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Extramural Research

Final Report: Endocrine Disruptors and Testis Development

EPA Grant Number: R827405
Title: Endocrine Disruptors and Testis Development
Investigators: Skinner, Michael K.
Institution: Washington State University
EPA Project Officer: Fields, Nigel
Project Period: August 1, 1999 through July 31, 2002
Project Amount: $534,583
RFA: Endocrine Disruptors (1999)
Research Category: Economics and Decision Sciences , Endocrine Disruptors

Description:

Objective:

The objective of this research project was to develop a better understanding of how endocrine disruptors influence testis development and function. Of particular interest were the cell-cell interactions that regulate testis growth, size, and morphogenesis that directly influence male fertility and sperm production. Sertoli cells are the epithelial cells responsible for the onset of embryonic testis development and maintenance of spermatogenesis in the adult testis. Preliminary research demonstrated that two families of paracrine growth factors directly influence testis development and function. The hypothesis tested was that endocrine distruptors affect locally produced paracrine growth factors that are essential for testicular cell growth and differentation during embryonic and postnatal testis development, which directly influences male fertility and sperm production in the adult. Abnormal testis development and male infertility caused by endocrine disruptors may, in part, be because of inappropriate control of testicular cell growth and differentiation during development. Preliminary studies indicated that the transforming growth factor (TGF) families are critical for embryonic testis growth. Preliminary studies also indicated that the neurotropin family of factors (i.e., NT3) has a critical role in the morphogenesis of testis development (i.e., sex cord or seminiferous tubule formation). The experimental approach consisted of the following specific objectives: (1) investigate the effects of endocrine disruptors on the TGF families during testis development; (2) investigate the effects of endocrine disruptors on the neurotropin growth factor family during testis development; and (3) investigate the physiological effects of these endocrine disruptors during testis development on male fertility.

The completion of these studies has provided insight into the mechanisms by which endocrine disruptors influence testis growth and function. Testicular cell growth and differentiation are essential for embryonic, prepubertal, pubertal, and adult testis function. Abnormal control of critical cell-cell interactions after treatment with endocrine disruptors is anticipated to result in male infertility. Inappropriate expression of the growth factors or receptors will result in subfertile males. Therefore, observations from the current proposal will provide an understanding of the actions of endocrine disruptors on testis development and function. Information obtained will determine how environmental toxins with estrogenic (e.g., methoxychlor [MXC]) and antiandrogenic (e.g., vinclozoline) activities may impair male fertility by adversely affecting gonadal neurotropins and TGFs.

Summary/Accomplishments (Outputs/Outcomes):

1. The methods developed to assess the TGF gene expression and protein expression have been utilized along with the embryonic testis organ culture system (developed to examine the effects of MXC on mRNA levels). MXC at 0.2 µM suppresses TGF-alpha, TGF-beta, and epidermal growth factor receptor mRNA levels. The actions of MXC and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on testis development have been established. Currently, the actions of vinclozoline are being examined.

2. The methods developed to assess neurotropin gene expression have been utilized to examine the actions of MXC. MXC was found to suppress NT3 and trkC mRNA levels. This correlated with the ability of MXC to block cord formation in the developing testis and influence cell migrations. This is being investigated more thoroughly, and the actions of vinclozoline are being examined.

3. In vivo experiments with exposure of gestating mothers to MXC demonstrated the effects on the F1 generation. A reduction in sperm function and increased spermatogenic cell apoptosis were demonstrated. Preliminary observations of F2 and F3 generations indicate that this effect may be transgenerational. Studies show that this may be because of the methylation status of the germline.

Technical Advances

1. We developed an embryonic testis organ culture system to assess the effects of endocrine disruptors on organ morphogenesis and tissue function. This was used to demonstrate that endocrine disruptors MXC and vinclozolin can alter early embryonic testis development at the time of sex determination and testis morphogenesis.

2. We demonstrated that in vivo endocrine disruptor (e.g., MXC and vinclozolin) exposure for a transient period at this time of sex determination caused an effect in the adult of reduced spermatogenic capacity and male fertility. Therefore, embryonic exposure caused delayed adult disease states of subfertility.

3. We provided the first documentation of an epigenetic transgenerational effect of an endocrine disruptor on male fertility. A transient embryonic exposure to MXC or vinclozolin induced a methylation-mediated reprogramming of the male germline, which caused subfertility and spermatogenic defects in the F1, F2, F3, and F4 generation animals. Therefore, an exposure at a critical developmental period of a gestating mother can cause an irreversible transgenerational phenotype for successive generations. This increases the potential risk associated with endocrine disruptor exposure. Mechanistically, this appears to be because of altered methylation states of imprinted genes that modify the germline. We must assess what diseases, other than male fertility, could be affected.

