Neonatal exposure to genistein disrupts ability of female mouse reproductive tract to support preimplantation embryo development and implantation

Abstract

Female mice treated neonatally with the phytoestrogen genistein (50 mg/kg/day) have multioocyte follicles, lack regular estrous cyclicity, and are infertile even after superovulation. To determine the cause of their infertility, we examined oocyte developmental competence and timing of embryo loss. Eggs obtained by superovulation of genistein-treated or control females were equally capable of being fertilized in vitro and cultured to the blastocyst stage. However, if eggs were fertilized in vivo, retrieved at the pronucleus stage, and cultured, there was a significant reduction in the percentage of embryos from genistein-treated females reaching the blastocyst stage. When these blastocysts were transferred to pseudopregnant recipients, the number of live pups produced was similar to that in controls. Preimplantation embryo development in vivo was examined by flushing embryos from the oviduct and/or uterus. Similar numbers of one-cell and two-cell embryos were obtained from genistein-treated and control females. However, significantly fewer embryos (<50%) were obtained from genistein-treated females on postcoital Days 3 and 4. To determine if neonatal genistein treatment altered the ability of the uterus to support implantation, blastocysts from control donors were transferred to control and genistein-treated pseudopregnant recipients. These experiments demonstrated that genistein-treated females are not capable of supporting normal implantation of control embryos. Taken together, these results suggest that oocytes from mice treated neonatally with genistein are developmentally competent; however, the oviductal environment and the uterus have abnormalities that contribute to the observed reproductive failure.

FIG. 1.

Confocal microscopy of metaphase II-arrested eggs immunostained for α-tubulin and DNA. Examples of normal-appearing meiotic spindles in eggs from control mice (A) and from Gen-treated mice (B). Example of abnormal spindle from Gen-treated mouse (C). Original magnification ×400.

FIG. 2.

Graph of embryo progression during 4 days in vitro. Fertilized eggs from mice treated neonatally with Gen (n = 15 mice [373 embryos]) or with corn oil alone (n = 11 mice [231 embryos]) were followed up for 4 days in culture to determine progression of development. A) The percentage of embryos that progressed to each stage was determined for each mouse, and the mean ± SE is plotted. The asterisk denotes statistical significance at P < 0.05 (Wilcoxon test). PN, pronuclear stage; 2C, two-cell stage; 4–8C, four- to eight-cell stage; Mor, morula stage; Blast, blastocyst stage. B) Percentage of embryos at the four-cell (4C) or five- to -eight-cell (5–8C) stage of development 48 h after hCG administration. Overall, 211 embryos from control mice and 356 embryos from Gen-treated mice were evaluated. There were significantly fewer five- to eight-cell embryos in the Gen-treated group than in the control group (P < 0.0001, Fisher exact test).

FIG. 3.

Graph of embryo development during 4 days in vivo. The mean ± SE number of embryos retrieved from control or Gen-treated pregnant mice 24, 48, 72, and 92 h after hCG administration is shown. The number of mice per group is indicated at the base of the column. The asterisk denotes statistical significance at P < 0.05 (Wilcoxon test).

FIG. 4.

Schematic diagram of the experimental design. All female mice were superovulated, and egg-cumulus cell masses were collected 14 h after hCG administration for in vitro fertilization (IVF) and immunohistochemistry (IHC) experiments. For embryo development, superovulated female mice were bred to males, and embryos were collected from the oviduct and/or uterus at the times indicated above each developmental stage. Embryos were then placed in culture to continue development to the blastocyst stage. Embryo transfers were performed using blastocysts derived from one-cell embryos. Developmental stages inside the oviduct represent in vivo progression, and developmental stages outside the oviduct represent in vitro progression.