Neonatal exposure to technical methoxychlor alters pregnancy outcome in female mice.

This study was designed to determine the ability of female mice who were exposed neonatally to the pesticide methoxychlor (MXC) to mate, ovulate, and become pregnant upon reaching sexual maturity. One-day-old female mice (5 to 8/group) were exposed daily by intraperitoneal (ip) injection for 14 d to either sesame oil or 10 microg estradiol-17beta or 0.1, 0.5 or 1.0 mg MXC suspended in sesame oil. The MXC exposures corresponded to 14 to 71, 68 to 357, or 135 to 714 mg/kg body weight, respectively. Three months later, female mice were placed with proven breeder males and checked daily for vaginal plugs. Mated female mice were sacrificed 18 d after the appearance of a vaginal plug to evaluate pregnancy. Uteri were examined for the presence of living fetuses and/or resorption sites. Ovaries were removed and prepared for histologic evaluation and tabulation of corpora lutea. All mice from all three MXC-treated groups did in fact mate, in comparison with only one of those exposed neonatally to estradiol. Increasing the dose of MXC produced a decreased number of pregnant animals at 18 d following mating. The mean number of live fetuses/litter was reduced in the 0.5 and 1.0 mg MXC-treated groups. Corpora lutea were significantly reduced in ovaries from only the 1.0 mg MXC group and the estradiol group. No effects of treatment were seen at 0.1 mg MXC. It is concluded that neonatal exposure to MXC does not interfere with mating. Instead, significant alterations are seen in initiating and/or maintaining pregnancy. The deleterious effects on pregnancy may be due to the influence of neonatal MXC treatments on the hypothalamic-pituitary-ovarian axis as well as on possible alteration of the uterine environment.

The effects of methoxychlor on early sea urchin development.

Methoxychlor (MXC) is a widely used pesticide which has been found in water sources near agricultural sites. Embryos of aquatic organisms are likely to encounter MXC due to land runoff. The sea urchin embryo (Strongylocentrotus purpuratus) was used as a model system to document the effects of MXC on early development up to the pluteus stage. Fertilized eggs and embryos were exposed to several concentrations (0.1, 1.0, 10, and 100 ppm) of the pesticide in both chronic and acute exposure regimens. With chronic exposure, percentages of embryos completing normal first cleavage decreased with increased concentrations of MXC, and subsequent cleavages became even more irregular in that blastomeres divided asymmetrically and asynchronously. Ten parts per million MXC allowed development through the hatched blastula stage, whereas embryos in 100 ppm MXC did not hatch. In acute exposure trials, fertilized eggs were pulsed (i.e., exposed for brief durations) to MXC for 30, 60, or 90 min. The MXC was then washed out. Recovery of normal development was proportional to the amount and duration of MXC exposure. Development was delayed in embryos exposed to 100 ppm MXC for 30 or 60 min. The embryos exposed to 100 ppm MXC for 90 min were abnormal as early as the four-cell stage, and by 72 hr more than 90% had abnormal gut development, indicating disruption of gastrulation. These data show that MXC exposure resulted in retardation of cleavage and abnormal gastrulation, basic morphogenetic processes.

Decreased superovulation in adult mice following neonatal exposures to technical methoxychlor.

To examine the effects of technical methoxychlor (MXC) on superovulation, neonatal mice received intraperitoneal (i.p.) injections of either sesame oil, 10 micrograms of estradiol 17 beta, or 0.1, 0.5, or 1 mg of technical MXC. At 2 and 4 months, half of the mice received a superovulatory regimen of 10 IU pregnant mare's serum gonadotropin followed by 10 IU human chorionic gonadotropin. The mice were sacrificed 15 to 20 h later, the number of ovulated oocytes were counted, and the ovaries were removed for histology. In the lowest MXC dose, the ovaries appeared normal and at 2 months, ovulated the same number of oocytes as controls. Estradiol or the highest two MXC doses induced ovarian atrophy. Following gonadotropin injections, these ovaries also ovulated oocytes. However, the number of oocytes recovered from experimental mice exhibited a time- and dose-dependent decline, and by 4 months, their number was significantly reduced. Neonatal exposures to MXC reduces ovulatory rates and ovarian functions in adults.

