2005 Progress Report: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters

EPA Grant Number: R830766
Title: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters
Investigators: Gaido, Kevin
Institution: The Hamner Institutes
EPA Project Officer: Hahn, Intaek
Project Period: February 24, 2003 through February 23, 2006 (Extended to March 31, 2007)
Project Period Covered by this Report: February 24, 2005 through February 23, 2006
Project Amount: $725,736
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text |  Recipients Lists
Research Category: Human Health , Children's Health , Health Effects , Health

Objective:

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 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 me n. 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 the normal rat’s 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 the 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 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 are less sensitive indicators 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. We will identify molecular markers of incomplete cytokinesis in gonocytes in the rat and use these markers to refine our understanding of the relationship between dose levels of DBP and the number of affected gonocytes. 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.

On postnatal days (pnd) 3–5, MNG in DBP-exposed testes became mitotically active but seemed unable to complete mitosis, as indicated by abnormal mitotic figures. To gain insights into the molecular events regulating the response of developing germ cells to DBP, we are establishing a Sertoli-germ cell co-culture using pnd 5 rat testes. To better preserve Sertoli cell structure, which is crucial for germ cell development, and to increase cell viability, we are using a mixture of proteins comprising the basal membrane (Matrigel) as a co-culture overlay. After establishing the doubling time and characterizing the Sertoli-germ cell ratio by immunostaining with markers specific for each cell type, co-cultures will be treated with mono-n-butyl phthalate (the active metabolite of DBP) and assessed for proliferation and apoptosis. These co-cultures will also be treated with testosterone and stem cell factor to establish the role of these factors on germ cell survival.

Future Activities:

The studies for the subsequent reporting period will focus on identification of key molecular pathways which are disrupted by DBP and lead to the development of multinucleated gonocytes. Effect of DBP on mitosis and apoptosis on gd 16-17 will be assessed at low doses of in utero DBP and di(2-ethylhexyl) phthalate (DEHP) exposure. Fate of the genetically abnormal MNG during postnatal testicular development in the rat will be determined. Sertoli-germ cell co-cultures will be used for establishing whether mono-n-butyl phthalate alters proliferation and apoptosis of these cells. These co-cultures will also be used to assess the role of testosterone and stem cell factor on survival of mono-n-butyl phthalate-exposed germ cells.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 22 publications 3 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article 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)
  • Abstract from PubMed
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  • Abstract: Toxicologic Pathology
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  • Other: Toxicologic Pathology
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  • Journal Article 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)
  • Full-text: Biology of Reproduction-Full Text HTML
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  • Abstract: Biology of Reproduction
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  • Other: Biology of Reproduction-Full Text PDF
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  • Supplemental Keywords:

    risk assessment, health effects, vulnerability, animal, mammalian, organism, age, chemicals, toxics, biology, pathology, measurement methods, plastics,, RFA, Health, Scientific Discipline, 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 risk

    Progress and Final Reports:

    Original Abstract
  • 2003 Progress Report
  • 2004 Progress Report
  • 2006
  • Final Report