mice for spermatozoa development from the stem cells is more or less constant (about 6 weeks). Accordingly, the fertilizing spermatozoa were at the following stages of development at the time of irradiation: epididymal sperm, condensing spermatids, round spermatids, pachytene spermatocytes, leptotene/zygotene spermatocytes and A/intermediate spermatogonia respectively (Meistrich et al. 1978, Muller et al. 1999). Data shown in Table 1 and Fig. 1 suggest that {gamma}-irradiation affects all the stages of spermatogenesis cycle in the male mice, for inducing MN. As seen, the frequencies of MN increased for all mating times post irradiation of male mice significantly different from controls (P<0.01). The increase in MN frequency from the first until the sixth week might indicate high sensitivity of spermatogonial cells to {gamma}-irradiation. The MN observed in embryos generated from irradiated male mice may be due to the presence of translocations as well as mitotic recombination involving inversions induced in a chromosome that affects chromosome pairing and meiotic segregation in male mice leading to aneuploid lagging chromosomes (Mozdarani & Salimi 2006) and unstable chromosomal aberrations such as dicentrics and acentric fragments in zygotes, expressed as MN. Radiation-induced DNA damage in spermatozoa may be transmitted to the next generation without being selected out at fertilization, because it has previously been shown that spermatozoa can retain a high fertilizing ability even after a high dose of irradiation (Kamiguchi & Tateno 2002). Damaged testicular cells not removed by apoptosis rely on DNA repair for their genome integrity to be preserved (Sailer et al. 1995). In mice, ionizing radiation has been shown to affect sperm DNA and lower development rate of the blastocysts (Ahmadi & Ng 1999). In an investigation on IVF rate of mouse eggs with sperm after X-irradiation at various spermatogenesis stages, Mastuda et al. (1985) have shown that the number of fertilized eggs seemed to remain constant almost at control level until the fourth week after X-irradiation reaching to a minimum level in the sixth week. The response to radiation exposure is very much dependent on the developmental stage of germ cells during which this exposure takes place. These changes are explained in terms of the differential sensitivity of cells to killing and aberration induction in the different phases of the cell cycle. Results obtained in the present study, shown in Fig. 1, are in agreement with other previous reports indicating radiosensitivity of all cell lineage in the spermatogenesis process (Collins et al. 1992, Tusell et al. 1995, Hasegawa et al. 1997, Kamiguchi & Tateno 2002, Cordelli et al. 2003).

The results presented indicate that the frequency of MN in preimplantation embryos generated from paternally and maternally irradiated mice was significantly higher than controls unirradiated (Tables 1 and 2, Fig. 2) and those embryos generated from male only irradiated mice (P<0.05). This effect might be due to DNA damage induced in spermatogenic cycle as well as preovulatory stage oocytes that are left unrepaired and transmitted to the zygote. However, there is evidence showing the repair capacity of male and female germ cells of mammals for DNA damage during gametogenesis. Repair of u.v. damage has been observed at all stages of germ cell maturation (Brazill & Masui 1978, Yoichi & Izuo 1989).

