Paternal Environmental Exposures and Reproductive Outcomes: A Comparison of In Vitro and In Vivo Fertilization
Deborah Ann Hansen PhD
St. Louis VA Medical Center John Cochran Division, St. Louis, MO
St Louis, MO
Funding Period: May 2010 - April 2013

BACKGROUND/RATIONALE:
Dioxin like compounds, known as poly aromatic hydrocarbons (PAH) result from incineration, incomplete fossil fuel combustion, and diesel truck exhaust and are relevant in deployment settings such as the exposure to Agent Orange in Vietnam, fossil fuels and combustibles during Gulf War I, and the open air burn pits of Afghanistan and Iraq. PAHs exert their effects through the aryl hydrocarbon receptor (AHR) which is also vital to normal physiologic processes such as cell growth, death, and differentiation. Nine hundred children with Spina Bifida, of Agent Orange exposed Vietnam veterans receive VA service connected benefits. Deployed Gulf War veterans reported higher rates of birth defects.

OBJECTIVE(S):
The primary objective characterizes the effect of male PAH exposure on parameters of natural mating and in vitro fertilization (IVF) through the segregation of seminal fluid factors from male germ cell effects. The secondary objective determines the role of AHR in relevant biochemical processes such as stress response pathways.

METHODS:
Laser microdissection (LMD) isolated stage specific male germ cell sub-populations. qRT-PCR validated stage specific LMD success. Western blot and immuno fluorescence confirmed protein expression. Cigarette smoke condensate (CSC) was used as a representative complex mixture that included AHR ligands. LMD with qRT-PCR elicited in vitro and in vivo baseline expression of stress response genes by cell stage and indicators of male germ cell oxidative stress. Immunofluorescent staining localized AHR and ARNT in epididymal sperm and oocytes. Apoptotic cells were identified using the TUNEL assay. Selected parameters of the AHR knockout male reproductive phenotype were defined. C57BL6 AHR responsive mice and AHR knockout mice (Taconic Farms) were used.

FINDINGS/RESULTS:
Stage specific gene expression paralleled published data and validated LMD success. CSC exposure resulted in a 0.5 fold and 0.8 fold decline of protamine 1 and 2 (Prm1 and Prm2); a 1.5 fold rise in superoxide dismutase 1 (Sod1) in round spermatids, a 1.8 fold rise in superoxide dismutase 2 (Sod2) in spermatogonia, and a 1.2 fold and 0.8 fold rise in glutathione perioxidase 4 (Gpx4) in spermatogonia and round spermatids. AHR and ARNT were present in the oocyte in a stage specific manner. AHR localized to the principal piece of sperm flagella. There was no increase in TUNEL assays nor Cyp1A1 expression in CSC exposed and non exposed males. The AHR KO male reproductive phenotype included decreased fertilization, reduced sperm counts, increased teratospermia, and increased spermatocyte apoptosis.

IMPACT:
This study has the potential to a) describe selected mechanisms of action of male reproductive toxicants, b) inform preconception counseling and reproductive decision making in the setting of paternal risk, c) establish a framework which can provide near real time information to inform risk reduction strategies and target prevention, and d) describe the reproductive phenotype of the AHR knockout male mouse. AHR loss of function will inform AHR gain of function by impacting the same cells, tissues, and pathways.

PUBLICATIONS:

Journal Articles

1. Esakky P, Hansen DA, Drury AM, Moley KH. Cigarette smoke condensate induces aryl hydrocarbon receptor-dependent changes in gene expression in spermatocytes. Reproductive toxicology (Elmsford, N.Y.). 2012 Dec 1; 34:(4):665-76.
2. Esakky P, Hansen DA, Drury AM, Moley KH. Molecular Analysis of Cell Type-Specific Gene Expression Profile During Mouse Spermatogenesis by Laser Microdissection and qRT-PCR. Reproductive sciences (Thousand Oaks, Calif.). 2012 Aug 30.

Conference Presentations

1. Hansen DA, Esakky P, Moley KH, Lee RK. Paternal Environmental Exposures and Reproductive Outcomes: A Comparison of in Vitro and in Vivo Fertilization. Poster session presented at: National Institute of Nursing Research Scientific Symposium; 2010 Sep 30; Bethesda, MD.
2. Hansen DA, Moley KH. Paternal Environmental Exposures and Reproductive Outcomes: A Comparison of in Vitro and in Vivo Fertilization. Poster session presented at: Utah-Florence International Symposium on the Genetics of Male Infertility; 2010 Feb 4; Salt Lake City, UT.

Thesis / Dissertation

1. Hansen DA. Spermatogenesis: Laser Microdissection Optimization. [Dissertation]. ProQuest Dissertations & Theses Database; 2010. UMI: 3432378 p.
2. Hansen DA. Spermatogenesis: Laser Microdissection Optimization. [Dissertation]. University of Missouri - Saint Louis; 2010.

DRA: Military and Environmental Exposures
DRE: Etiology
Keywords: Adverse events, Deployment Related, Genomics, Reproductive Care (Gynecological)
MeSH Terms: none