Cellular reprogramming in the F3 mouse with paternal F0 radiation history.

PURPOSE: In the mouse, paternal F0 acute irradiation of Type B spermatogonia produces biological effects in offspring, including altered signalling kinase activities and protein levels. It was hypothesized that these effects represented cellular reprogramming that would alter the response of somatic cells in these offspring to an acute ionizing radiation exposure. MATERIALS AND METHODS: Nineteen-day-old third generation (F3) CD1 mice with and without an acute 1.0 Gy paternal F0 radiation history each received an acute dose of 1.0 Gy from attenuated 137C n-rays. Kidney PKC and MAPK activities, and p53 protein levels were evaluated immediately following F3 irradiation. The same endpoints and DNA damage were evaluated in kidney-derived fibroblast primary cell cultures 3 weeks post-irradiation. RESULTS: Kidneys had significantly decreased PKC and MAPK activities and p53 protein levels related to F0 irradiation and increased PKC and MAPK activities following F3 irradiation irrespective of F0 radiation history. Kidney-derived fibroblasts had significant changes or strong trends for all selected endpoints based upon cross-interaction of F0 radiation history with F3 irradiation. Comet assays demonstrated significantly increased DNA damage in fibroblasts related to F0 irradiation and increased DNA damage following F3 irradiation. However, significantly decreased F3 irradiation damage was demonstrated based upon cross-interaction of F0 radiation. CONCLUSIONS: The data suggest that irradiation of paternal F0 Type B spermatogonia resulted in cellular reprogramming causing offspring with this radiation history to have altered responses to acute somatic n-irradiation.

Heritable effects of paternal irradiation in mice on signaling protein kinase activities in F3 offspring.

We evaluated F3 mouse offspring from paternal F0 attenuated 137Cs gamma-irradiation (1.0 Gy) for heritable effects on gene products that can modulate cell proliferation rate and that may be markers for genomic instability. The F3 generation was selected for evaluation as a stringent test for heritability of effects from paternal F0 germline irradiation. Male CD1 mice were bred 6 weeks after irradiation so that the fertilizing sperm were type B spermatogonia at the time of irradiation. The resulting F1 males were bred to CD1 females to produce F2 four-cell embryos. The F2 embryos with a radiation history were paired with 'control' CD1 four-cell embryos that were heterozygous for the neo transgene. These F2 XY-XY chimeras, consisting of cells derived from both an embryo with a paternal F0 radiation history and a control embryo, were transferred to foster mothers, raised to adulthood and bred to produce F3 offspring. F3 offspring were evaluated for hepatic activities of receptor tyrosine kinase, protein kinase C and MAP kinase and for protein levels of nuclear p53 and p21(waf1). All three protein kinase activities were altered and nuclear levels of p53 and p21(waf1) protein were higher in the group of offspring that included F3 offspring with a paternal F0 radiation history than in littermates in the neo-positive control group. To our knowledge, this is the first observation in the descendants of paternal germline irradiation of effects on signal protein kinase activities and downstream nuclear target proteins that can influence cell proliferation rates.

Paternal effects from methamidophos administration in mice.

In this study, the mouse was used to evaluate paternal germline exposure to the organophosphate methamidophos for its potential to produce adverse effects on spermatozoa and in the offspring. There have been reports that organophosphate exposure can increase abnormal sperm morphology in mice. However, effects transmitted to the offspring following paternal exposure have not been reported previously. The maximum tolerated dose (MTD) was 7.5 mg kg(-1) body weight and this dose resulted in no deaths, although blood plasma cholinesterase activity was still decreased. Males were euthanized 4 weeks after an acute intraperitoneal injection of methamidophos (0.5, 3.75, 5.0, and 7.5 mg kg(-1) body wt) and the number of spermatids per gram testes and sperm morphology were analyzed. In this study, abnormal sperm morphology on a per group basis exhibited a dose-response significantly related to increased methamidophos exposure as indicated by regression analysis and a nested ANOVA (p < 0.0001). Preimplantation embryos that were conceived 6 weeks after paternal methamidophos exposure (5 mg kg(-1) body wt) exhibited a significant increase in cleavage arrest. Fertility of males was also affected as shown by a decrease in the number of two- to four-cell embryos per male (postexposure week 6) and an increase in the number of degenerated embryos (postexposure weeks 4-6). We conclude that methamidophos may have the potential to produce transmissible adverse embryonic effects following an acute paternal germline exposure.

Influence of different forms of fluid shear stress on vascular endothelial TGF-beta1 mRNA expression.

