Figure 1.
Critical importance of local (unknown) factors in the regulation of testosterone production by fetal Leydig cells in the rat during the masculinization programming window (A) and downstream effects of impairment of testosterone production by fetal exposure to dibutyl phthalate (DBP; 500 mg/kg/day from e13.5–e21.5) (B).
Figure 2.
Effect of in utero 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).
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.
Figure 3.
Effect of in utero exposure of rats to vehicle (control) or dibutyl phthalate (DBP: 500 mg/kg/day) on steroidogenic enzyme and anti-Müllerian hormone gene expression in testes at e21.5.
(A) cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), (B) Steroidogenic acute regulatory protein (StAR), (C) cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), (D) hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3β-HSD), (I) Anti-Müllerian hormone (Amh). Values are Means ± SEM for 19–22 animals per group (minimum of 5 litters per group). *p<0.05, **p<0.01, in comparison with respective control. (E–F) Immunohistochemistry for Cyp11a1 on e21.5 testis sections isolated from control (E) and DBP-500-exposed (F) animals. (G–H) Immunohistochemistry for 3β-HSD on e21.5 testis sections isolated from control (G) and DBP-500-exposed (H) animals. (J–K) Immunohistochemistry for Amh on e21.5 testis sections isolated from control (J) and DBP-500-exposed (K) animals. Scale bars E–H = 20 µm.
Table 1.
An overview of SF-1, COUP-TFII and SF-1/COUP-TFII binding sites in the promoters of StAR, Cyp11a1, Cyp17a1, Hsd3b1 and Amh.
Figure 4.
Altered COUP-TFII expression in fetal rat Leydig cells after in utero exposure to vehicle (control) or to different doses of dibutyl phthalate (DBP) and the relationship to intratesticular testosterone levels at e21.5.
(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.
Figure 5.
Age-dependent alteration in COUP-TFII expression in fetal rat Leydig cells in vehicle-exposed control rats and after in utero exposure to dibutyl phthalate (DBP; 500 mg/kg/day).
(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.
Figure 6.
Altered COUP-TFII expression in fetal rat Leydig cells after in utero 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.
(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.
Figure 7.
Altered COUP-TFII expression in fetal rat Leydig cells after in utero exposure to vehicle (control), to dexamethasone (Dex; 100 µg/kg/day) to dibutyl phthalate (DBP; 500 mg/kg/day) or combined DBP + Dex and the relationship to intratesticular testosterone levels at e21.5.
(A) Triple immunofluorescence for SMA (blue), 3β-HSD (red) and COUP-TFII (green) on testis sections from representative vehicle (control) and DBP±Dex-exposed animals on higher power images. Note that exposure to Dex alone resulted in increased occurrence of COUP-TFII-immunopositive fetal Leydig cells (arrows) compared with controls and that combined exposure to DBP-500 + Dex or exposure to DBP-500 alone resulted in most Leydig cells being immunopositive for COUP-TFII (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 groups shown in panel A. Values are Means ± SEM for 3–6 animals per treatment group (minimum of 3 litters per group). ***p<0.001, in comparison with respective control; other comparisons are indicated by capped lines. (C) Corresponding intratesticular testosterone levels for the treatment groups in panel A. Values are Means ± SEM for 19–22 animals per 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.
Figure 8.
Effect of in utero 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.
(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 (Cyp11a1), (F) Steroidogenic acute regulatory protein (StAR), (G) cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), (H) Anti-Müllerian hormone (Amh) 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.
Figure 9.
Effect of in utero 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.
(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.
Figure 10.
COUP-TFII expression in fetal Leydig cells in human fetal testis samples from late 1st trimester (A), early 2nd trimester (B) and late 2nd trimester (C).
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.