There is a possibility that the early mouse embryo has an excision repair capacity for damage in DNA of the male gamete before fertilization, after the sperm enters the egg cytoplasm (Yoichi & Izuo 1989). However, the extent of radiation damage induced both in preovulatory oocytes and spermatozoa might be so high that repair machinery of oocyte fails to repair majority of damages. The result of this study is consistent with the finding of other investigators regarding sensitivity of preovulatory stage to ionizing radiation for induction of chromosomal aberrations in mice (Reichert et al. 1975, Tease & Fisher 1986, de Boer & van der Hoeven 1991, Jacquet et al. 2005). Therefore, the increase of MN yield in pre-embryos might be explained in terms of DNA damages induced in cells forming the zygotes. It might be possible that DNA damage induced by ionizing radiation in the male (or female) gamete once transmitted to the embryo triggers genomic instability that results in the generation of new chromosomal damage (and hence new MN) at each cell division (Weissenborn & Streffer 1988, Niwa & Kominami 2001). Also, since chromosomes in dividing cells in spermatogenesis cycle and preovulatory oocytes are in replicated form, radiation-induced DNA damage especially double-strand breaks (DSBs) may convert to chromatid breaks. However, even if DNA damage is repaired in repair proficient cells, signaling of a single DSB triggers the cell to make a genomic rearrangement at the crossover points of a looped chromatin domain, possibly a transcription factory (Bryant 1998). If incomplete, the rearrangement leads to chromatid breaks associated with cancer predisposition. Rearrangements may also occur between sister chromatids leading to an inversion adjacent to the break site (Bryant 1998, Bryant & Mozdarani 2004). Completion of this rearrangement would lead to a transmissible inversion, a possible carcinogenic event. An alternative mechanism might also be involved in the process of radiation induced genomic instability that is transmissible over many generations of cells leading to increased occurrence of such genetic effects among the progeny of irradiated cell after many generations (Little 1998). In a study using HPRT mutant subclones, Little et al. (1997) have shown an indirect relationship between mutational instability and chromosomal instability. These authors suggested that some cells in a mutationally unstable population may develop chromosomal instability during proliferation of the paternal population from a single surviving cell (Little et al. 1997). However, either mechanism involved in transmission of genomic instability to preimplantation embryos leads to increased frequency of MN in embryonic cells. Increased frequency of MN following mating of irradiated male mice with unirradiated female mice at weeks 1 and 2 after irradiation (Fig. 1), where fertilizing spermatozoa were in the epididymal sperm and spermatid stages at the time of irradiation respectively, might be justified with the latter mechanism. Therefore, genomic instability, increased cancer incidence, elevated mutation rates in the germ line, and somatic tissues of the offspring (Dubrova 2003, Antoshchina et al. 2005, Dasenbrock et al. 2005, Uma Devi & Satyamitra 2005, Vorobtsova 2006), lethal and teratogenic events (Pils et al. 1999) from maternal as well as paternal irradiation may be inherited by their preimplantation embryos and offspring.

The effect of vitamin C treatment on the frequencies of MN in preimplantation embryos generated by irradiated male alone and parents is considerable for all study groups. As summarized in Tables 1 and 2 and shown in Figs 1 and 2, decrease in the frequencies of MN is significantly different compared with irradiated only group (P<0.01). However, reducing effect of vitamin C was not as much as observed for those embryos generated by irradiated male in the presence of vitamin C and mated with unirradiated female, so that DRF calculated for parental irradiated is about 2 compared with DRF of about 3 for paternal irradiation when the frequencies of MN in total embryonic cells was considered for calculation. This observation might not prove the inefficacy of vitamin C of ridding the cells of excessive amounts of free radicals induced by radiation, but might be indicative that presence of unrepaired or misrepaired DNA damage in each gamete can lead to an increased rate of chromosomal abnormalities in subsequent embryos.

Generally, the protective effect of ascorbic acid can be explained by scavenging ROS due to its antioxidant property. Several other studies have established the radioprotective value of vitamins C, E, and carotenoids in protecting normal cells (Dura Kovic 1993). Administration of ascorbic acid protected mice against radiation induced lethality (Guaiquil et al. 2001). It has been reported that the addition of vitamin C in a single dose before irradiation reduced the level of DNA damage to normal cells (Bergsten et al. 1990). Also the prevention in radiation-induced MN as well as apoptosis was found in human lymphocytes when vitamin C was added at low concentration before exposure of the cells to radiation (Konopacka & Rzeszowska-Wolny 2001, Mozdarani & Ghoraeian 2008). Direct interaction of ascorbic acid with DNA might be another explanation for its protective effect. A marked protective effect on DSB was observed with ascorbic acid (Yoshikawa et al. 2006). Yoshikawa et al. (2003) have shown that ascorbic acid in millimolar concentration induces condensation in the higher order structure of giant DNA. Therefore, changes induced by ascorbic acid in the higher order structure of DNA may be closely associated with its ability to protect against DSB. Protective effect of vitamin C on the fertilizing spermatozoa in the first week post irradiation that was in epididymal sperm stage at the time of irradiation (Fig. 1A and B) might be partly due to radical scavenging and antioxidation mechanism of vitamin C in reducing radiation induced free radicals in the vicinity of DNA molecules of spermatozoa, hence reducing damage to DNA. It is believed that differentiating spermatids and spermatozoa are not able to repair their DNA damages as potent as somatic or other germ cells (Mastuda & Tobari 1989). Moreover, vitamins work as coenzymes meaning they help enzymes facilitate necessary molecules, perhaps involved in cellular repair processes. One or all of these mechanisms may be involved in the protective effects of vitamin C against radiation induced DNA damage in mouse gametes leading to lower frequencies of MN induced in preimplantation embryos generated by mating of irradiated mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Declaration of interest
 Acknowledgements
 References
 