This study investigated the effects of long-term exposure of steady (19 dyne/cm2), 1-Hz non-reversing pulsatile (19+/-6 dyne/cm2) and 1-Hz purely oscillatory (0+/-19 dyne/cm2) shear stress on endothelial transforming growth factor-beta1 (TGF-beta1) mRNA expression. Cultured bovine aortic endothelial cells (BAECs) were systematically exposed to the three flow conditions for periods of 2, 6, 12 and 24 h, and relative differences in TGF-beta1 mRNA levels were measured by semi-quantitative RT-PCR. In response to steady shear stress, TGF-beta1 mRNA levels normalized to no-flow controls were 1.24, 1.42, 1.30 and 1.47 at the 2-, 6-, 12- and 24-h time points, respectively. In response to non-reversing pulsatile flow, these levels were 1.49, 1.64, 1.64 and 1.73, while the respective transcript levels for oscillatory flow were 1.33, 1.12, 1.12 and 1. 93. These results indicate that BAEC TGF-beta1 mRNA was up-regulated with the kinetics of the up-regulation faster for steady and non-reversing pulsatile flow than for oscillatory flow. Given the preferential localization of early atherosclerotic lesions in arterial regions exposed to low and/or oscillatory shear stress and the implication of TGF-beta1 as an athero-protective gene, these results are consistent with the notion that regions transiently exposed to oscillatory flow may be particularly prone to atherosclerosis.

Gap junction intercellular communication mediates the competitive cell proliferation disadvantage of irradiated mouse preimplantation embryos in aggregation chimeras.

Gap junction intercellular communication (GJIC) is thought to play a role in the growth modulation that occurs within cell populations. An example of heterologous growth inhibition (competitive cell proliferation disadvantage) occurs within mouse aggregation chimeras comprised of irradiated and nonirradiated cleavage-stage embryos. The goal of this investigation was to test the hypothesis that GJIC participates in the competitive cell proliferation disadvantage that is expressed by the irradiated embryo in aggregation chimeras. Specifically, we tested the capacity of the GJIC inhibitor 18 alpha-glycyrrhetinic acid (AGA) to inhibit competitive cell proliferation disadvantage in heterologous aggregation chimeras that were comprised of one embryo that was irradiated with 1.0 Gy of (137)Cs gamma rays and then paired with one nonirradiated embryo. We found that AGA successfully inhibited fluorescent dye transfer between irradiated and nonirradiated embryos in heterologous chimeras. Chronic exposure to AGA prevented competitive cell proliferation disadvantage in these radiation chimeras, while exposure to AGA for the first 15 h of culture (prior to gap junction development) did not prevent competitive cell proliferation disadvantage. An unexpected observation was the apparent lack of any effect of inhibiting GJIC by exposure to AGA on blastocyst formation and cell number allocation in the two principal stem cell lineages of the preimplantation mammalian embryo, trophectoderm and inner cell mass.

In vitro fertilization rate of mouse oocytes with spermatozoa from the F1 offspring of males irradiated with 1.0 Gy 137Cs gamma-rays.

Previous studies suggest that the spermatozoa from acutely irradiated male mice exhibit a reduced fertilization rate in vitro with the maximum decrease occurring for spermatozoa produced 6 weeks after irradiation (Y. Matsuda et al., Mutation Res. 142 (1985) 59-63). We have found that spermatozoa from unirradiated F1 males conceived 6 weeks after paternal F0 irradiation also exhibit a significantly reduced fertilization rate in vitro. After acute 137Cs gamma-irradiation yielding an absorbed dose of 1.0 Gy, adult CD1 F0 male mice were mated at weekly intervals with unirradiated female CD1 mice. Unirradiated adult males from F1 litters conceived 5 and 6 weeks after paternal F0 irradiation were allowed to mature. Their epididymal spermatozoa were evaluated for in vitro fertilization rates using oocytes from unirradiated 8-12-week-old CD1 females. The mean fertilization rate for spermatozoa from F1 males conceived 5 weeks after paternal F0 irradiation (80.74 +/- 15.74 SD %, n = 5) did not differ significantly from the control fertilization rate (89.40 +/- 10.94 SD %, n = 8). However, the fertilization rate for spermatozoa from F1 males conceived 6 weeks after paternal F0 irradiation (56.14 +/- 21.93 SD %, n = 5) was significantly less than the fertilization rate for control spermatozoa (p < 0.006) or for that of the F1 males conceived 5 weeks after paternal F0 irradiation (p < 0.04). These data suggest that spermatozoa obtained 6 weeks after paternal F0 irradiation can transmit a decrease in fertilization rate to the F1 generation males as well as exhibit decreased fertilization rate themselves when tested directly in vitro.

