Variation in commercial rodent diets induces disparate molecular and physiological changes in the mouse uterus

Abstract

Although ovarian estrogen, estradiol-17beta, is a key modulator of normal reproductive functions, natural and synthetic compounds with estrogen-like activities can further influence reproductive functions. Plant-derived phytoestrogens specifically have received much attention because of associated health benefits. However, a comprehensive understanding of the beneficial and/or detrimental impacts of phytoestrogen consumption through commercial rodent diets on uterine biology and early pregnancy at the molecular level remains largely unexplored. Using multiple approaches, we demonstrate here that exposure of adult female mice to a commercial rodent diet with higher phytoestrogen levels facilitates uterine growth in the presence or absence of ovarian estrogen, alters uterine expression of estrogen-responsive genes, and advances the timing of implantation compared with a diet with lower phytoestrogen levels. The finding that variability in phytoestrogen content in commercial rodent diets, both within and between brands, influences experimental results stresses the importance of this investigation and raises caution for investigators using rodents as animal models.

Fig. 1.

Commercial rodent diets alter uterine growth responses. (A) Adult ovariectomized mice show comparable body weights after 8 weeks of feeding the Purina diet 5V00 or 5001 (P > 0.05). (B and C) Mice consuming the Purina 5001 diet have markedly higher uterine wet weights than those on the Purina 5V00 diet (*, P < 0.005). (D–G) Uterotrophic assay with adult ovariectomized mice on the respective diets treated with oil, E₂, P₄, or E₂ plus P₄. Numbers within the bar indicate the number of mice examined (*, P < 0.05; **, P < 0.005).

Fig. 2.

Uterine BrdUrd incorporation in ovariectomized mice on the Purina diet 5V00 or 5001 with or without steroid hormone treatment. When examined at 6 h, mice on the diet 5001 with higher phytoestrogen levels showed increased uterine epithelial BrdUrd incorporation even in the absence of E₂, albeit at lower levels as compared with E₂ treatment (A). After 24 h of E₂ treatment, luminal and glandular epithelia showed comparable BrdUrd labeling in mice on either diet (B). Increased BrdUrd incorporation in uterine stromal cells was noted at 24 h of P₄ treatment in mice on the 5001 diet (C). In contrast, mice on the same diet showed reduced stromal cell BrdUrd labeling in response to an injection of P₄ or P₄ plus E₂ after 2 days of priming with P₄ (D). These experiments were repeated in three mice in each group with similar results. Representative photographs of longitudinal uterine sections are shown at ×400. le, luminal epithelium; ge, glandular epithelium; s, stroma.

Fig. 3.

Commercial rodent diets alter estrogen-responsive gene expression. (A and B) Comparative RT-PCR detection of LF, LIF, PR, ERα, and sFRP2 mRNAs in ovariectomized mice consuming the 5V00 or 5001 diet. Data are presented as fold induction and are relative to values from mice on the low-phytoestrogen diet 5V00. Values represent the mean ± SEM of three to four independent RNA samples. (C and D) In situ hybridization of LF in uteri of ovariectomized females on different diets at 6 and 24 h after exposure to steroid hormones. Representative dark-field photomicrographs of longitudinal uterine sections are shown at ×40. myo, myometrium; le, luminal epithelium; ge, glandular epithelium; s, stroma.

Fig. 4.

Commercial rodent diets exert differential effects on uterine PR and ERα protein distribution in ovariectomized mice treated with E₂. Representative immunohistochemical staining of longitudinal uterine sections is shown at ×400. Up-regulation of PR (A and B) and down-regulation of ERα (C and D) protein accumulation was noted in ovariectomized mouse uteri fed the 5001 diet with higher phytoestrogen levels. These experiments were repeated on three mice in each group with similar results. le, luminal epithelium; ge, glandular epithelium; s, stroma.

Fig. 5.

Commercial rodent diets alter the window of uterine receptivity for implantation. (A) Higher dietary phytoestrogen levels advance the timing of the attachment reaction. Numbers within the bar indicate the number of mice with blue bands per mice examined. (B) Representative photographs of uteri with or without implantation (blue bands) on the respective diets. (C) Recovered unimplanted blastocysts from mice consuming the low phytoestrogen diet showing normal morphology. (Scale bar, 50 μm.) (D) Dietary phytoestrogens alters the uterine responsiveness to native E₂ in initiating implantation in delayed implanting mice. Numbers within the bar indicate the number of mice with blue bands per mice examined. (E) In situ hybridization of HB-EGF in uteri devoid of blue bands at 2200 hours on day 4 of pregnancy on the respective diets. Representative dark-field photomicrographs of longitudinal uterine sections are shown at ×40. Arrow indicates the site of the blastocyst. myo, myometrium; le, luminal epithelium; ge, glandular epithelium; s, stroma.