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

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

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

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

COUP-TFII expression in fetal Leydig cells in human fetal testis samples from late 1^st trimester (A), early 2^nd trimester (B) and late 2^nd trimester (C).

<p>Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on human fetal testis sections. SC = seminiferous cords. Scale bars = 50 µm. (D) Quantification of the percentage of COUP-TFII positive fetal Leydig cells in samples shown in panels A–C. Values are Means ± SEM for 3–11 samples per treatment group. *p<0.05, **p<0.01, in comparison with respective control; other comparison is indicated by capped line.</p

Effect of <i>in utero</i> exposure of mice to vehicle (control), dibutyl phthalate (DBP 500 mg/kg/day) or to diethylstilbestrol (DES 100 µg/kg on e11.5, e13.5, e15.5 and e17.5) on COUP-TFII immunoexpression in fetal Leydig cells at e18.5.

<p>(A–B, E–F) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on testis sections from representative vehicle (control; A, E), DBP-exposed (B) and DES-exposed (F) animals. Scale bars = 50 µm. Asterisks indicate blood vessels. Arrows in F indicate COUP-TFII-positive Leydig cells. (C–D) Quantification of the percentage of COUP-TFII positive fetal Leydig cells (C) and corresponding intratesticular testosterone levels (D) in control and DBP-exposed animals. Values are Means ± SEM for 7 animals per group (minimum of 3 litters per group). (G–H) Quantification of the percentage of COUP-TFII positive fetal Leydig cells (G) and corresponding intratesticular testosterone levels (H) in control and DES-exposed animals. Values are Means ± SEM for 6–9 animals per group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control. NS = not significant.</p

An overview of SF-1, COUP-TFII and SF-1/COUP-TFII binding sites in the promoters of <i>StAR</i>, <i>Cyp11a1</i>, <i>Cyp17a1</i>, <i>Hsd3b1</i> and <i>Amh</i>.

*<p>: “weak” COUP-TFII binding site.</p><p>StAR = steroidogenic acute regulatory protein; Cyp11a1 = cytochrome P450, family 11, subfamily a, polypeptide 1; Cyp17a1 = cytochrome P450, family 17, subfamily a, polypeptide 1; Hsd3b1 = hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1; Amh = anti-Müllerian hormone; SF-1 = steroidogenic factor-1; COUP-TFII = chicken ovalbumin upstream promoter transcription factor II; bp = basepair.</p