Developmental Programming: Does Prenatal Steroid Excess Disrupt the Ovarian VEGF System in Sheep?

Abstract

Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR-3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T- and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype.

Keywords: PCOS; VEGF; ovary; sheep; testosterone.

FIG. 1

Representative images of VEGFA, VEGFB, FLT1, KDR, and FLT4 immunostaining in primordial/primary, preantral, and antral follicles. The first left column represents the results of Western blot analysis conducted in ovarian homogenates (verification of antibody specificity). The next column represents negative controls of immunohistochemistry. VEGFA, vascular endothelial growth factor A; VEGFB, vascular endothelial growth factor B; FLT1, vascular endothelial growth factor receptor 1 (fms-related tyrosine kinase 1); KDR, vascular endothelial growth factor receptor 2 (kinase insert domain receptor); FLT4, vascular endothelial growth factor receptor 3 (fms-related tyrosine kinase 4); G, granulosa cells; TE, theca externa; TI, theca interna; O, oocyte. Bar = 25 μm. Preantral follicles: VEGFA and FLT4: Day 140 (d140); VEGFB, FLT1, and KDR: 10-mo-old (10mo).

FIG. 2

Relative expression of immunopositive area (%) of VEGFA and VEGFB in fetal ovaries on Days 90 (d90) and 140 (d140) as well as in the ovaries of 10- (10mo) and 21-mo-old (21mo) sheep born from control females. Differences among groups were assessed using analysis of variance followed by the Duncan multiple range test. Different letters denote significant differences in the immunopositive area among different follicle types (P < 0.05); no significant differences were found among different days within a follicle type. VEGFA, vascular endothelial growth factor A; VEGFB, vascular endothelial growth factor B.

FIG. 3

Relative expression of immunopositive area (%) of FLT1, KDR, and FLT4 in fetal ovaries on Days 90 (d90) and 140 (d140) as well as in the ovaries of 10- (10mo) and 21-mo-old (21mo) sheep born from control females. Differences among groups were assessed using analysis of variance followed by the Duncan multiple range test. Different letters denote significant differences in the immunopositive area among different follicle types (P < 0.05); no significant differences were found among different days within a follicle type. FLT1, vascular endothelial growth factor receptor 1 (fms-related tyrosine kinase 1); KDR, vascular endothelial growth factor receptor 2 (kinase insert domain receptor); FLT4, vascular endothelial growth factor receptor 3 (fms-related tyrosine kinase 4).

FIG. 4

Representative images of the ovarian arteries within the hilum in Fetal Day 140 (d140) and 10-mo-old animals. A–C) Fetal ovaries from untreated mothers (controls). D–F) Fetal ovaries from T-treated mothers. G–I) Fetal ovaries from DHT-treated mothers. Ovarian hilum in control ovaries shows cross sections of ovarian arteries filled with red blood cells (A–C). Ovarian hilum in T- (D–F) and DHT-treated (G–I) groups shows that ovarian arteries in the hilum are larger than in the control group. J–K) The 10-mo-old ovaries from sheep born from control females. L) Arterial tracing of one vessel. Parameters assessed included measurements of arterial area (AA, orange), length (AL, green), and width (AW, yellow), lumen area (LA, dark blue), lumen length (LL, purple), and lumen width (LW. light blue). Numbers in the figure indicate measurement values in μm for diameter or μm² for area. Each number is underlined by a color line corresponding to the color in the image. Difference between arterial and lumen area provided wall area. Bar = 100 μm. T, testosterone; DHT, dihydrotestosterone.

FIG. 5

Cumulative area distributions (μm²) of arteries in the hilum of fetal ovaries on Day 140 (left panel) and in the ovaries of 10-mo-old (right panel) sheep born from control (blue), T-treated (black) and DHT-treated (gray) females. T, testosterone; DHT, dihydrotestosterone. Differences among groups were assessed using a linear mixed model after natural log (base e) transformation.