Summary

A primary observation was the transgenerational effects of a transient in utero exposure to the endocrine disruptors MXC and vinclozolin on testis function and gametogenesis. Studies demonstrated the ability of MXC and vinclozolin to promote a transgenerational phenotype in spermatogenic cells. Treatment of gestating mothers had no major effect on sex determination or gross testis histology throughout development. When animals were exposed in utero E8-E15, spermatogenic defects were observed in the pubertal and adult F1 generation. Treated animals had an increase in spermatogenic cell apoptosis. Similar results were observed at postnatal day 20 or day 60 for both endocrine disruptors. In addition to this decreased spermatogenic cell survival, sperm motility and morphology also were found to be impaired. Interestingly, animals exposed in utero at E15-E20 (i.e., E20 around birth) to the same dose of endocrine disruptor had no spermatogenic cell defects. Therefore, only exposure in the E8-E15 period of development had effects on later adult spermatogenesis, and if exposure was past E15 of testis development, no effect was observed. This correlates with critical processes such as germ cell remethylation, cord formation, and sex determination during the E10-E15 period, compared to the growth phase after E15.

Previous studies of endocrine disruptor actions on the testis that exposed rats after E15 likely did not see effects because of missing the important E10-E15 period. These previous studies have determined that transient embryonic exposure (i.e., E8-E15, but not E15-E20) to an endocrine disruptor can influence male reproduction (i.e., spermatogenic capacity and sperm viability) in the F1 generation. The primary objective was to determine if the effects of exposure of endocrine disruptors on the F1 generation could be passed to subsequent generations. The male rats of the F2 generation (derived from the animals discussed above) were examined. Interestingly, the same increased apoptosis rate in spermatogenesis was identified in the F2 generation as the F1 generation. A three- to fivefold increase in apoptosis was observed in P20, P60, and P120 male rats. Epididymal sperm were analyzed from P60 and P120 rats, and an approximately 20 percent decrease in sperm motility and forward movement also was observed. Observations were similar made with the F2 generation of vinclozolin-treated animals at both P20 and P60 ages of development. Analysis of the F3 and F4 generation demonstrated similar effects on spermatogenesis and sperm, with a similar effect in sperm motility with treated animals. Therefore, exposure of a gestating mother at the critical time of sex determination and testis morphogenesis (i.e., cord formation) appears to cause a germline effect, causing a decrease in spermatogenic capacity and sperm viability that is transgenerational in the male. This experiment was repeated with four different gestating mothers, and similar results were observed up to the F4 generation. An outcross of an affected treated male with a wild type untreated female reduced slightly the magnitude of the effect, but some aspects of the phenotype (i.e., increased apoptosis) remained.

It currently is speculated that transient exposure at the critical time of testis development is crucial, and that a phenomenon such as an imprinted methylation state of the germline may be involved. This intriguing observation of a transgenerational effect of an endocrine disruptor through the male germline was examined mechanistically. Recently, it has become evident that the epigenetic effects of agents on processes such as DNA methylation, DNA acetylation, and general chromatin structural changes can have profound effects on cellular function and differentiation. This can have significant effects on both health and disease states. These epigenetic changes can reprogram mammalian development and influence critical processes such as germline imprinting.

More recently, the potential effects of environmental toxins and endocrine disruptors being directed through these epigenetic processes have been appreciated. The ability to influence embryonic development and affect the F1 generation can be through factors such as genomic imprinting or specific effects on parameters such as steroid receptor expression. Although no specific studies have examined the potential transgenerational effects of endocrine disruptors through specific epigenetic mechanisms, the ability to have an epigenetic cause of transgenerational effects in mammals has been demonstrated. Presumably, this is mediated through germline mutations that reprogram the germline, which then is expressed in successive generations.

The results demonstrated the effects of an endocrine disruptor in the male germ cells that is transgenerational. The hypothesis that this was because of an epigenetic phenomenon through a DNA methylation of the male germline was investigated. Gestating mother rats were exposed to 100 mg/kg MXC from E8-E15, and on postnatal day 6, rat testis was collected for analysis. DNA was isolated and methylation state determined with methylation-sensitive restriction enzymes and the polymerase chain reaction (PCR). For this analysis, 13 sets of primers specific for general methylation sites were used, and from this analysis approximately 10-15 different PCR products with methylation alterations were detected after MXC treatment. This observation supports the proposal that epigenetic effects of MXC through methylation changes in the testis does occur. An examination of one of the large gels demonstrated some polymorphisms between different animals with the Sprague Dawley rats used. For example, one of the replicates of the 200 mg/kg-treated animals did not show the methylation change. To eliminate this, an inbred Fisher rat strain was used in future analysis. Analyses have shown less polymorphisms and detection of more consistent methylation changes. The research investigated the mechanism of action of the endocrine disruptors through potential epigenetic changes in the germline. Although it is known, a general demethylation occurs early in embryonic development for somatic cells.

