Ethylene glycol monomethyl ether (EGME) exposure of male mice produces a decrease in cell proliferation of preimplantation embryos.

In this study using an aggregation chimera assay we examined male mice exposed to a nonmutagenic reproductive toxicant, EGME, for the transmission of impaired viability to their progeny preimplantation embryos. Prior to their aggregation into pairs, one of the embryos was labeled with a viable dye fluorecein isothiocyanate (FITC) to determine the relative cellular contribution from each partner embryo when chimeras were dissociated 30 to 35 h later (2 to 3 cell cycles). Direct cell-cell contact of embryos derived from exposed males and embryos from control males creates a competitive situation that has been shown to confer a cell proliferation disadvantage to the embryo from an exposed parent. The cell proliferation disadvantage is expressed as a "proliferation ratio": number cells from an experimental embryo/total chimera cell number. Male mice were exposed to EGME by gavage for 5 days with 0, 50, 200, 750, or 1500 mg/kg and were serially mated with unexposed female mice for the next 7 weeks. Proliferation ratios were significantly decreased in the 50, 200, and 750 mg/kg dose groups at week 4, which corresponds to the pachytene spermatocyte stage of spermatogenesis. Proliferation ratios were also significantly decreased in the 1500 mg/kg group at week 5. Due to transient infertility in this dose group, there were not sufficient numbers of embryos to evaluate for week 4. These results indicate that male mice exposed to EGME transmitted adverse effects to their progeny embryos that were expressed as an embryonic cell proliferation disadvantage in the chimera assay.

Effects of maternal exposure to trichloroethylene (TCE) on cell proliferation in the mouse preimplantation embryo.

Pregnant mice were treated with trichloroethylene (TCE) at doses ranging from 0.01 micrograms/kg body weight to 483 mg/kg body weight when embryos were traversing the pronuclear stages of development. No treatment-related effect was seen on total number of embryos recovered from oviducts at the 2 to 4 cell stage of development. The 4-cell embryos were tested for cellular proliferative disadvantage by the embryo chimera assay in which embryos from treated mothers (experimental embryos) are paired with embryos from nontreated mothers (control embryos) to form aggregation chimeras. No significant cell proliferation decreases were observed for any of the experimental embryos across all doses examined.

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay.

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.

A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice.

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.

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

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.

Male reproductive toxicity and recovery associated with acute ethoxyethanol exposure in rats.

Effects of 2-ethoxyethanol (EE) on semen parameters in male rats were investigated employing an animal model that allowed assessment of toxicity and recovery in the same animal. Prior to exposure, 70-d-old Long-Evans hooded males were placed with ovariectomized, hormonally primed females on several occasions and their copulatory behaviors were monitored and scored. At 100 d of age, these males were mated with females that were sacrificed 15 min postejaculation. The semen sample was recovered from the female reproductive tract and scored for sperm motility, sperm count, and abnormal sperm morphology. Following this preexposure baseline assessment, the males were intubated with 0, 936, 1872, or 2808 mg EE/kg for 5 consecutive days. The males were mated weekly for the next 14 wk. Copulatory behaviors were monitored and ejaculated semen samples analyzed on wk 1, 4, 7, 10, and 14. The males were sacrificed at wk 16 and the testes and epididymides were processed for histological evaluation. Data analyses indicated that EE produced a rapid decline in sperm counts in the two highest groups, with most of the males becoming azoospermic by wk 7. The males in the low dose group also exhibited a significant decrease in sperm counts at this week. Additionally, there was a significant increase in abnormal sperm morphology at wk 7 in the low-dose males. Partial or complete recovery was apparent in the sperm parameters by wk 14, as evidenced by an increase in sperm counts and a decrease in abnormal morphology and further supported by epididymal and testicular histological assessment at wk 16. At sacrifice, there were no significant differences between groups on body weights, organ weights, or epididymal sperm counts, except for a significant depression of epididymal weight in the middle dose group. While high doses of EE produced a decline in sperm counts starting after the first week of exposure, the early spermatid-late spermatocyte stages, represented by mature spermatozoa in the wk 7 ejaculates, appeared to be particularly sensitive to this compound. Moreover, most of the males exhibited recovery following this acute dosing regimen.