Impaired cell proliferation in mice that persists across at least two generations after paternal irradiation.

Irradiation of male F0 mice 6 to 7 weeks prior to mating causes significant changes in the proliferation of F1 and F2 embryonic cells. These changes are revealed as a competitive cell proliferation disadvantage in chimera assays when the affected embryo is paired with a normal embryo in an aggregation chimera. This effect has been observed previously to be transmitted to F1 embryos for absorbed doses from 0.01 to 1.0 Gy; 0.01 Gy is about 100-fold lower than detectable using conventional germline mutation assays. However, until now there has been no reported cross-generation heritability. We now report that this competitive cell proliferation disadvantage persists without degradation in the F2 generation of embryos when F0 males received 1.0 Gy from gamma irradiation 6 and 7 weeks prior to conception of F1 males.

Expression and function of amphiregulin during murine preimplantation development.

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-alpha significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-alpha, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-alpha in these embryos.

Transmitted proliferation disadvantage from mouse oocytes labeled in vivo with [3H]thymidine: radiosensitive target considerations.

This study was conducted to test the hypothesis that a nuclear target is involved in the embryonic cell proliferation disadvantage transmitted by irradiated mouse oocytes and detected by the chimera assay. In this assay, the cells from the irradiated embryo exhibit a competitive cell proliferation disadvantage when they are challenged by direct cell-cell contact with cells from a normal embryo in an aggregation chimera. Here, six pregnant CD-1 mice received a total of 1.85 TBq tritiated thymidine (TdR) delivered by multiple intraperitoneal injections during days 13-15 postconception. Six more pregnant mice were sham-injected to provide control embryos. Sixty randomly selected female progeny were mated at 47 days of age and their 4-cell embryos tested in the chimera assay. The mean proliferation ratio (PR, number of cells from the experimental embryo divided by total cell number of the chimera) for experimental chimeras was 0.45 +/- 0.02 SE (n = 43), which was significantly less than the mean PR of 0.49 +/- 0.01 SE (n = 47; p = 0.02) for control chimeras. This entire experiment was repeated, with similar results. A comparison for TdR confined to the nucleus (i.e., mean beta-ray range is only 0.7 microm) with the relationship for uniform irradiation by 137Cs gamma-rays demonstrates that these two very different modes of dose delivery produce essentially identical PRs. These results in vivo suggest a nuclear DNA target for embryonic cell proliferation disadvantage consistent with our previous findings in vitro.

Comparative effects of essential and nonessential metals on preimplantation mouse embryo development in vitro.

It is well recognized that deficiencies of essential trace elements during early development can result in structural abnormalities and/or embryonic death. Recently, there has been increasing interest in the concept that small excesses of essential metals can also have negative effects on the developing embryo. We hypothesized that, with respect to toxicity, metals with similar physico-chemical properties would act by similar mechanisms to influence the preimplantation embryo. In the current study we investigated the influence of four essential (Cu, Mn, Fe, Zn), and eight nonessential (Cr, Hg, Pb, V, Al, Ag, Cd, As) metals on mouse preimplantation embryonic development. Two cell stage mouse embryos were cultured for 72 h in media containing varying metal concentrations (0.05 - 200 microM). Embryo cell differentiation and proliferation were respectively assessed by scoring for blastocyst formation and final embryo cell number. Both nonessential and essential metals were embryotoxic at relatively low concentrations. However, in contrast to our expectations, at similar molar concentrations, redox active essential metals were less toxic than non-redox active nonessential metals. These data suggest that direct metal binding to critical membrane sites and/or intracellular ligands, including protein and nucleic acids, may trigger abnormal development and death prior to metal-associated oxidative damage.

Enhancing effect of maternal zinc deficiency and 137Cs gamma-irradiation on the frequency of fetal malformations in mice.

