Figure 1.
Total number of genes differentially up- or downregulated by 1 nM and 10 nM BPA after 8, 14 and 21 days of exposure.
Genes were selected with a fold change cutoff ≥1.5 (p-value <0.05). The global expression change compared with control cells was time but not dose dependent.
Figure 2.
Distribution of differentially expressed genes per altered function.
Genes significantly up- or downregulated after cell exposure to BPA for 8 days (blue), 14 days (red), or 21 days (green), and genes common to all time points (black) were examined and classified per function according to Gene Ontology. The results comprise a specific pattern of BPA toxicity, similar for all time points. The amplitude of the toxicity is given by the scale representing the number of modulated genes for each type of altered function. A – 1 nM BPA. B – 10 nM BPA.
Figure 3.
Comparative analysis of canonical pathways significantly altered by 1 nM BPA at D8, D14 and D21.
The x-axis depicts gene ratios within a dataset mapping to the considered pathway (see Methods for calculation). A Fisher's exact test was used to determine a p-value representing the significance of these associations (p<0.01). In all cases, the ratios increased with exposure time.
Figure 4.
Diagram of the main stages of meiotic recombination and the corresponding stages of the meiotic prophase (L = leptotene, Z = zygotene and P = pachytene).
The 120 genes showing a fold change ≥1.5 (p value ≤0.05) in BPA-treated cultures compared with controls, and involved in events of the meiotic prophase, were classified according to their function. This figure shows that the main functions of the first meiotic prophase are altered by BPA. Genes having several functions appear several times in this figure.
Figure 5.
The top-ranked Ingenuity network identified within the group of 120 genes preceding meiotic divisions, involved in the first meiotic prophase, or essential to meiosis that were found to be differentially expressed in our datasets.
This literature-based network shows the high level of connections between genes linked to DNA DSB repair in our datasets, considering all BPA doses and time points. The indicated values of fold change (induction or repression) are the maximal values found in the time course with 1 nM and 10 nM BPA. The red nodes are upregulated, the green nodes are downregulated.
Table 1.
Microarray gene expression validation by qRT-PCR.
Figure 6.
Effects of BPA on the percentages of leptotene and diplotene stages.
A– percentage of leptotene stage increased at all doses and time points. The increase was at the limit of significance for 1 nM at D8 (p = 0.06) and significant for D14 and D21 at the two doses (p<0.05). B – diplotene stage percentage decreased at all doses and time points. The increase was not significant for 1 nM at D8 and was significant for D14 and D21 at the two doses (p<0.05). Each bar represents the mean ±SD (n = 3 cultures for D8 and D14, and 2 cultures for D21) versus control. C = control culture, 1 and 10 = 1 nM and 10 nM BPA, respectively.
Figure 7.
Effect of BPA on the pachytene stage progression.
The pachytene index (PI) decreased at both dose and time points (p<0.05). C = control culture, 1 and 10 = 1 nM and 10 nM BPA, respectively. Each bar represents the mean ±SD (n = 3 cultures for D8 and D14, and 2 cultures for D21) versus control.
Figure 8.
Pictures of abnormal spermatocyte nuclei cultured in the presence of BPA and stained with the anti-SCP3 antibody.
A– leptotene nucleus with abnormally long stretches of axial cores without indication of polarization; B – pachytene nucleus with total asynapsis; C – pachytene nucleus with pulverized synaptonemal complexes, proving apoptosis; D – diplotene nucleus with univalents (short arrows) and fragments (long arrows). Bar = 10 µm.
Figure 9.
Changes of synaptonemal complex (SC) abnormality percentages during the pachytene stage in BPA-cultured spermatocytes.
A– the percentage of nuclei with asynapsed SC increases at each dose and time point (p<0.05). B – the percentage of nuclei with pulverized SC (apoptotic nuclei) increases at each dose and time point (p<0.05). Each bar represents the mean ±SD (n = 3 cultures for D8 and D14, and 2 cultures for D21) versus control. C = control culture, 1 and 10 = 1 nM and 10 nM BPA, respectively.