Animals
NMRI mice aged 6–8 weeks were purchased from Razi Institute (Karaj, Iran). Male mice were housed singly in plastic cages; females were housed 4–5 per cage at least for 1 week before being used for experiments. The animals were housed and maintained on a 12 h light:12 h darkness cycle at 20–24 °C and 60–70% relative humidity. Mice were fed with standard breeding granulated diet and water ad libitum. Females and males were randomly assigned to control or treatment group and mated overnight after the induction of superovulation in females using i.p. injection of 10 international units (IU) of pregnant mare's serum gonadotropin folligon (PMSG; Intervet, Boxmeer, The Netherlands) followed by injection of 10 IU of human chorionic gonadotropin (HCG; Organon, Oss, The Netherlands) with a 42–48 h intervals. This study was approved by the Ethical Committee of the Tarbiat Modares Universiy and animals were treated according to the university regulations. A flow diagram of the experimental protocol is shown in Fig. 3.


Figure 3
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  		Figure 3 A flow diagram showing experimental protocol used in various stages of the study. VP+, vaginal plaque positive (pregnant mice); PMSG, pregnant mare's serum gonadotropin; HCG, human chorionic gonadotropin.

 
Vitamin C treatment
L-Ascorbic acid, known as vitamin C (Darupakhsh, Tehran, Iran), was administered i.p. one hour before irradiation at a dose of 100 mg/kg. Also a control group receiving only vitamin C was considered for possible genotoxic effect of the dose used in the study. The rationale for using this concentration of vitamin C (100 mg/kg) was based on previous experiences (El-Nahas et al. 1993, Konopacka & Rzeszowska-Wolny 1998, Mozdarani & Nazari 2007). It was shown that this concentration could effectively reduce radiation-induced chromosomal aberrations and MN in bone marrow cells as well as in preimplantation embryos following whole body {gamma}-irradiation.

{gamma}-Irradiation and coupling
Paternal (male only) irradiation
Male mice were whole body irradiated alone or in the presence of vitamin C with 4 Gy {gamma}-rays generated from a cobalt-60 source (Theratron II, 780 C, Canada) at a dose rate of 1.32 Gy/min, with source sample distance=82 cm, field size: 20x20 cm at room temprature (23±2 °C). Four days after {gamma}-irradiation, irradiated male mice were mated with superovoulated non-irradiated females in successive 6 weeks at weekly intervals. Three to five irradiated mice were assigned for coupling in each experimental group. Two unirradiated female mice were transferred with an irradiated male to a cage overnight to mate. The next morning female mice were checked for VP. A VP positive female was considered as a pregnant mouse (Fig. 3). A control unirradiated or only vitamin C treated animals were assigned for experimental groups. Because all experiments were repeated three times, in order to kill fewer mice, two control groups were assigned for two corresponding time points for each experiment, so that there was a control for all experimental groups. The sampling time for controls was similar to the corresponding treated experimental group. However, the number of embryos and embryonic cells analysed as control well correspond with other experimental groups.

Parental (male and female) irradiation
Male mice were irradiated alone or after vitamin C injection as described above. Female mice were whole body irradiated alone or in the presence of vitamin C at a dose of 4 Gy, with irradiation condition described earlier, ~18–20 h after PMSG injection and 24 h prior to HCG injection. Four days after {gamma}-irradiation, irradiated male mice, for successive 6 weeks at weekly intervals, were mated with superovulated irradiated females (Fig. 3). Three to five irradiated male mice were assigned for coupling in each experimental group. Two irradiated female mice were transferred with an irradiated male to a cage overnight to mate. The next morning a VP positive female was considered as a pregnant mouse. A control unirradiated or only vitamin C treated animals were assigned for experimental groups as described for the male only irradiated group. All experiments were repeated three times.

