Grantee Research Project Results
Final 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 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 the 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.
Summary/Accomplishments (Outputs/Outcomes):
Exposure of male rats to some phthalate esters during development results in altered expression of genes that may be involved in germ cell proliferation and survival. Our studies revealed that exposure to 500 mg/kg/day di-n-butyl phthalate (DBP) from gestation day (gd) 12 to 20 significantly increased the number of multinucleated germ cells (gonocytes) in the fetal rat testis. In humans, multinucleated gonocytes (MNG) are believed to be precursors of testicular germ cell cancer, which is the most common cancer in young men. Our immunostaining with markers for proliferation and apoptosis indicated that MNG in the fetal rat testes exposed to DBP were neither proliferating nor undergoing apoptosis on gd 17–21. On gd 21 many gonocytes in DBP-exposed testes were joined by intercellular bridges, which were not observed in control testes. These bridges were detected by electron microscopy only, and could not be seen by light microscopy without specific markers. In normal rat testis, intercellular bridges have been occasionally observed on gd 17 (but not gd 21) and are believed to result from incomplete cytokinesis during gonocyte mitosis, which normally occurs in the rat on gd 16. Gonocyte multinuclearity may result from cytoplasmic confluence of gonocytes joined by intercellular bridges following their enlargement or from plasma membrane fusion. We hypothesize that DBP interferes with division of gonocytes resulting in persistence of these bridges several days after mitosis on gd 16. Persistent bridges and lack of support from Sertoli cells due to altered cell-cell contact between Sertoli cells and gonocytes in DBP-exposed fetal testes may cause an increase in the number of MNG following exposure to DBP.
The effect of DBP exposure on fetal development of gonocytes was most apparent on gd 21. Using morphology of MNG as an endpoint, we obtained a dose-response relationship between the number of MNG per section and several dose levels of DBP, including the highest estimated human exposure level (0.1 mg/kg/day). Statistical significance of DBP treatment on occurrence of MNG was achieved at the 100 mg/kg/day dose level. At this dose level the dose-response curve exhibited an effect of threshold. Although MNG develop in the normal fetal rat testis, exposure to DBP in utero increased the number of these genetically altered cells in a dose-dependent manner. Counts of MNG were based on morphological criteria, which is a less sensitive indicator of DBP response as compared to stereological characteristics of the fetal testis such as the testis volume and total cell number. For these parameters, significance of DBP treatment was reached at 50 and 30 mg/kg/day dose levels, respectively. Persistent bridges in DBP-exposed gonocytes suggest that incomplete cytokinesis contributes to the development of MNG. Intercellular bridges between developing male germ cells have been reported in the human fetal testis. Given the high level of conservation of cellular processes underlying testicular development in humans and rats, markers of incomplete cytokinesis will aid in the assessment of MNG occurrence in the developing human testis.
Using a stereological approach, we established that gestational exposure to 500 mg/kg/day DBP starting on gd 12 significantly inhibited proliferation of somatic cells in the fetal testis. The decrease in the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells was first detected on gd 17, which was the earliest time point examined in our study, and persisted on gd 20–21. The overall shape of curves representing kinetics of cell proliferation in control and DBP-exposed fetal testes suggests that our inability to detect a significant difference on gd 18 and 19 was due to variability of the control fetus data rather than recovery of cell proliferation in DBP-exposed testes. Consistent with decreased cell proliferation, testis cell number on gd 17 and 19–21 was significantly lower in the exposed testes as compared to control. The rate of cell proliferation and the total cell numbers were in good agreement in regard to representation of the fetal testis growth and consistent with previously reported fetal and early postnatal Sertoli and Leydig cell numbers in the rat. Collectively, our terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and BrdU data indicate that the smaller testis in DBP-exposed rat fetuses resulted from a decrease of somatic cell proliferation rather than apoptosis. Testis volume was the least sensitive cellular parameter since significant reduction in volume was achieved only after cell number decrease for several days. After birth, all cellular parameters examined in our study returned to control values. A lack of significantly increased apoptosis in the DBP-exposed testes on gd 17–21 indicates that the observed sharp postnatal increase in the number of proliferating cells was not a compensatory response for apoptosis in somatic cells during the fetal period of development. The staining pattern of BrdU-positive cells was similar in control and treated testes on all gd examined in this study. Thus, it is unlikely that cell proliferation in treated testis decreased because of death of specific progenitor somatic cells at earlier gestation days that were not assessed in our studies.
Our data confirm the previous notion that certain testicular responses to DBP exposure in utero are reversible. So far the reported reversible effects include reduced fetal testicular testosterone, the collapsed fetal Sertoli cells cytoskeleton, and altered expression of smooth muscle actin in fetal peritubular myoid cells. The stereological approach to quantify cellular outcomes in the fetal testis following exposure to DBP was more sensitive than measuring weights of fetal testes and revealed DBP-induced effects at the 30 mg/kg/day dose level. Data obtained by this approach will be useful for developing a mechanistic model that incorporates cellular responses in the fetal testis following exposure to various DBP dose levels.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 22 publications | 3 publications in selected types | All 3 journal articles |
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Boekelheide K, Kleymenova E, Liu K, Swanson S, Gaido KW. Dose-dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n-butyl) phthalate. Microscopy Research and Technique 2009;72(8):629-638. |
R830766 (Final) |
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Howroyd P, Hoyle-Thacker R, Lyght O, Williams D, Kleymenova E. Morphology of the fetal rat testis preserved in different fixatives. Toxicologic Pathology 2005;33(2):300-304. |
R830766 (2004) R830766 (2005) R830766 (Final) |
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Kleymenova E, Swanson C, Boekelheide K, Gaido KW. Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact. Biology of Reproduction 2005;73(3):482-490. |
R830766 (2005) R830766 (Final) |
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Supplemental Keywords:
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.