Neonatal phytoestrogen exposure alters oviduct mucosal immune response to pregnancy and affects preimplantation embryo development in the mouse

Abstract

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are "posteriorized" as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.

FIG. 1.

Increased oviductal IgA in genistein-treated mice. A) IgA levels in oviducts of control and genistein-treated mice on Pregnancy Days 2 and 4. Means ± SEM were plotted. White bars, controls; black bars, genistein-treated. *P < 0.05. B) Representative actin blot of oviduct samples from control or genistein-treated mice, as indicated, on Pregnancy Day 4.

FIG. 2.

A–D) PAS/Alcian blue stain. Two-cell embryos (e) present in oviductal lumen in A and B. Note increased Alcian blue staining in epithelium (arrow) and lumen (asterisk) in B and D. E–H) Factor VIII immunohistochemistry; blood vessels indicated (arrowheads). A, C, E, and G are controls; B, D, F, and H are genistein-treated. Bar = 100 microns.

FIG. 3.

Expression of Cxcl15, Slpi, Cd44, and Ltf in oviduct following neonatal genistein-treatment and in control FRT. A) Relative expression of the indicated genes in the oviducts of control and genistein treated mice at indicated time points. Mean relative expression ± SEM were plotted. d5, PND5; d22, PND22; 8h, before ovulation; 15h, after ovulation; 48h, Pregnancy Day 2; 96h, Pregnancy Day 4. White bars, controls; black bars, genistein-treated. *P < 0.05. B) Relative expression of the indicated genes in FRT in adult controls. ov, oviduct; ut, uterus; cx, cervix; v, vagina.

FIG. 4.

Expression of Cxcl15, Cd44, Slpi, and Ltf in control or genistein-treated mice following breeding to vasectomized males. Oviducts were collected 96 hours post hCG and breeding. Mean relative expression ± SEM plotted. White bars, controls; black bars, genistein-treated. *P < 0.05.

FIG. 5.

Inhibition of alterations in gene expression by estrogen receptor antagonist. Expression of Pitx1, Cxcl15, Slpi, and Cd44 in oviduct of control (Co), ICI, genistein (Gen), or genistein plus ICI (G+I)-treated mice on Pregnancy Day 4. Mean relative expression ± SEM plotted. *P < 0.05; line above groups denotes the comparison.

FIG. 6.

Alterations in embryo development in vivo following neonatal genistein-treatment. A) Mean percentage of surviving embryos in each category flushed from the FRT at indicated time points are plotted. n = 46–133 embryos from 4 mice/group/time point. B) Blastocyst cell allocation plotted as mean number of cells/embryo or TE:ICM ratio/embryo ± SEM. n = 27–35 embryos pooled from 6 mice/group. *P < 0.05. mor, morula; blasts, blastocysts; ICM, inner cell mass; TE, trophectoderm. White bars, controls; black bars, genistein-treated.