Mammary Gland Morphology and Gene Expression Differ in Female Rats Treated with 17β-Estradiol or Fed Soy Protein Isolate

Abstract

Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy infant formula, with those of 17β-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were fed AIN-93G diets containing casein or SPI beginning on postnatal d 30. Rats were ovariectomized on postnatal d 50 and treated with 5 μg/kg/d E2 or vehicle for 14 d. Microarray analysis revealed that E2 treatment altered expression of 780 genes more than or equal to 2-fold (P < 0.05), whereas SPI feeding altered expression of only 53 genes more than or equal to 2-fold. Moreover, the groups had only 10 genes in common to increase more than or equal to 2-fold. The combination of SPI feeding and E2 altered expression of 422 genes and reversed E2 effects on many mRNAs, including those involved in the c-myc signaling pathway, cyclin D1, and Ki67. ERα binding to its response element on the Tie-2/Tek and progesterone receptor promoters was increased by E2, but not SPI, and this promoter binding was suppressed by the combination of E2 + SPI for the Tie-2/Tek promoter but increased for the progesterone receptor promoter (P < 0.05). SPI reduced the ratio of epithelial to fat pad area and E2 + SPI reduced both epithelial and fat pad area (P < 0.05). These data suggest that SPI is only minimally estrogenic in the rat mammary gland even in the absence of endogenous estrogens.

Feeding soy protein isolate and treatment with estradiol have different effects on mammary gland morphology and gene expression in weanling male and female rats

“…Time-impregnated SpragueDawley rats (Harlan Industries, Indianapolis, IN) were fed AIN-93G diets at a level of 20% protein, with casein as the sole protein source. Corn oil replaced soybean oil (43,44,45). After birth, dams continued to be fed AIN-93G throughout lactation.…”

“…Blood was collected and frozen at Ϫ20°C until analysis. One abdominal mammary gland from each animal was frozen at Ϫ80°C, another was used for preparation of whole mounts (46) and determination of mammary gland morphology by light microscopy by a trained veterinary pathologist (L. Hennings) as described previously (35,45). In brief, the mammary tissues were fixed flat and defatted in acetone for a minimum of 48 h. Tissues were then rehydrated through a series of alcohols, stained overnight with carmine alum, then dehydrated through a series of alcohols, and cleared in xylene.…”

“…Chromatin immunoprecipitation (ChIP) was performed as previously described (45). Briefly, chromatin was extracted with the ChIP-It Enzymatic Express kit (ActiveMotif, Carlsbad, CA) from pools of 300 mg of minced male mammary tissues following the manufacturer's protocol.…”

“…DNA was eluted in 50 l AM2 and reverse cross-linked by addition of 50 l reverse cross-linking buffer and heating at 95°C followed by treatment with proteinase K then proteinase K stop solution. As previously published (45), ER binding to estrogen response elements (ERE) was analyzed in estrogen-responsive elements in the first exon of the progesterone receptor gene Pgr and the fourth intron of the ER␣ gene Esr1. The 5=-untranslated region of Aldh3a2 was utilized as a negative control.…”

“…These studies provide a closer parallel with consumption of soy infant formula. Dietary exposure to SPI in ovariectomized adult female rats was associated with no morphological change other than a decrease in epithelial/fat pad area and an overlap of only 1% in gene expression change between estradiol (E2) and SPI, suggesting that SPI has minimal estrogenic actions in the adult female mammary gland even in the absence of endogenous estrogens (45). Exposure to SPI continuously through gestation and early development, indirectly via the dam and directly following weaning, has been shown to decrease the number of TEBs, increase the number of lobules, and reduce mammary adipose cell size in female rat mammary.…”