Response of adult murine uterine epithelium to 50% methoxychlor.

This study was designed to assess the response of uterine epithelia of adult mice to a 4-week exposure of 50% methoxychlor (MXC) to ascertain whether significant changes were induced by 50% MXC that might compromise future implantation. Sexually mature virgin female mice were exposed to 0.1, 0.5, 1.0, 2.5, or 5.0 mg MXC via oral gavage for 5 consecutive days for 4 weeks. Controls received either sesame oil or 25 micrograms estradiol-17 beta (E-17 beta) also by gavage. At sacrifice, segments from each uterine horn were prepared for morphometric studies or for transmission electron microscopy. Results revealed a dose-dependent increase in the heights of uterine epithelial cells. Epithelial cell heights of the two groups treated with the highest doses of the pesticide were similar to that of the E-17 beta-treated group. Electron microscopy revealed increased vacuolization and swelling of mitochondria in cells of the 2.5 and 5.0 mg treated groups when compared to either of the control groups. In addition, there were effects on the number and size of microvilli in the uterine epithelial cells. The present study clearly demonstrates that a 4-week exposure of adult female mice to 50% MXC elicits significant estrogenic and toxic effects on the uterine epithelium.

Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations.

The purpose of this study is to examine the effects of the pesticide methoxychlor (MXC) on the ultrastructural appearance of the different cellular components of the mouse ovary. Sexually mature (7- to 8-week) virgin female CD-1 mice were exposed to 5.0 mg MXC (50% technical grade) via oral gavage for 5 consecutive days each week for 4 weeks. Control groups received either 0.025 mg estradiol-17 beta (E-17 beta) or the sesame oil vehicle for the same time period. Twenty-four hours following the final exposure, animals were sacrificed. Ultrastructural observations revealed increased lipid accumulation in interstitial cells and theca cells of both estradiol-treated and 5.0 mg MXC-treated mice. This would suggest that these cells are unable to synthesize and secrete steroids. Thus, this commonly employed pesticide appears to closely mimic those effects on the female ovary induced by estrogen.

Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies.

This study was designed to assess whether exposure to the estrogenic pesticide methoxychlor (MXC) during pregnancy would affect reproductive parameters not only in female offspring exposed prenatally, but also in those of a subsequent litter. Mice were exposed via oral gavage to 7.5, 5.0, or 2.5 mg technical grade MXC (50%) or 0.025 mg estradiol-17 beta (E-17 beta) from days 6 to 15 of pregnancy. Following delivery, female offspring (F1a) were cross-fostered and sacrificed at 8 weeks of age. Mothers exposed during their first pregnancy were allowed to mate again and their second set of offspring (F1b) were similarly evaluated to detect any latent effects from the initial exposure. Mice exposed to 7.5 mg MXC were unable to carry their litters to term. Results revealed a significant increase in the length of gestation of mice exposed to both E-17 beta and 5.0 mg MXC. A larger percentage of atretic follicles appeared in the ovaries of F1a females exposed prenatally to 5.0 mg MXC when compared to controls. Females from the F1b litter displayed a significant advance in time of vaginal opening, an apparent residual effect of MXC from a mother exposed during a previous pregnancy.

Effects of methoxychlor on the reproductive system of the adult female mouse. 1. Gross and histologic observations.

The purpose of this study is to examine the effects of the pesticide methoxychlor (MXC) on the reproductive system of the adult female mouse. Sexually mature (7- to 8-week) virgin female CD-1 mice were exposed to 1.25, 2.5, or 5.0 mg MXC (50% technical grade) via oral gavage for 5 consecutive days each week for either 2 or 4 weeks. Control groups received either 0.025 mg estradiol-17 beta (E-17 beta) or the sesame oil vehicle for the same time period. Vaginal smears were taken daily, and weights were recorded weekly. Twenty-four hours following the final exposure, animals were sacrificed. Ovaries and reproductive tracts were removed and weighed. One ovary from each animal was prepared for light microscopic evaluation. Results revealed a dose dependency of MXC in inducing persistent vaginal estrus (PVE). Ovaries of MXC-exposed and E-17 beta-exposed animals weighed significantly less than the sesame oil controls. In addition, there was an increase in the number of atretic large follicles in the E-17 beta group and in those mice treated with the two highest doses of MXC, indicating a potential reduction in the immediate fertility of the animal. Thus, this commonly employed pesticide appears to mimic closely those effects on the female reproductive system induced by estrogens.

