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

Altered COUP-TFII expression in fetal rat Leydig cells after <i>in utero</i> exposure to vehicle (control) or to 500 mg/kg/day dibutyl phthalate (DBP) from e19.5-e20.5 (late treatment window) and the relationship to intratesticular testosterone levels at e21.5.

<p>(A–D) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on testis sections from representative vehicle (control) and DBP-exposed animals on high resolution tiled images (A and C) and at higher power (B and D). Asterisks in panel D indicate Leydig cell aggregates that are predominantly immunopositive for COUP-TFII. SC = seminiferous cords. Scale bars in A and C = 200 µm, in B and D = 20 µm. (E) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in animals from the treatment groups shown in panels A–D. Values are Means ± SEM for 8–10 animals per treatment group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control. (F) Corresponding intratesticular testosterone levels for the treatment groups in panels A–D. Values are Means ± SEM for 18–20 animals per group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control.</p

Altered COUP-TFII expression in fetal rat Leydig cells after <i>in utero</i> exposure to vehicle (control) or to different doses of dibutyl phthalate (DBP) and the relationship to intratesticular testosterone levels at e21.5.

<p>(A–B) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on testis sections from representative vehicle (control) and DBP-exposed animals. Note that major persistence of COUP-TFII expression in fetal Leydig cells is observed after exposure to DBP-100 and DBP-500 (100 and 500 mg/kg/day, respectively), whereas DBP-20 (20 mg/kg/day) had a much smaller effect. Asterisks indicate Leydig cell aggregates that are predominantly immunopositive for COUP-TFII. SC = seminiferous cords. Scale bar A = 200 µm, B = 20 µm. (C) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in vehicle (control) and DBP-exposed animals using tiled high resolution images as shown in panel A. Values are Means ± SEM for 6–9 animals per treatment group (minimum of 3 litters per group). **p<0.01, ***p<0.001, in comparison with respective control; other comparisons are indicated by capped lines. (D) Corresponding intratesticular testosterone levels for the treatment groups in panels A and B. Values are Means ± SEM for 4–21 animals per group (minimum of 3 litters per group). **p<0.01, ***p<0.001, in comparison with respective control.</p

Effect of <i>in utero</i> exposure of rats to vehicle (control) or dibutyl phthalate (DBP: 500 mg/kg/day) on age-dependent changes in intratesticular testosterone levels per 10⁶ fetal fetal Leydig cells (A), Leydig cell number per testis (B), Leydig cell nuclear volume (C) and Leydig cell cytoplasmic volume (D).

<p>Values in A are Means ± SEM for 5–12 animals at each age (minimum of 3 litters per group). Values in B–D are Means ± SEM for 4–8 animals in each group (minimum of 3 litters per group). *p<0.05, **p<0.01, ***p<0.001, in comparison with respective controls; other comparisons are indicated by capped lines.</p

Effect of <i>in utero</i> exposure of rats to vehicle (control) or to diethylstilbestrol (DES 100 µg/kg on e13.5, e15.5, e17.5, e19.5 and e20.5) on COUP-TFII immunoexpression in fetal Leydig cells at e21.5.

<p>(A) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on testis sections from representative vehicle (control) and DES-exposed animals. Note that in controls occasional fetal Leydig cells are COUP-TFII-immunopositive (arrow) whereas exposure to DES resulted in a 3-fold increase in the % of COUP-TFII-immunopositive Leydig cells (asterisks). SC = seminiferous cords. Scale bar = 20 µm. (B) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in animals from the treatment group shown in panel A. Values are Means ± SEM for 3–13 animals per treatment group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control. (C) Corresponding intratesticular testosterone levels for the treatment groups in panel A. Values are Means ± SEM for 13–25 animals per group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control. (E) cytochrome P450, family 11, subfamily a, polypeptide 1 (<i>Cyp11a1</i>), (F) Steroidogenic acute regulatory protein <i>(StAR</i>), (G) cytochrome P450, family 17, subfamily a, polypeptide 1 (<i>Cyp17a1</i>), (H) Anti-Müllerian hormone (<i>Amh</i>) gene expression in testes from control and DES-exposed males at e21.5. Values are Means ± SEM for 11–24 animals per group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control. NS = not significant.</p

Age-dependent alteration in COUP-TFII expression in fetal rat Leydig cells in vehicle-exposed control rats and after <i>in utero</i> exposure to dibutyl phthalate (DBP; 500 mg/kg/day).

<p>(A–B) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on fetal testis sections from vehicle (control) and DBP-exposed animals. Arrows in A indicate examples of individual Leydig cells positive for COUP-TFII whereas asterisks indicate DBP-induced aggregates of Leydig cells which are predominantly COUP-TFII-immunopositive. SC = seminiferous cords. Scale bar A = 20 µm, B = 200 µm. (C) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in vehicle (control) and DBP-exposed animals using tiled high resolution images as shown in panel B. Values are means ± SEM for 5–8 animals at each age (minimum of 3 litters per group). ***p<0.001, in comparison with respective control; other comparisons are indicated by capped lines.</p