Sequences of the primers used to amplify first-strand cDNAs for mos, emi2, cyclin E and cdk2.

<p>*<b>AT:</b> annealing temperature.</p

Identification of Sertoli cells and germ cells in co-cultures of PS with Sertoli cells on day 3, by using a monoclonal antibody against vimentin conjugated with a red fluorochrome, Alexa 647.

<p>a: Representative flow cytometry analysis of co-cultured cell populations on day 3: Distribution of vimentin-positive diploid Sertoli cells (R3), vimentin-positive tetraploid Sertoli cells (R4), and vimentin-negative germ cells (R2) according to their ploidy (Hoechst 33342). The high specificity of the anti-vimentin antibody was shown by the near complete disappearance of R3 and R4 regions, when co-cultured cells were incubated with non immune mouse IgG conjugated with Alexa 647 as a control (b). Vimentin-positive diploid Sertoli cells (R3) and vimentin-positive tetraploid Sertoli cells (R4) were sorted on glass slides and examined for Hoechst (c, f) and vimentin (d, g) by fluorescent confocal microscopy. e and h are the transmission pictures corresponding respectively to c, d and f, g. Isolated Sertoli cells (c, d, e) and doublets of Sertoli cells (f, g, h) (as highlighted by the double blue spots showing the two nuclei labelled by Hoechst 33342) were identified in R3 and R4 regions respectively. All bars represent 10 µm.</p

Detection of Mos (A), Emi2 (B), Cyclin E (C) and Cdk2 (D) by Western blotting in PS and RS.

<p>Fifty µg of PS and RS were resolved by SDS-PAGE (10% gel). For each protein are presented (i) the Western blot (WB) highlighting the specific bands: 39 and 43 kDa for Mos, 71 kDa for Emi2, 51 kDa for Cyclin E (40 and 33 kDa are low molecular weight derivatives) and 34 kDa for Cdk2, and (ii) the staining of the nitrocellulose membranes with Ponceau Red S (NM-PR) assessing the blotting of proteins. The molecular weights of each specific band are indicated on the left of the WB. Locations of molecular mass standards are shown on the left of NM-PR.</p

Relative levels of Mos (A) or Emi2 (B) or Cyclin E (C) or Cdk2 (D) in the different populations of rat testis meiotic cells (young PS (yPS), middle to late PS (m+lPS), SII, RS).

<p>Each point is the mean±SEM of n experiments. Different letters indicate statistically significant differences (p<0.05 or p<0.01).</p

Expression of the mRNAs of mos, emi2, cyclin E and cdk2 in total germ cells (GC), PS and RS.

<p>18S served as a loading control. Specific mRNA/18S mRNA ratios are indicated under each band. sm (size marker).</p

Fluorescent immunocytochemical staining of Mos, Emi2, CyclinE and Cdk2 in elutriated PS and RS.

<p>A) Immunocytochemical localization of Mos and Emi2 in PS and RS. Mos and Emi2 are present in PS (a, a′), and in RS (b, b′). e, f (reaction with non immune rabbit IgG) and e′, f′ (reaction with non immune goat IgG) are the corresponding controls. c, d, g, h, c′, d′, g′ and h′ are the transmission pictures corresponding to a, b, e, f, a′, b′, e′ and f′ respectively. B): Immunocytochemical co-localization of Cyclin E and Cdk2 in PS and RS. Cyclin E (a, a′) and Cdk2 (b, b′) are present in PS and RS (c, c′: merge) respectively. d, d′, e and e′ are controls for Cdk2 (reaction with non immune mouse IgG) which were examined for green or red fluorescence. f, f′, g and g′ are controls for Cyclin E (reaction with non immune rabbit IgG) which were examined for red or green fluorescence. h and h′ are the transmission pictures corresponding to a, b, c and a′, b′, c′ respectively. i and i′ correspond to d, e and d′, e′; j and j′ correspond to f, g and f′, g′ respectively. All bars represent 10 µm.</p

Comparative analysis of canonical pathways significantly altered by 1 nM BPA at D8, D14 and D21.

<p>The x-axis depicts gene ratios within a dataset mapping to the considered pathway (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106245#s2" target="_blank">Methods</a> 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.</p

Diagram of the main stages of meiotic recombination and the corresponding stages of the meiotic prophase (L = leptotene, Z = zygotene and P = pachytene).

<p>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.</p

Pictures of abnormal spermatocyte nuclei cultured in the presence of BPA and stained with the anti-SCP3 antibody.

<p><b>A</b>– leptotene nucleus with abnormally long stretches of axial cores without indication of polarization; <b>B</b> – pachytene nucleus with total asynapsis; <b>C</b> – pachytene nucleus with pulverized synaptonemal complexes, proving apoptosis; <b>D</b> – diplotene nucleus with univalents (short arrows) and fragments (long arrows). Bar = 10 µm.</p

Total number of genes differentially up- or downregulated by 1 nM and 10 nM BPA after 8, 14 and 21 days of exposure.

<p>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.</p