Chlordecone-induced follicular toxicity in mouse ovaries.

The effect of the pesticide, chlordecone, on murine follicular development was examined. Female CD-1 mice were exposed to chlordecone for 5 consecutive days for each of 4 consecutive weeks (0.25 mg/day). Controls received sesame oil vehicle or estradiol-17 beta (E-17 beta; 0.1 mg/day) since chlordecone has been ascribed estrogenic activity. Animals were sacrificed 24 h following the final exposure. Ovaries were removed, serially sectioned, and stained. Follicles were classified as small, medium, or large and were tabulated. Twice as many medium-sized follicles were found in the E-17 beta-treated mice as in both the chlordecone-exposed and sesame oil control groups. Both pesticide- and E-17 beta-exposed mice displayed a much higher percent of atresia in the large follicles; however, there were more actual healthy, large follicles in the E-17 beta group. Thus, both chlordecone and E-17 beta induced increased atresia among large follicles, which could be due to the estrogenicity of these agents. However, a decreased pool of healthy large- and medium-sized follicles occurred in chlordecone-treated mice, a condition not seen in E-17 beta-treated mice. Thus, the pool of potentially ovulatory follicles is reduced in the pesticide-treated animals.

Ovulatory response of chlordecone (Kepone)-exposed mice to exogenous gonadotropins.

The present study assessed the ability of the murine ovary to ovulate in response to exogenous gonadotropins following exposure to an estrogenic pesticide chlordecone (Kepone). Sexually mature virgin female CD-1 mice were exposed by oral gavage to either 0.062 mg, 0.125 mg or 0.25 mg (2, 4 or 8 mg/kg, respectively) chlordecone for 5 consecutive days for 2, 4, or 6 weeks. Control groups received either 0.1 mg estradiol-17 beta (E-17 beta) or the sesame oil vehicle for the same period. During the final week of exposure all experimental and control animals were treated with a superovulatory regimen of PMSG and hCG. The results revealed that the lower 2 chlordecone doses (0.062 and 0.125 mg) had highly variable effects on the ovulatory responses. The highest chlordecone dose (0.25 mg), however, produced a significant and progressive decrease in the ovulatory responses when compared to both E-17 beta and vehicle controls. Since ovulation was progressively impeded in mice exposed to 0.25 mg chlordecone, this high chemical dose may have exerted a direct effect on the ovary since sufficient exogenous gonadotropins were available to stimulate ovulation.

Galactose inhibition of ovulation in mice.

Clinical evidence suggests an association between galactosemia and premature ovarian failure. In the present study, adult female mice were fed a diet consisting of 50% galactose for either 2, 4, or 6 weeks. At all times there was a decrease in the normal ovulatory response, as evidenced by a reduction in the number of corpora lutea when compared with controls. Additionally, the exposure of galactose-treated mice to a superovulatory regimen of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) failed to induce an increased ovulatory response. Morphologic alterations, such as the increase in interstitial tissue and the appearance of lipofuscin, coupled with the failure to respond to exogenous gonadotropins, suggest that the reduced ovulatory response may be occurring at the level of the ovary. This effect, however, is reversible with cessation of galactose treatment.

Benzo(a)pyrene inhibits ovulation in C57BL/6N mice.