It is well established that in mammals transitory zinc (Zn) deficiency during embryogenesis can have a negative influence on fetal development. Similar to Zn deficiency, maternal exposure to low levels of ionizing radiation during the first day of pregnancy has been shown to negatively affect preimplantation embryo development, and higher doses of maternal irradiation during late stages of embryogenesis can result in malformations. Here we report the effect of transitory maternal Zn deprivation combined with low dose irradiation during embryogenesis on fetal outcome. Pregnant mice were acutely dosed with 0.00, 0.05, 0.10, or 0.25 Gy of gamma-radiation the day after mating (GD0), or with 0.00 or 0.50 Gy on GD8. Mice irradiated on GD0 were either fed a low Zn diet (0.4 microgram Zn/g) for 48 h prior to mating through GD0 or GD5 and then switched to a control diet (50 micrograms Zn/g) through GD18, or they were fed the control diet throughout gestation. Dams irradiated on GD8 were either fed the low Zn diet from GD0 through GD10 and then switched to the control diet, or they were fed the control diet throughout gestation. Zn deprivation did not influence any of the maternal or embryonic/fetal parameters measured in the cohorts fed the low Zn diet through GD0. In contrast, groups fed the low Zn diet through GD5 or GD10 had lower mean maternal body weights, fewer live fetuses/litter, and a higher incidence of resorptions than controls. Fetuses from dams fed the low Zn diet through GD10 were smaller and shorter, and had a higher frequency of malformations than controls. Irradiation on GD8 under adequate Zn conditions had no effect on any parameter, but GD8 irradiation during maternal Zn deficiency had an additive effect on the frequency of fetal malformations.

Mouse immature oocytes irradiated in vivo at 14-days of age and evaluated for transmitted effects using the aggregation embryo chimera assay.

A previous study using the mouse-preimplantation-embryo-chimera assay demonstrated a reproducible transmitted effect (proliferation disadvantage observed in early embryos) from females irradiated as 49-day-old adults using 0.15 Gy of gamma rays and then mated seven weeks later, i.e., embryos were from oocytes that were immature at time of irradiation. Because mouse immature oocytes are known to be much more radiosensitive to cell killing in juveniles than in adults, a follow-on study was performed here using 14-day-old juvenile mice. In contrast to adults, the exposure of juveniles to 0.15 Gy of gamma rays did not result in a detectable transmitted proliferation disadvantage when animals were mated 7 or 12 weeks later. This observation is discussed in light of previous studies on mouse immature oocytes and embryo chimeras.

Radiosensitive target in the mouse embryo chimera assay: implications that the target involves autocrine growth factor function.

Mouse preimplantation embryos express at least two functional cell surface growth factor receptors that are radiosensitive in other cell types, the epidermal growth factor receptor (EGF receptor) and the insulin-like growth factor I receptor (IGF-I receptor). These embryos also express ligands that bind to and activate these receptors, including transforming growth factor alpha (TGF-alpha) and insulin-like growth factor II (IGF-II), which bind to the EGF receptor and IGF-I receptor, respectively. Embryo-expressed IGF-II and TGF-alpha increase embryo cell number--a measure of proliferation rate--and stimulate blastocoele formation--a measure of cell differentiation--allowing the embryo to self-modulate cell proliferation and morphogenesis into a blastocyst (Paria and Dey, Proc. Natl. Acad. Sci. USA 87, 4756-4760, 1990; Dardik and Schultz, Development 113, 919-930, 1991; Rappolee et al., Genes Dev. 6, 939-952, 1992). In this work, we tested the hypothesis that IGF-I receptor and/or EGF receptor function may be impaired to produce the radiation-induced competitive cell proliferation disadvantage that is expressed by irradiated embryos that are aggregated with nonirradiated embryos in chimeras. Cleavage-stage embryos were irradiated with 137Cs gamma rays (0.5 or 1.0 Gy) and paired with nonirradiated same-stage embryos to form groups of chimeras that were cultured in control medium or medium containing IGF-II, insulin, EGF or TGF-alpha. The cell proliferation disadvantage expressed by the irradiated embryos within chimeras was completely eliminated by IGF-II or insulin. In contrast to the rescue action of IGF-II or insulin in chimeras, neither EGF nor TGF-alpha could prevent the cell proliferation disadvantage exhibited by irradiated embryos paired with nonirradiated embryos in chimeras. For irradiated conventionally cultured zona-enclosed embryos, IGF-II and TGF-alpha did not increase mean embryo cell number significantly, although both IGF-II and TGF-alpha did increase blastocoele formation significantly. Collectively, these results support the following conclusions: (1) Ligands for the IGF-I receptor can rescue irradiated embryos from competitive cell proliferation disadvantage in chimeras, while ligands for the EGF receptor cannot; (2) IGF-I receptor function and EGF receptor function are affected differently by ionizing radiation with respect to competitive cell proliferation and are affected similarly by ionizing radiation with respect to blastocoele formation; (3) EGF receptor-dependent stimulation of competitive cell proliferation and cell differentiation are affected differently by ionizing radiation in preimplantation embryos.

Evidence that murine preimplantation embryos express aryl hydrocarbon receptor.

