Grantee Research Project Results
2003 Progress Report: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters
EPA Grant Number: R830766Title: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters
Investigators: Gaido, Kevin
Institution: The Hamner Institutes
EPA Project Officer: Aja, Hayley
Project Period: February 24, 2003 through February 23, 2006 (Extended to March 31, 2007)
Project Period Covered by this Report: February 24, 2003 through February 23, 2004
Project Amount: $725,736
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
The objective of this research project is to identify key molecular and cellular events associated with germ cell development in the rat testis that are targets for endocrine active chemicals following in utero exposure.
Progress Summary:
Exposure of male rats to some phthalate esters during development results in the altered expression of genes that may be involved in germ cell proliferation and survival. To better understand germ cell development and to identify the cellular processes in germ cells that are affected by in utero exposure to di-n-butyl-phthalate (DBP), we assessed cell proliferation and apoptosis in testes from male rat fetuses and pups from dams exposed to 500 mg/kg/day DBP from gestation day (gd) 12 to gd 21. Tissue sections of testes collected on gd 17-gd 21 and postnatal days (PNDs) 1, 2, and 5 were examined. Cell proliferation was assessed by immunostaining with antibodies against proliferating cell nuclear antigen and 5-bromo-2-deoxyuridine (BrdU). Apoptosis was measured by terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labeling (TUNEL) assay. Multinucleated gonocytes developed in the fetal rat testis exposed to DBP. These cells were neither proliferating nor undergoing apoptosis. The lack of active mitosis makes it highly unlikely that the multinuclearity in these cells resulted from incomplete cytokinesis during mitosis. The multinucleated gonocytes migrated to the basal membrane and entered into mitosis on PND 3-PND 5, as did normal gonocytes. This finding suggests that molecular mechanisms governing postnatal migration and the initial stages of mitosis are unaffected by developmental DBP exposure. On PND 5, the multinucleated gonocytes exhibited abnormal mitotic figures characterized by an increased number of chromosomes.
Exposure to DBP in utero resulted in smaller fetal rat testes as determined by stereological measurements, including size and total cell number. TUNEL and BrdU data indicated that the smaller size of the DBP-exposed testis resulted from a cumulative decrease in proliferation of Sertoli cells and interstitial cells over several days, but did not result from cell death. The size of the testis and the rate of cell proliferation returned to their control levels after birth. The decreased cell proliferation during fetal development, however, irreversibly altered seminiferous tubule formation in the DBP-exposed testes.
Histological examination of haematoxylin and eosin slides showed that Sertoli cells in affected tubules were altered by developmental exposure to DBP. The vimentin cytoskeleton had collapsed in DBP-exposed Sertoli cells. The Sertoli cells also had convoluted plasma membranes and had lost contact with germ cells, as determined by immunostaining with cadherins (the plasma membrane glycoproteins mediating calcium-dependent cell adhesion). Immunostaining data were supported by electron microscopy observations that confirmed the retraction of extended Sertoli cell processes in DBP-treated testis. Altered contacts between Sertoli cells-gonocytes were apparent on gd 17 and persisted until gd 21.
Our studies demonstrated that although proliferation of fetal germ cells appeared unaffected by developmental exposure to DBP, such exposure significantly decreased proliferation of all other cell types in the fetal rat testis. The effect of this exposure on cell proliferation was reversed upon birth and cessation of DBP treatment. DBP dosing, however, ended at birth during a period when Sertoli cells normally are actively proliferating. If proliferating Sertoli cells are direct targets for DBP, as suggested by our data, and DBP exposure continued through the neonatal period, then the cellular consequences of such exposure may become more severe.
We determined that the number of seminiferous tubule cross sections is a robust endpoint predictive of a possible adverse outcome. Studies are now in progress to establish dose-response relationships for this and other cellular endpoints in the dose range, from 0.1 (approximate level of human exposure) to 100 mg/kg/day DBP.
Future Activities:
We will focus on the identification of key molecular pathways, which disruption by in utero exposure to DBP led to development of multinucleated gonocytes. The development of multinucleated gonocytes and overall effect of DBP on cell proliferation in the fetal testis will be assessed at low doses of in utero DBP exposure. The fate of the genetically abnormal multinucleated gonocytes during neonatal development will be determined. Testes explants from normal and in utero DBP-exposed male rat fetuses will be established and treated with testosterone and stem cell factor to establish the role of these factors on germ cell survival.
Journal Articles:
No journal articles submitted with this report: View all 22 publications for this projectSupplemental Keywords:
sensitive subpopulation, teratogen, reproductive development, children's health, phthalate esters, gene expression, apoptosis, cell proliferation, endocrine active chemical, androgen, germ cell development, rat testis, Sertoli cells, risk assessment, health effects, vulnerability, animal, mammalian, organism, age, chemicals, toxics, biology, pathology, measurement methods, plastics., RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, POLLUTANTS/TOXICS, Health Risk Assessment, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, Risk Assessments, endocrine disruptors, Environmental Microbiology, Physical Processes, Biochemistry, Endocrine Disruptors - Human Health, Biology, fetal exposure, altered gene expression, germ cell vulnerability, molecular mechanisms, endocrine disrupting chemicals, exposure, altered sexual development, EDCs, exposure studies, developmental biology, gestational exposure, animal models, fetal development, mice, reproductive processes, fetal genocyte degeneration, phthalates, human health riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.