Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day; e15.5–e18.5) on testis weight (A), and penis length (B) at Pnd25 and Pnd75 (adulthood).

<p>Values are means ± SEM for N = 6–20 from a minimum of three litters. ***p<0.0001, in comparison with respective control.</p

Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day; e15.5–e18.5) on bodyweight (A), and anogenital index (B) at Pnd25 and Pnd75 (adulthood).

<p>Values are means ± SEM for N = 6–20 from a minimum of three litters. **p<0.01 in comparison with respective control.</p

Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day; e15.5–e18.5) on serum testosterone levels at Pnd25 and Pnd75 (adulthood).

<p>Values are means ± SEM for N = 4–13 animals from a minimum of two litters.</p

Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day) on (A) testicular PGE₂ levels at e17.5 and (B) intra-testicular testosterone content at e17.5.

<p>Values are means ± SEM for N = 5 for the top graph and N = 7–28 for the bottom graph, from a minimum of 3 litters. **p<0.01, in comparison with respective control.</p

Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day; e15.5–e18.5) on anogenital index (A), bodyweight (B) and testis weight (C) at e21.5.

<p>Values are means ± SEM for N = 7–45 from a minimum of three litters ***p<0.001 in comparison with respective control.</p

Anogenital distance (AGD) and intratesticular testosterone (ITT) in rats at e21.5 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Only treatments which included the masculinization programming window (MPW) resulted in a significant reduction in AGD in animals (A), whereas ITT was maximally reduced when treatment included the late (e19.5–e20.5) window (B). Values are Means ± SEM for 18–39 animals from 3–7 litters per group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group; ^bp<0.001 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-500 full treatment window group.</p

Contribution of small and large Leydig cell aggregates to the total Leydig cell aggregate area per testis in e21.5 rat testes after <i>in utero</i> exposure to vehicle (control) or dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Values are Means ± SEM for 8–15 animals from 3–5 litters per treatment group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group (except p<0.05 when Dex is compared with DBP-500 late window treatment); ^bp<0.05 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-750 full treatment window group.</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) (A, B) or intratesticular testosterone (ITT) at e21.5 (C, D) or between AGD and ITT at e21.5 (E, F) in animals exposed <i>in utero</i> to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex during all treatment windows (A, C, E), or during the full treatment window (e13.5–e20.5) only (B, D, F).

<p>Dysgenesis is negatively correlated with AGD irrespective of treatment period (A, B) whereas all other correlations were affected by the treatment period (full details in text).</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) at postnatal day (pnd) 8 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e21.5.

<p>(A) Average size of the 3 largest Leydig cell aggregates is shown as Means ± SEM for 8–17 animals from 3–6 litters. ***p<0.001, in comparison with controls; other comparisons are indicated by capped lines. (B) Correlation between dysgenesis and anogenital distance (AGD) in pnd8 animals.</p

Immunohistological analysis of focal dysgenetic areas in rat testes exposed to vehicle (control) or dibutyl phthalate (DBP).

<p>(A–B) Double immunofluorescence for 3β-HSD (blue) and Sox-9 (red) on e21.5 testis sections from (A) vehicle (control) and (B) DBP-exposed (750 mg/kg/) animals, illustrating focal dysgenesis in which Leydig cell aggregates contain ectopically localized Sertoli cells. Green depicts DAPI nuclear counterstain. SC = seminiferous cords. Scale bar = 20 µm. (C1–C6) Example of sections stained for 3β-HSD (brown) used for Leydig cell aggregate analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030111#pone-0030111-g003" target="_blank">Figure 3</a>). Arrows indicate large Leydig cell aggregates, asterisks indicate seminiferous cords. Scale bar = 200 µm. (D–E) Double immunohistochemistry for 3β-HSD (blue) and SMA (brown) on postnatal day (pnd) 8 testis sections from (D) vehicle (control) and (E) DBP-exposed (500 mg/kg/) animals, illustrating focal dysgenesis after DBP-exposure, with large Leydig cell aggregates and malformed seminiferous cords and intratubular Leydig cells (arrows). Scale bar = 50 µm.</p