Original ContributionsMethoxychlor given in the periimplantation period blocks sexual arousal in male mice
Introduction
At the present time, the problem of environmental chemicals that exhibit estrogenic activities is of great concern. Numerous reviews indicate that estrogens and environmental chemicals with estrogenic activity cause endocrine-disrupting effects, including altered reproductive functions [1], [2], in various animals. The nonsteroidal pesticide methoxychlor (MXC), which is a DDT substitute currently in use, is a proestrogen that is activated in vivo into an active estrogenic metabolite [3].
Our previous studies indicated that exposures of immature neonatal mice to either commercial or purified MXC induce both short- and long-term effects on the reproductive system of both sexes. In newborn females, MXC induced precocious maturation of the reproductive tract and damaged the reproductive organs in adults. In immature males, MXC caused a significant inhibition of accessory organ development and decreased plasma testosterone levels [4], [5], [6], [7].
It has also been shown that not only postnatal, but also prenatal stages of rodent development may be susceptible to natural estrogens or ecoestrogens. In utero exposure of pregnant mice at Days 11 through 17 to ethinyl estradiol impaired Leydig cell development and reduced Sertoli cell number, resulting in altered spermatogenesis in the offspring [8]. Moreover, pregnant mice treated with ethinyl estradiol on Days 11 through 13 produced offspring with significantly more cryptorchid testes [9]. Some rat studies indicate that ecoestrogenic effects appear even earlier, especially during the preimplantation and early postimplantation stages of embryo development [10], [11], [12].
One study indicated that MXC alters normal preimplantation embryo development in mice, and with higher doses, blocks implantation completely [13]. Also, gonadotropin treatments before mating, which increase endogenous levels of estradiol and alter the estradiol/progesterone ratio in rodents [14], causes premature ovulation [15], and increases the mutation rate in mouse preimplantation embryos [16]. Similar treatment of mice [16], rats [14], [17], and stoat females [18] before mating affected the rate and timing of preimplantation embryo development.
Most of these studies either examined the short-term effects of estrogenic chemicals on early embryonic development and the implantation process, or utilized the postnatal period or late postimplantation prenatal stage of development as a reference [19]. However, whether these chemicals target the early stages of embryonic development and alter sexual behavior in adult offspring is still not known. Nevertheless, there is evidence that the earliest stages of embryonic development in mammals are vulnerable to changes in their environment and that these changes may exert long-term effects on the offspring.
Our work with rats [20] and other work with mice [21] indicate that manipulation of preimplantation embryos, such as embryo transfer and embryo cryobanking, results in long-lasting physiologic effects and behavior changes in the offspring. Such data implies that the earliest stages of embryonic development in mammals may be a possible target for ecoestrogens.
Days 5 through 7 of gestation are highly significant in mouse embryo development because implantation occurs at about 4.5 d after ovulation. At this stage, embryonic cells have differentiated into an inner cell mass, and there is formation of trophectoderm and neurulation [22]. However, the biologic role of estrogen receptor (ER) in the inner cell mass of preimplantation and implanting embryos remains unknown. Furthermore, very little is known about the long-term effects of MXC on mammalian behavior. One study showed that fetal exposure of mice to MXC alters the urine-marking (territorial) behavior of male offspring. These changes were believed to be due to binding of MXC with ER in the developing brain [23]. This information becomes especially significant in view of the fact that ER has been detected in fetal mouse gonads [24], and preimplantation and implanting embryos [25], [26], and that MXC accelerates premature expression of ER genes in the uterine epithelium of newborn mice [27].
The premise of this study is that exposures to MXC could not only impact sensitive animals very early in their development and target the periimplantation embryo specifically, but may also induce long-term effects on sexual behavior and reproductive function in adults. Because the effects of MXC in males are less well known than its action in females, we investigated the long-term effects of exposure of the periimplantation embryo to this pesticide. Also, because the ER may be the target of environmental ecoestrogens, we administered the estrogenic pesticide MXC to mice on Days 5 through 7 of pregnancy. The endpoints of this study were to determine whether these exposures to MXC would lead to long-term effects on reproductive functions, female-induced sexual behavior and arousal, and the subsequent neuroendocrinologic responses in adult male offspring.
Section snippets
Treatment of animals
Sexually mature female mice (8 to 10 weeks old) of the ICR strain were used in this experiment. The animals were kept in experimental animal laboratory facilities in Novosibirsk, Russia, under standard animal laboratory conditions with a light regimen of 10 h light and 14 h dark. Females were given food and water ad libitum and paired with males of the same strain to induce pregnancy. The females were individually caged overnight with males and examined for vaginal plugs early the next morning.
Results
Pregnancy was considered successful if at least one pup developed to term. All 6 females with vaginal plugs in the untreated group became pregnant. One olive oil-treated female with a vaginal plug was not pregnant. The pregnancy rate in this group was 80%. In the group exposed to 1 mg MXC, only 40% of females with a vaginal plug delivered pups. In contrast, in the estradiol-treated group, none of the females became pregnant. Following parturition, there were no visible clinical signs or
Discussion
Mice not exposed to MXC, but either untreated or injected with olive oil, exhibited a pregnancy rate of 80 to 100%. Although the administration of 1.0 mg (33 mg/kg body weight) MXC on Days 5 through 7 of pregnancy did not completely prevent implantation, the pregnancy rate was reduced to 40%. There were no live births when estradiol was administered to mice during the same time of pregnancy. These results suggest that the pregnancy rate in mice is relatively resistant to the effects of this
Acknowledgments
This study was supported in part by Grant No. 27.82, awarded to Dr. Sergei Ya. Amstislavsky by the Program Russian Universities Grant for Basic Research.
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