The underlying mechanism of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced alterations during development is thought to be mediated by a cytosolic aryl hydrocarbon receptor (Ahr). The specific role of Ahr-mediated changes in gene expression during embryonic development has not been elucidated. Recently, we reported that TCDD directly affects preimplantation embryo development in vitro, by accelerating differentiation of the blastocyst. In the work reported here, we provide evidence which suggests that Ahr mRNA and protein are present in mouse preimplantation embryos. Our results suggest that the embryo transcribes, rather than maternally--inherits Ahr mRNA. In addition, culturing embryos in medium with an Ahr antisense oligodeoxynucleotide resulted in a significantly lower incidence of blastocyst formation as well as mean embryo cell number. Results from this work suggest that Ahr may function in embryonic cell differentiation and proliferation independent of its known function in mediating TCDD toxicity in other systems.

Epidermal growth factor receptor function in early mammalian development.

We review here the data indicating a role for epidermal growth factor receptor (EGF receptor) signalling in early mouse development. Embryonic development of the metazoan embryo generally begins with the formation of a cystic structure and epithelial layers that subsequently form anlagen of the definitive body parts and organs. For the mammalian embryo, this cystic structure is a blastocyst whose wall consists of trophectoderm, the first epithelium to develop during mammalian embryogenesis. The onset of expression and function of EGF receptors is coincident with the onset of trophectoderm development. Modulating EGF receptor expression and function modulates trophectoderm differentiation, leading to the hypothesis that functional EGF receptors participate in the induction of trophectoderm development and perhaps of other embryonic epithelial derivatives such as nervous tissues.

Influence of antioxidants on cadmium toxicity of mouse preimplantation embryos in vitro.

To test the hypothesis that the developmental toxicity of cadmium (Cd) is due in part to oxidative damage, embryos were cultured in medium containing 0.0, 1.0, 3.0, or 6.0 microM Cd with or without various antioxidants for 72 h. Ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and glutathione (GSH) were all effective at ameliorating 1.0 microM Cd-induced embryotoxicity. For embryos cultured in medium containing either 3.0 or 6.0 microM Cd, GSH was effective at ameliorating Cd toxicity while the other antioxidants tested were ineffective. Pretreating embryos with antioxidants for 24 h prior to exposing them to Cd and antioxidants did not significantly alter the previously observed improvement with the exception that pretreatment with GSH virtually eliminated Cd-induced embryotoxicity between 1.0 and 6.0 microM Cd. A 4-h exposure to GSH prior to culture in Cd markedly improved embryo development suggesting that GSH taken up during pretreatment can provide protection against Cd-induced embryotoxicity. This work supports the hypothesis that the developmental toxicity of Cd is in part due to oxidative damage that can be modulated by select antioxidants.

Effect of a metallothionein antisense oligonucleotide on embryo development.

The effect of a metallothionein (MT) antisense oligodeoxynucleotide (ODN) on mouse preimplantation embryo development was investigated. Preimplantation embryos were cultured for 72 h and examined following exposure to either an MT antisense or sense ODN. Blastocyst formation (cavitation) and embryo cell number were lower in embryos exposed to the MT antisense ODN than in controls or in embryos exposed to the MT sense ODN. In embryos cultured in medium containing free nucleotides, cavitation frequency was not affected, although mean embryo cell number was lower than in controls. Combined, this work shows that an antisense ODN against MT can significantly affect blastocyst formation of preimplantation embryos; some, but not all, of the observed effects on embryo cell number may have been due to nucleotide toxicity.

Periconceptional zinc deficiency affects uterine 3H-estradiol binding in mice.

To better define the mechanisms by which zinc (Zn) deficiency influences periconceptional development, we examined the effects of this developmental insult on uterine estrogen metabolism. CD-1 mice were assigned to 1 of 3 groups (Low Zn, LZ; Control, C; or Replete, R) and fed either a low Zn (< or = 0.3 microgram Zn/g) or control diet (47 micrograms Zn/g) 5 days prior to gestation day (GD) 0 and continuing up to GD 4 during early pregnancy. Mice in the R group were fed the low Zn diet until GD 1 after which they were fed the control diet. Uterine 3H-estradiol binding in vivo was measured on GD 2, GD 3, and GD 4. Binding was similar among groups on GD 2 and GD 3, but was lower on GD 4 in LZ mice than in C and R mice (61% of control value). On GD 4, uterine 3H-estradiol binding in vitro was measured and was lower in LZ mice than in C and R mice (63-74% of control values); the reduction in binding was due to lower receptor number. Thus, Zn deficiency can result in a reduction in uterine estradiol receptors and estradiol binding.