Successful female reproductive function requires follicle growth, ovulation, and formation of the corpus luteum. Treatment of C57BL/6N mice with a single intraperitoneal injection of benzo(a)pyrene in doses ranging from 1 to 500 mg/kg produced a dose- and time-dependent decrease in the number of corpora lutea. This effect on the number of corpora lutea is most pronounced at 1 week after treatment, with a threshold of about 1 mg/kg, and an ED50 of 1.6 mg/kg. By 2 weeks after treatment partial recovery of follicle growth and ovulation occurred, as indicated by an increase in the ED50 to 20 mg/kg. Complete recovery of normal corpora lutea number occurs in mice treated with less than 100 mg/kg by 3 weeks after treatment, with little change in the ED50 noted between 3 and 4 weeks post-treatment, 78 mg/kg at both times. Mice treated with 100 or 500 mg/kg did not recover normal corpora lutea number over the course of this experiment. These data indicate that acute exposure to benzo(a)pyrene, and perhaps other polycyclic aromatic hydrocarbons, may have a transient adverse effect on follicle growth, ovulation, or formation of corpora lutea. A consequence of this effect, transient infertility, has been observed previously when exploring the effect of polycyclic aromatic hydrocarbons on murine reproduction.

Effects of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) on gonadal development in the chick embryo: a histological and histochemical study.

Fertile white Leghorn chicken eggs were exposed via intravitelline injections to dosages of 5.0, 10.0, or 20 mg 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) in olive oil prior to incubation. Control embryos received only the olive oil vehicle. Eggs were placed in a forced-draft incubator for either 5 or 12 days. Embryos were removed and their gonadal areas prepared for histological or histochemical evaluation. Histological examination of DDT-exposed 5-day embryos revealed no significant differences in the number of primordial germ cells aggregating in the gonadal area and in the localization of acid and alkaline phosphatase activity. Embryos exposed to DDT for 12 days revealed significant alterations in both ovaries and testes. The testes of DDT-exposed embryos consisted of mostly stroma with fewer seminiferous cords than controls while ovaries of exposed embryos contained a larger number of distended medullary cords as well as a difference in the distribution of these cords when compared to controls. There was an increased alkaline phosphatase activity in the stromal cells of female gonads. Increased amounts of alkaline phosphatase activity found in the stroma at 12 days might be due to a DDT-induced stimulation of these cells to differentiate more rapidly. Acid phosphatase activity was found in the secondary sex cords of control 12-day ovaries, but was much reduced or absent in those of pesticide-exposed embryos. These results indicate that a single dosage of DDT administered to a chick embryo prior to incubation does not affect early stages of gonadal development but that effects on both ovaries and testes occur 12 days following exposure.

Early mammalian embryonic development.

This report examines the major events occurring during the first month of development in the human embryo. Developmental events are detailed, beginning with cleavage of the zygote and ending with the formation of the three primary germ layers. Certain aspects of early human development are compared with that in lower forms of vertebrates to emphasize species differences in these basic embryological processes. A discussion of the formation of identical twins is included, since it has its inception during the first month of development.

Acid and alkaline phosphatase activity in migrating primordial germ cells of the early chick embryo.

Little information is available concerning enzyme activity in primordial germ cells (PGCs) of the early chick embryo. The present study is designed to examine the disposition of alkaline and acid phosphatase activity in the PGCs during their migration into the developing gonads of the early chick embryo. White Leghorn chick embryos were sacrificed at daily intervals from 1 to 6 days of incubation. Following sacrifice the embryos were fixed, dehydrated, and embedded in glycol methacrylate (GMA). Alkaline and acid phosphatases were demonstrated by the simultaneous diazo-coupling method. The embryonic tissues at the different ages were examined for PGCs and the histochemical reactions for alkaline and acid phosphatases in these cells evaluated. Acid phosphatase activity did not appear within PGCs until 3 days of incubation, and then in only a few PGCs in the active phase of their migration in the dorsal mesentery, suggesting that there is no large wave of degeneration of these cells during migration. Alkaline phosphatase activity was observed as early as 2 days of incubation in PGCs during the passive phase of their migration in extraembryonic blood vessels. Alkaline phosphatase-positive PGCs in the active phase of migration were also found in the dorsal mesentery; however, the cellular localization of this enzyme differed from that observed in the passively migrating PCGs, indicating that there are alterations in the metabolic activities of these cells during the active and passive phases of migration.