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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 (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.

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.

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.

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 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.

High-energy, high-charge nuclei may contribute substantially to the yearly equivalent dose in space flight from galactic cosmic radiation (GCR) at solar minimum. The largest single heavy-ion component is 56Fe. We used the mouse embryo chimera assay to test 512 MeV/u 56Fe nuclei for effects on the rate of proliferation of embryonic cells transmitted by sperm from irradiated mice. Male CD1 mice were acutely irradiated with 0.01, 0.05 or 0.1 Gy (LET, 184 keV/micron; fluence, 3.5 x 10(4)-3.3 x 10(5) nuclei/cm2; average dose rate, 0.02 Gy/min) at the Lawrence Berkeley Laboratory BEVATRON/BEVALAC Facility in Berkeley, CA. Irradiated males were bred weekly for 7 weeks to nonirradiated females and their four-cell embryos were paired with control embryos, forming aggregation chimeras. After 30-35 h of culture, chimeras were dissociated to obtain "proliferation ratios" (number of cells contributed by the embryo from the irradiated male/total number of cells in the chimera). Significant dose-dependent decreases in proliferation ratios were obtained across all three dose groups for postirradiation week 2 (P0.05 to P0.003). The 0.01- and 0.05-Gy dose groups also produced significant decreases in proliferation ratios for postirradiation week 1 (P0.05 to P0.01) and the 0.05-Gy dose group produced significant decreases in proliferation ratios for postirradiation week 6 (P0.05). Postirradiation weeks 1, 2 and 6 correspond to irradiation of epididymal sperm, testicular spermatids and spermatogonia, respectively. We calculate that only about 5% of sperm in the 0.1-Gy, 2.5% in the 0.05-Gy and 0.5% in the 0.01-Gy dose groups sustained direct hits from 56Fe nuclei. However, up to 47% of sperm during postirradiation weeks 1 and 2 transmitted proliferation ratios that were at or below one standard deviation from control mean proliferation ratios. Morphometry on sectioned testes showed a significant log-linear dose response for cell killing of type B spermatogonia, which are the most radiosensitive stage of spermatogenesis and which would have been tested as mature sperm during postirradiation week 6. We conclude that amplification from secondary radiation produced in the mouse and/or from diffusible chemical products arising from hit sperm and adjacent cells contributed to the high incidence of transmitted effects on proliferation of embryonic cells.

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

The first epithelium to appear during mammalian embryogenesis is the trophectoderm, a polarized transporting single cell layer that comprises the wall of the blastocyst. The trophectoderm develops concurrently with blastocoele fluid production as the morula develops into a blastocyst. The process whereby the morula becomes a fluid-filled cyst is called 'cavitation', which can be regarded as the first functional expression of the trophectoderm phenotype. The outer layer of cells of the morula comprise the nascent trophectoderm and are already morphologically polarized prior to the onset of cavitation. A major working hypothesis in the field of mammalian embryogenesis is that such polarization is a prerequisite for the initiation of cavitation. To test this hypothesis we examined morulae for their ability to cavitate during treatments that modify morphological polarity in nascent trophectoderm cells. These treatments included ouabain, different concentrations of extracellular K and Na, cytochalasins and colcemid. Ouabain and extracellular K and Na affect the activity of Na/K-ATPase, which has been implicated in the maintenance of morphological polarity of nascent trophectoderm cells. Cytochalasins and colcemid also modify apical-basal polarity of nascent trophectoderm cells and impair cavitation. The endpoints that were monitored included incidence of cavitation, rate of cavitation and the status of morphological polarity of nascent trophectoderm cells. Collectively, all of these treatments indicate that there is a functional association between an asymmetric distribution of organelles along the apical-basal axis of nascent trophectoderm cells and the ability of the embryo to produce nascent blastocoele fluid efficiently. In addition, the preimplantation embryo appears to possess two mechanisms for accumulating blastocoele fluid.

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

It would be useful to have a clear definition of Male-Mediated Developmental Toxicity (MMDT) in order to make suggestions or recommendations for its investigation in the laboratory, but this not available. For the purposes of this document, MMDT will be considered to include all factors contributed by the male parent to the conceptus that can result in other-than-normal development.