Research Grants/Fellowships/SBIR

Final Report: Developmental Exposure to Endocrine Disruptors: Fertility and Gene Expression Profiles

EPA Grant Number: R827402
Title: Developmental Exposure to Endocrine Disruptors: Fertility and Gene Expression Profiles
Investigators: Zacharewski, Timothy , Chou, Karen , Saama, Peter
Institution: Michigan State University
EPA Project Officer: Fields, Nigel
Project Period: September 1, 1999 through July 31, 2002
Project Amount: $738,712
RFA: Endocrine Disruptors (1999) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals



It has been proposed that exposure to estrogenic chemicals during critical periods of development leads to decreased sperm counts and sperm quality in sexually mature offspring. Although controversial, this hypothesis is mechanistically feasible and is supported by epidemiological and experimental studies. The objective of this research project was to examine the effect of diethylstilbestrol (DES), genistein (GEN), and ethynyl estradiol (EE) on sperm counts and quality following developmental exposure. The hypothesis that developmental exposure to DES, GEN, and bisphenol A compromises sperm quality and decreases sperm counts by modulating estrogen receptor-mediated gene expression will be tested by examining the effect of developmental exposure to DES, GEN, and EE on developmental landmarks, sperm quality and counts, and testicular gene expression.

Summary/Accomplishments (Outputs/Outcomes):


B6D2-F1 mice were exposed to DES, GEN, and EE by gavage to the dam during gestation and via lactation until weaning at postnatal day 21. Sperm quality was assessed in developmentally exposed male offspring at 15 and 45 weeks using sperm motion analysis and in vitro fertilization assays. In addition, pups were monitored for several developmental landmarks (i.e., body weight, anogenital distance). Changes in gene expression in the testis were examined in control and treated animals at 3, 15, and 45 weeks using a customized cDNA microarray enriched for genes expressed in the mouse testis. Microarray technology provides a high-capacity assay to simultaneously monitor the modulation of expression of hundreds of genes and to identify treatment-related, gene-expression changes. Significant effects on gene expression were analyzed using several statistical approaches.

Developmental exposure to DES, GEN, and EE affected sperm quality and sperm counts, but in different ways. For example, DES compromised fertility by decreasing sperm counts and quality. In contrast, GEN significantly increased fertility in the high-dose group with no significant treatment-related effects on body weight, anogenital distance, seminal vesicle weight, or testis weight. The doses used in the study were considerably higher than the estimated human intake. EE (0, 0.1) decreased sperm concentrations and the number of motile sperm, but there was no treatment effect on in vitro fertilizing ability.

To investigate the effects of developmental exposure of DES, GEN, and EE on testicular gene expression, a cDNA microarray was constructed that was enriched with genes expressed in the testis. In addition, several computational resources were developed to assist with the analysis and storage of microarray data. This included an automated and customizable program to correct, filter, and normalize raw microarray data captured using GenePix, a commonly used microarray image analysis application. Several statistical methods were investigated to maintain awareness of new methods and to evaluate if these methods were superior or comparable to those proposed in the original application. Microarray analysis tools were subsequently used to identify early and latent alterations in the expression of genes involved in estrogen signaling (estrogen receptor alpha), steroidogenesis (steroidogenic factor 1, 17alpha-hydroxylase/C17,20-lyase, P450 side chain cleavage, steroidogenic acute regulatory protein, and scavenger receptor class B1), lysosomal function (LGP85, saposin), and regulation of testicular development (testicular receptor 2, inhibin/activin beta C, Hoxa10) in DES-treated animals. Examination of the same genes by real-time polymerase chain reaction revealed thatGEN had no effect on their expression. The results suggest that the effects elicited by GEN are not similar to an estrogen receptor agonist. In addition, the effect of gestational and lactational exposure to DES, GEN, and EE on ovulation and egg fertilizing ability in sexually mature female offspring was investigated. Although analysis of the results are ongoing, preliminary examination indicates that DES, GEN, and EE can affect the ovulatory response in female offspring.

