Reproductive abnormalities in adult male mice following preimplantation exposures to estradiol or pesticide methoxychlor.

Adult females of ICR strain of mice were bred, separated into different experimental groups, and treated as follows. On Days 2-4 of pregnancy, the mice received daily subcutaneous injections of either 0.05 ml sesame oil (vehicle) or same volume of 5.0mg of purified methoxychlor (MXC) suspended in the vehicle. Another group received a single subcutaneous injection of 1.0 microg of estradiol-17beta (E) on Day 2 of pregnancy only. Male offspring were tested at 3 and 6 months of age. At 3 months, E or MXC did not alter the weights of seminal vesicles, preputial glands, or testes, although after exposure for 30 min to a female in estrus behind a partition, testosterone levels were significantly reduced in treated males in comparison to control males exposed to the same partition test. At 6 months, the preputial glands and testes weight remained unchanged, while the seminal vesicles were significantly heavier in E- and MXC-treated males. Same partition tests again revealed that in E and MXC groups, testosterone levels remained significantly lower in comparison to control males. MXC or E exposures during preimplantation appear to induce long-term effects on the sexual development in 3 and 6 month-old-males by compromising their sexual arousal and altering seminal vesicles weights in the older group.

Preimplantation exposures of murine embryos to estradiol or methoxychlor change postnatal development.

Long-term effects of in vivo exposures to proestrogen methoxychlor (MXC) or estradiol-17beta (E) were studied during early pregnancy (preimplantation) in ICR mice. Pregnant dams received either subcutaneous injections of 1 microg of E on Day 2 of pregnancy only (vaginal plug = Day 1), or 5.0mg of MXC on Days 2-4 of pregnancy in sesame oil. Pregnant control mice were treated with the vehicle only. Litter size, postnatal survival, sex ratio at birth, and anogenital distance (AGD) in offspring of both sexes were examined, as well as vaginal opening in female offspring. High mortality rate was recorded in MXC-exposed offspring due to infanticide. Exposures to either E or MXC did not change sex ratio at birth, but the litter size was smaller in the former group. On postnatal Day 21, male pups exposed to either E or MXC at preimplantation stage exhibited shorter AGD than the controls, with the change most pronounced after MXC treatments. AGD in female offspring was unaffected after MXC exposures, but E treatments produced longer AGD in the females than that recorded in the controls. Preimplantation exposures to E or MXC also accelerated sexual maturation as significantly more females exhibited precocious vaginal opening at weaning. Our study shows that exposures to MXC or E at preimplantation stages cause long term alteration of sexual development during weaning in offspring of both sexes. Also, MXC treatments retarded both growth and weight of both sexes of offspring, in comparison to controls.

Preimplantation mouse embryo development as a target of the pesticide methoxychlor.

Effects of methoxychlor (MXC) and estradiol-17beta (E) were studied in mouse preimplantation embryos. Pregnant mice received s.c. injections of sesame oil only, 10 microg E, or 0.5 mg purified (95%) MXC on Days 2-4 of pregnancy (plug = Day 1). Another group received a single dose of 2.5 microg E on Day 2 only. Based on the average weight of pregnant females, 10 microg of estradiol was equivalent to 0.33 mg/kg of bw, 2.5 microg of estradiol was equivalent to 0.082 mg/kg of bw, and the 0.5-mg dose of MXC was equivalent to 16.5 mg/kg of bw. All embryos were collected for analyses on Day 4. MXC and both estradiol-17beta doses suppressed embryonic development to blastocyst, decreased embryo cell numbers, and caused abnormal blastocyst formation. The high estradiol-17beta dose significantly increased the percent degenerating embryos and caused a tube-locking effect, with retention of embryos in the oviduct. In contrast to estradiol-17beta, MXC at the dose used in this study did not alter tubal transport of embryos. Also in contrast to estradiol-17beta, MXC increased the percentage of nuclear fragmentation and micronuclei. In preimplantation embryos, MXC and estradiol-17beta both suppressed embryo development. MXC effects were, however, different from those of estradiol-17beta, indicating a difference in mechanism of action, possibly due to cytotoxicity and induction of apoptosis.

The pesticide methoxychlor disrupts the fusion of myoblasts into myotubes in skeletal muscle cell culture.

We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 muscle cell culture. Various concentrations of MXC or beta-estradiol (E) were added to the culture media. MXC (100 microM) disrupted myoblast fusion into myotubes, but 10 microM MXC or 10 microM E had no effect. Correlated with the diminished size of the myotubes, the clustering of acetylcholine receptors (AChRs) was inhibited by 100 microM MXC, but not by 10 microM MXC or 10 microM E. However, since clusters of AChR receptors did form, the postsynaptic clustering mechanism remained intact. Since E did not disrupt myoblast fusion into myotubes or the clustering of AChRs, we conclude that the abnormality induced by MXC is mediated by a mechanism of action that is independent of E. We believe this to be the first demonstration that MXC induces abnormal effects in the process of muscle development in skeletal muscle cell culture.

Altered behaviors in male mice, male quail, and salamander larvae following early exposures to the estrogenic pesticide methoxychlor.

Numerous publications show that methoxychlor (MXC), in use today as a DDT substitute, is a reproductive toxicant; it produces deleterious effects on the structure and function of the reproductive organs in exposed species. Exposure of mice (33 mg/kg body weight) to purified (95%) MXC at the time of implantation, or injection, of 5 mg MXC into freshly laid quail eggs prior to artificial incubation, altered sexual arousal and sexual behavior in adult males of both species. When placed near a plastic partition with an estrus female behind it, the MXC-exposed male mice showed no sexual arousal, spent less time near the partition, and exhibited lower testosterone levels. Similarly, adult quail males that were exposed to MXC during incubation showed a lack of sexual interest and copulatory behavior when presented with a receptive female. Some males showed a longer latency period before mounting the female, while others did not show any sexual interest at all. Exposure of salamander embryos to purified MXC at or above 0.3 microM (0.1 mg/l) induced precocial hatching of embryos and reduced the startle response and the distance traveled in response to startle. Exposing hatched larvae to MXC for 3 days also resulted in a blunted startle response. Due to the blunted startle response and decreased avoidance travel, the exposed salamander larvae appear more susceptible to predation and these effects may contribute to amphibian population declines. The results of these studies indicate that developmental exposures to environmental chemicals with hormonal activities produce undesirable behaviors that may affect population dynamics and survivability of exposed species.