BACKGROUND/RATIONALE:
Birth defects and pregnancy loss are among the reproductive outcomes reported following paternal exposures to poly aromatic hydrocarbons (PAHs) in military settings. PAHs are highly toxic, widely dispersed environmental hazards that activate the aryl hydrocarbon receptor (AHR). AHR is essential to xenobiotic metabolism and cellular processes such as growth, death, and differentiation.

OBJECTIVE(S):
Specific aim 1 detected the trans-generational effects of paternal exposures to PAHs. Specific aim 2 interrogated stress response pathways. Specific Aim 3 targeted embryonic AHR activation and glucose transport following fertilization with PAH exposed males.

METHODS:
Research design. The EPA rodent dominant lethal assay (DLA) was modified by: a) expanding reproductive endpoints; b) segregating germ cell effects from seminal fluid effects; and c) incorporating molecular methods. Back extrapolation, with timed sequential mating, created a repeated measures design which described the impact by spermatogenic cell stage. C57BL6 mice were exposed to cigarette smoke condensate (CSC) as a representative complex mixture of carcinogens, mutagens, and particulate matter and paralleled military exposures. The spermatogenic phenotype of the AHR knockout (AHRKO) mouse informed cells, tissues, and pathways impacted by AHR. Laser microdissection (LMD), quantitative real-time PCR (qRT-PCR), and protein analysis via western blot (WB), immunofluorescence (IF), and confocal microscopy interrogated stress response pathways. Spermatogenic factors were segregated from seminal fluid factors via in vitro fertilization (IVF). Endpoints included sperm motion measures, fertilization, blastocyst progression,16.5 embryo day and term litter size, weight, length, morphology, and histopathology. Females were non-exposed.

FINDINGS/RESULTS:
Pure germ cell stages were captured via LMD. QRT-PCR confirmed expression of melanoma antigen A4 (Magea4 p<0.001) in spermatogonia, heat shock protein A 2 (Hspa2 p<0.001) in spermatocytes; and c) protamine 2 (Prm2 p<0.001) in spermatids as markers of mitosis, meiosis, and nuclear condensation respectively. Protein analysis confirmed corresponding proteins.
LMD and cell stage specific antioxidant defenses.
Baseline stage specific stress response expression was established. Superoxide dismutase 2 (Sod2) and testis specific glutathione peroxidase 4 (Gpx4) were significantly higher (p<0.01 respectively) in spermatocytes and round spermatids. Sod2 converts free radicals to hydrogen peroxide which is degraded by Gpx4. Heat shock protein 90 (Hsp90aa1) was highest in spermatocytes (p<0.001). Hsp90aa1 is an AHR chaperone which predominates in the testes, and is highly inducible under stress.
AHR, toxicology, and cell cycle regulation and anti-oxidant defenses.
Seven daily Intra-peritoneal injections of CSC 40 ug in 0.6% DMSO in C57BL6 males increased Gpx4 (p<0.01) and Sod2 (p<0.001) in LMD isolated spermatocytes. CSC in cell culture, with and without AHR blockade, resulted in the 25 fold down regulation of Cyp1A1 (p < 0.05) with AHR inhibition. Sod2 over-expression persisted with AHR antagonist and was accompanied by a three-fold up-regulation (p < 0.05) of nuclear factor-erythroid 2-related factor 2 (Nrf2) which coordinates the rapid response to oxidative stress.
AHR and spermatogenesis
Seminiferous tubule degradation was present in the AHRKO testes. Reduced markers of Sertoli cell-Sertoli cell-germ cell adhesion (Testin >100 fold, p<0.01), spermatogonial proliferation (Magea4 4 fold, p <0.05), germ cell maturity (Hspa2, Prm1, Prm2), and oxidative stress (Nrf2, Sod2, Ucp2) were also present. One AHR response element (AHRE) was found within MAGEA4 which suggested AHR contributed to MAGEA4 regulation. Thirty five core AHRE were found within 9Kb of the Testin start site, two of which were in the region of maximal promoter activity. Because this suggested that Testin was highly regulated by AHR, we wondered if CSC would disrupt the blood testes barrier (BTB). Continine (a metabolite of nicotine) and benzo(a)pyrene (a component of CSC) antibodies were found in the testicular fluid of mice treated for 35 days.
AHR and reproductive measures
Because sperm are exposed to drugs and toxins via the seminal fluid, we wanted to know if AHR was present in mature sperm. We identified AHR in the acrosome and flagella of sperm from wild type mice which was absent in the AHRKO sperm. This is significant because the flagellum contains the energy, structure, and motility required for fertilization. IVF with AHRKO sperm significantly reduced fertility (48% vs. 80.6%, p = 0.013) accompanied by a non-significant increased progression to blastocysts.
AHR, in vivo toxicology, and IVF
C57 males were exposed to 400 ug CSC in 2% DMSO for 7, 14, and 28 days followed by IVF or natural mating. Increased rates of fertilization by IVF occurred at all time points in the CSC males, ranging from 68% to 76%. Conversely the progression to blastocyst stage embryos was decreased to 47% in CSC males when compared to the 60% blast rate from non-exposed males. Conversely 200 ug CSC for 35 days resulted in non-significant declines in motility and fertility, and non-significant increases in progressive motility and progression to blasts. There were no significant changes in fertility measures.
AHR, in vivo toxicology, and natural mating.
C57 males received CSC 400 ug (equivalent of 0.036 cigarettes per day) IP in 2% DMSO for the duration of one spermatogenic cycle (35 days) followed by mating at a 1:2 ratio with non-exposed females. The first female delivered 7 pups with no apparent defects. However all pups were dead the next day. The pups from the second female were macrosomic with fore and hind limb defects. The dose was decreased by half to determine the lowest observed effect level. While the 6.5 day post-natal weights were significantly higher (p=0.0149) in the pups from CSC treated males, the 16.5 day embryo weights and lengths were non-significant. However hydronephrosis and cardiac defects were found among 50% and 100% of the four randomly selected 16.5 day embryos respectively.

IMPACT:
If confirmed this information will have significant implications for deployment planning by limiting the duration of exposures in areas of reproductive risk.

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External Links for this Project

NIH Reporter

Grant Number: I01HX000206-01
Link: https://reporter.nih.gov/project-details/7750459

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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. [view]
2. Esakky P, Hansen DA, Drury AM, Moley KH. Modulation of cell cycle progression in the spermatocyte cell line [GC-2spd(ts) Cell-Line] by cigarette smoke condensate (CSC) via arylhydrocarbon receptor-nuclear factor erythroid 2-related factor 2 (Ahr-Nrf2) pathway. Biology of reproduction. 2014 Jan 1; 90(1):9. [view]
3. 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.). 2013 Mar 1; 20(3):238-52. [view]
4. Hansen DA, Esakky P, Drury A, Lamb L, Moley KH. The aryl hydrocarbon receptor is important for proper seminiferous tubule architecture and sperm development in mice. Biology of reproduction. 2014 Jan 1; 90(1):8. [view]

Conference Presentations

1. 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. [view]
2. 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. [view]

DRA: Military and Environmental Exposures
DRE: Etiology
Keywords: Adverse events, Deployment Related, Genomics
MeSH Terms: none