The effects of DES and GEN on mammary gland development also were assessed in developmentally exposed offspring. Paradoxical results exist that demonstrate opposite effects of prenatal and postnatal exposure to estrogens on mammary gland development. However, the effect of gestational and lactational exposure has not been described. Mammary gland whole mounts were examined on postnatal day 49 for epithelial growth (percent of fat pad occupied by epithelium), length of mammary epithelial tree from nipple to distal edge, number of terminal end buds ([TEBs], undifferentiated proliferating structures), and density of alveolar buds ([ABs], differentiated milk secreting lobules). There were no significant changes in body weight or anogenital distance in female offspring of DES or GEN-treated dams. Although there was a trend towards an increase in mammary gland growth and an increase in alveolar bud formation in DES-exposed offspring, the results were not significant because of large interlitter variability. We also did not detect any significant changes in mammary growth, TEBs, or ABs in GEN-exposed offspring. These results suggest that combined gestational and lactational exposure to GEN has little effect on mammary gland development at levels of exposure comparable to human intake.

Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other project views: All 66 publications 9 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Fielden MR, Zacharewski TR. Challenges and limitations of gene expression profiling in mechanistic and predictive toxicology. Toxicological Sciences 2001;60(1):6-10. R827402 (2001)
R827402 (Final)
not available
Journal Article Fielden MR, Halgren RG, Dere E, Zacharewski TR. GP3: GenePix post-processing program for automated analysis of raw microarray data. Bioinformatics 2002;18(5):771-773. R827402 (2001)
R827402 (Final)
not available
Journal Article Fielden MR, Halgren RG, Fong CJ, Staub C, Johnson L, Chou K, Zacharewski TR. Gestational and lactational exposure of male mice to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing ability in vitro, and testicular gene expression. Endocrinology 2002;143(8):3044-3059. R827402 (Final)
not available
Journal Article Fielden MR, Matthews JB, Fertuck KC, Halgren RG, Zacharewski TR. In silico approaches to mechanistic and predictive toxicology: An introduction to bioinformatics for toxicologists. Critical Reviews in Toxicology 2002;32(2):67-112.
abstract available  
R827402 (2001)
R827402 (Final)
not available
Journal Article Fielden MR, Fong CJ, Haslam SZ, Zacharewski TR. Normal mammary gland morphology in pubertal female mice following in utero and lactational exposure to genistein at levels comparable to human dietary exposure. Toxicology Letters 2002;133(2-3):181-191.
abstract available   full text available
R827402 (Final)
not available
Journal Article Fielden MR, Samy SM, Chou KC, Zacharewski TR. Effect of human dietary exposure levels of genistein during gestation and lactation on reproductive development and sperm quality in mice. Food Chemistry and Toxicology 2003;41(4):447-454. R827402 (Final)
not available
Journal Article Halgren RG, Fielden MR, Fong CJ, Zacharewski TR. Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones. Nucleic Acids Research 2001;29(2):582-588 R827402 (2001)
R827402 (Final)
R827401 (2001)
not available
Journal Article Saama PM. Sparse preconditioned iterative solvers for ill-posed mixed model equations. Society for Industrial and Applied Mathematics Journal on Numerical Analysis. R827402 (Final)
not available
Supplemental Keywords:

diethylstilbestrol, DES, genistein, bisphenol A, endocrine distruptors, genetic susceptability, pesticides, DDT, bioavailability, children, developmental biology, developmental toxicants, endocrine disrupting chemicals, EDCs, endocrine disruption, environmental hazard exposures, estrogen response, estrogenic chemicals, ethynyl estradiol, exposure, fertility, gene expression, genetic mechanisms, growth and development, human exposure, reproductive health, reproductive processes, rodent, sensitive populations, sperm count., RFA, Health, Scientific Discipline, Toxics, Ecology, Environmental Chemistry, Chemistry, Epidemiology, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, Biology, Endocrine Disruptors - Human Health, sensitive populations, estrogenic chemicals, bioavailability, endocrine disrupting chemicals, exposure, statistical analysis, children, fertility, developmental biology, genetic mechanisms, DDT, gene expression, human exposure, growth and development, estrogen response, reproductive processes, reproductive health, sperm count, rodent, developmental toxicants, environmental hazard exposures

Relevant Websites: Exit

Progress and Final Reports:
Original Abstract
2001 Progress Report