Effects of Continuous Bisphenol A Exposure from Early Gestation on 90 Day Old Rat Testes Function and Sperm Molecular Profiles: A CLARITY-BPA Consortium Study
Edward Dere, Linnea M. Anderson, Susan M. Huse, Daniel J. Spade, Elizabeth McDonnell-Clark, Samantha J. Madnick, Susan J. Hall, Luísa Camacho, Sherry M. Lewis, Michelle M. Vanlandingham, and Kim Boekelheide.
Toxicology and Applied Pharmacology (2018) DOI: https://doi.org/10.1016/j.taap.2018.03.021 PMID: 29596923
Bisphenol A (BPA) is a ubiquitous industrial chemical that has been identified as an endocrine disrupting compound (EDC). There is growing concern that early life exposures to EDCs, such as BPA, can adversely affect the male reproductive tract and function. This study was conducted as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) to further delineate the toxicities associated with continuous exposure to BPA from early gestation, and to comprehensively examine the elicited effects on testes and sperm. NCTR Sprague Dawley rat dams were gavaged from gestational day (GD) 6 until parturition, and their pups were directly gavaged daily from postnatal day (PND) 1 to 90 with BPA (2.5, 25, 250, 2500, 25,000, 250,000 μg/kg/d) or vehicle control. At PND 90, the testes and sperm were collected for evaluation. The testes were histologically evaluated for altered germ cell apoptosis, sperm production, and altered spermiation. RNA and DNA isolated from sperm were assessed for elicited changes in global mRNA transcript abundance and altered DNA methylation. Effects of BPA were observed in changes in body, testis and epididymis weights only at the highest administered dose of BPA of 250,000 μg/kg/d. Genome-wide transcriptomic and epigenomic analyses failed to detect robust alterations in sperm mRNA and DNA methylation levels. These data indicate that prolonged exposure starting in utero to BPA over a wide range of levels has little, if any, impact on the testes and sperm molecular profiles of 90 day old rats as assessed by the histopathologic, morphometric, and molecular endpoints evaluated.
Figure 1. Quantification of retained spermatid heads (RSH), homogenization resistant spermatid heads (HRSH) and TUNEL-positive germ cell nuclei at postnatal day 90.
Quantification of retained spermatid heads (RSH), homogenization resistant spermatid heads (HRSH) and TUNEL-positive germ cell nuclei at postnatal day 90 following continuous exposure to BPA from early gestation. Values indicated by the boxes are expressed as the mean ± SEM and data points for each litter are represented by the circles. Data for the consortium were analyzed using one-way ANOVA relative to the CLARITY-BPA vehicle group followed by Dunnett's multiple correction test and data from the high-dose group were analyzed using a one-tailed unpaired t-test relative to the concurrent vehicle group.
- Figure 1 (137 KB)
Figure 2. Quantification of BPA-elicited DMRs in sperm across doses.
Sperm DNA methylation in the consortium and 250,000 μg/kg/d high-dose groups were compared against their corresponding vehicle controls. Litters were used as the unit of replication for the analysis of the consortium dose groups, and pups as unit of replication for the high-dose study (n = 8 and n = 19, respectively; n represents the number of pups/litters analyzed). Further comparison between the two separate vehicle control groups was used to identify the background frequency of DMRs due to biological variability.
- Figure 2 (137 KB)
Figure 3. Monte Carlo analysis of sperm DNA methylation following continuous exposure to BPA from early gestation through PND 90.
Randomly selected BPA-treated and vehicle controls (n = 5 litters per group) were iteratively analyzed to identify aberrant DNA methylation; 100 iterations were conducted for the consortium dose groups, and 1000 iterations were conducted for the high-dose group (>10% methylation difference relative to concurrent vehicle control and q-value < 0.05).
- Figure 3 (441 KB)
Table 1. Body, testis and epididymis weights at postnatal day 90 following continuous BPA exposure from early gestation.
- Table 1 (37 KB)
Table 2. Summary of the Monte Carlo analysis providing an assessment of the robustness of the BPA-induced changes in sperm DNA methylation.
- Table 2 (52 KB)
- Figure S1: Hierarchical clustering of 10,000 randomly selected normalized probe intensities from the sperm transcriptomic analyses. (579 KB)
- Figure S2: Dose-response modeling of the 32 DMRs identified using the 4-parameter Brain-Cousens model. (198 KB)
- Figure S3: Dose-response modeling of the 32 DMRs identified using the 5-parameter Brain-Cousens model. (202 KB)
- Figure S4: Dose-response modeling of the 32 DMRs identified using the 4-parameter log-logistic model. (199 KB)
- Figure S5: Dose-response modeling of the 32 DMRs identified using the 5-parameter log-logistic model. (198 KB)