Recently, it has been shown that a demethylation and remethylation of the primordial germ cells occur during sex determination in the gonad. A reprogramming of the germline could account for the transgenerational phenomena and will be investigated in future research. Another objective of this research project was to isolate and identify the methylated genes affected by the endocrine disruptors. MXC-affected methylated PCR products were isolated, sequenced, and mapped to the appropriate chromosome and closest gene. Both were identified as uncharacterized genes, and this demonstrates the feasibility of identifying the methylated genes affected by the endocrine disruptors.


Journal Articles on this Report : 12 Displayed | Download in RIS Format

Other project views: All 16 publications 12 publications in selected types All 12 journal articles

Type Citation Project Document Sources
Journal Article Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and mate fertility. Science 2005;308(5727):1466-1469
abstract available  
R827405 (Final)
not available
Journal Article Anway MD, Memon MA, Uzumcu M, Skinner MK. Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis. Journal of Andrology 2006;27(6):868-879
abstract available  
R827405 (Final)
not available
Journal Article Cupp AS, Skinner MK. Actions of the endocrine disrupter methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth. Reproductive Toxicology 2001;15(3):317-326 R827405 (2001)
R827405 (Final)
not available
Journal Article Cupp AS, Tessarollo L, Skinner MK. Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: Role in seminiferous cords germ cell survival. Biology of Reproduction 2002;66(6):1838-1845. R827405 (Final)
not available
Journal Article Cupp AS, Uzumcu M, Skinner MK. Chemotactic role of neurotropin 3 in the embryonic testis that facilitates morphological male sex determination. Biology of Reproduction 2003;68(6):2033-2037. R827405 (Final)
not available
Journal Article Cupp AS, Uzumcu M, Suzuki H, Dirks H, Phillips B, Skinner MK. Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development. Journal of Andrology 2003;24(5):736-745. R827405 (2001)
R827405 (Final)
not available
Journal Article Levine E, Cupp AS, Skinner MK. Role of neurotropins in rat embryonic testis morphogenesis (cord formation). Biology of Reproduction 2000;62(1):132-142. R827405 (2001)
R827405 (Final)
not available
Journal Article Levine E, Cupp AS, Miyashiro L, Skinner MK. Role of transforming growth factor-α and the epidermal growth factor receptor in embryonic rat testis development. Biology of Reproduction 2000;62(3):477-490 R827405 (2001)
R827405 (Final)
not available
Journal Article Skinner MK, Anway MD. Seminiferous cord formation and germ-cell programming - Epigenetic transgenerational actions of endocrine disruptors. Ann N Y Acad Sci 2005;1061(Testicular Cell Dynamics and Endocrine Signaling):18-32
abstract available  
R827405 (Final)
not available
Journal Article Uzumcu M, Westfall SD, Dirks KA, Skinner MK. Embryonic testis cord formation and mesonephric cell migration requires the phosphotidylinositol 3-kinase signaling pathway. Biology of Reproduction 2002;67(6):1927-1935. R827405 (Final)
not available
Journal Article Uzumcu M, Dirks KA, Skinner MK. Inhibition of platelet-derived growth factor actions in the embryonic testis influences normal cord development and morphology. Biology of Reproduction 2002;66(3):745-753
abstract available  
R827405 (Final)
not available
Journal Article Uzumcu M, Suzuki H, Skinner MK. Effect of the anti-androgenic endocrine disruptor vinclozolin on embryonic testis cord formation and postnatal testis development and function. Reproductive Toxicology 2004;18(6):765-774.
full text available
R827405 (Final)
not available
Supplemental Keywords:

biology, environmental biology, cell biology, molecular biology, chemical pollutants, biochemistry, environmental exposure, endocrine disruptors, endocrine disrupting chemicals, EDCs, human health, children's health, environmental risk, health risk assessment, Northwest, EPA Region 10, molecular genetics, cell-cell interactions, chemical interference, developmental biology, embryonic development, fetal development, gonad morphology, male fertility, neurotropin growth, paracrine growth factors, physiology, postnatal development, Sertoli cells, sexual development., RFA, Health, Scientific Discipline, Geographic Area, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Children's Health, Molecular Biology/Genetics, Biology, Endocrine Disruptors - Human Health, EPA Region, neurotropin growth, cell-cell interactions, testis development, EDCs, endocrine disrupting chemicals, sexual development, sertoli cells, developmental biology, human exposure, human growth and development, physiology, fetal development, chemical interference, embryonic development, Region 10, gonad morphology, postnatal development, paracine growth factors

Progress and Final Reports:
Original Abstract
2000 Progress Report
2001 Progress Report
2002 Progress Report

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The 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.

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