It is worthy to mention that several authors used the dose of 4 Gy for studying radiation-induced genomic instability (Konopacka & Rzeszowska-Wolny 1998), in vitro cytogenetic studies on human and mouse germ cells (Tusell et al. 1995, Jacquet et al. 2005) and prenatal effects of {gamma}-irradiation (Bang et al. 2002).

Embryo recovery and slide preparation
At about 68 h post HCG injection, the pregnant females were killed by cervical dislocation method and their oviducts were flushed using a special flushing syringe (Supa, Tehran, Iran) filled with 37 °C incubated T6 medium (ingredients for pH of 7.2–7.4; NaCl (4.73 mg/ml), KCl (110 µg/ml), NaH2PO4 (50 µg/ml), MgCl2.6H2O (100 µg/ml), CaCl2.2H2O (260 µg/ml), NaHCO3 (2.10 mg/ml), Phenol Red (10 µg/ml), EDTA (6 µg/ml), glucose (1 mg/ml), and Na-pyruvate (30 µg/ml) purchased from Sigma; Penicillin G (60 µg/ml) and Streptomycin (50 µg/ml) from Seromed, Germany and Na-lactate (1.98 µg/ml) from Merck) under a stereomicroscope (Hund-Wetzlar, Wetzlar, Germany) to obtain 4–8-cell embryos. The collected morphologically normal embryos were transferred to fresh T6 medium supplemented with 15 mg/ml BSA (Sigma). Embryos then were transferred to Tyrode's acid [ingredients for pH 2.5; NaCl (8 mg/ml), KCl (2 mg/ml), MgCl2.6H2O (0.1 mg/ml), CaCl2.2H2O (0.25 mg/ml), glucose (1 mg/ml), and polyvinylpyrrolidone (4 mg/ml) all from Sigma) to remove the zona pellucida. This process was followed under a stereomicroscope (Nikon, Tokyo, Japan) to avoid damage to the blastomeres. Then embryos were transferred to a watch glass containing 1% sodium citrate (Sigma) as a hypotonic solution for 30 min. Embryos were placed on a pre-cleaned slide and fixed with a drop of fixative consisting of methanol and acetic acid (3:1; Merck). After leaving overnight at room temperature, slides were stained in 4% Giemsa (Merck) for 3 min and cells were analyzed under a light microscope (Nikon) at 400xmagnification to screen MN in blastomeres. All slides were randomized and evaluated by the same scorer blindly. Criteria described by Fenech et al. (2003) were followed for MN identification and scoring.

Statistical analysis
Data were statistically analyzed and the significance of any inter-group differences was evaluated with Mann–Whitney U-test to compare two groups and one-way ANOVA to compare three or more groups using SPSS (version 12) software (SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered as significant.


   Declaration of interest
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
Acknowledgements
 References
 
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


   Funding
 
This work was supported by Royan Institute and Tarbiat Modares University.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Acknowledgements
References
 
Authors would like to thank Dr M H Zahmatkesh for his help in {gamma}-irradiation of mice. Technical help from Ms M Salimi is highly appreciated.

Received February 16, 2008
First decision September 25, 2008
Revised manuscript received March 19, 2008
Accepted September 29, 2008

   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Acknowledgements
 References
 

Ahmadi A & Ng SC 1999 Fertilizing ability of DNA damaged spermatozoa. Journal of Experimental Zoology 284 696–704.[CrossRef][Web of Science][Medline]

Antoshchina MM, Riabchenko NI, Nasonova VA, Fesenko EV & Pelevina II 2005 The genome instability in the descendants of the Chinese hamster of the cells, irradiated by the low dose and by various intensities of gamma-radiation. Radiatsionnaia Biologiia, Radioecologiia 45 291–293.

Athar M, Abdula M, Sultana S & Favier A 1993 Free radicals and trace elements. Journal of Trace Elements in Experimental Medicine 6 65–73.

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