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

Abstract

Long-term effects of in vivo exposures to proestrogen methoxychlor (MXC) or estradiol-17β (E) were studied during early pregnancy (preimplantation) in ICR mice. Pregnant dams received either subcutaneous injections of 1 μg of E on Day 2 of pregnancy only (vaginal plug=Day 1), or 5.0 mg 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.

Introduction

Exposure of murine embryos during early stages of development (preimplantation) in vitro to such estrogenic compounds as pesticides methoxychlor (MXC), o,p′-DDT, and bispenol A (BPA) affects their development [1], [2], [3]. It has been shown also that MXC [4], [5] as well as estradiol [5], [6], [7] suppress preimplantation embryo development in mice in vivo and cause their retention in the oviducts (tube-locking effect).

On the other hand, in vivo exposure of mice to xenoestrogens either prenatally [8] or few days after birth [9], [10], [11] affects postnatal reproductive development in both sexes. Other experiments demonstrated that even a small augmentation of serum estradiol due to in utero position of male fetus between two female fetuses causes prostate enlargement and changes behavior in males, when compared with their male siblings positioned between males in uterus [12], [13].

Recently, only one study investigated the long-term, postnatal effects of exposing murine embryos in vitro to xenoestrogens during preimplantation stage of development. Using BPA as a model xenoestrogen, this study examined only such general effects as the rate of growth during weaning period [14]. Thus, the more specific effects of in vivo exposure of preimplantation embryos to estrogens/xenoestrogens on their sexual development after birth have not been adequately investigated in mice.

Reproductive anomalies have been found in animals born into ecosystems that are polluted by xenoestrogens [15]. Early prenatal stages of embryo development may be sensitive targets to these environmental pollutants because estrogen receptors (ER) were found in all blastomeres in mouse embryos throughout preimplanatation period of development [16], [17], [18]. Thus, it is possible, that in vivo exposure of preimplantation embryos to ecoestrogens may alter short- and long-term developmental processes in mice and other mammals.

Using mouse as a model species, we have shown earlier that pesticide MXC exposure during the early postimplantation period (Days 5–7) significantly decreases sexual arousal, with a concomitant decrease in testosterone levels in adult males [19], [20]. In our most recent study, in vivo exposures of preimplantation mouse embryos (Days 2–4) to MXC suppressed embryo development, increased rate of embryo abnormalities, and produced a high incidence of apoptosis in blastocysts [5].

The present paper addresses the question whether similar in vivo exposures to MXC of mouse embryos at preimplantation stages alter postimplantation sexual development in both sexes. The aim of this study was to examine the effects of the exposures of preimplantation embryos to MXC during in vivo development and to compare its effects with estradiol. Anogenital distance (AGD) in both sexes close to the end of the weaning period was used as a measure of masculinization/demasculinization. The presence of vaginal opening at weaning was also used an indicator of sexual maturation in female offspring. The body weight was used as a measure of the overall growth.