Figure 6.

Angiogenesis and luteinization are compromised after intrafollicular injection with an antibody against THBS1. Histological sections of ovaries shown in Figure 5 were immunostained for VWF (brown) to assess angiogenesis; hematoxylin counterstain. (A,B) Endothelial cell invasion into the granulosa cell layer of a control IgG-injected follicle (A) and THBS1 antibody-injected follicle (B) shows VWF+ cells within the granulosa cell (gc) layer. Arrows indicate VWF+ cells which have invaded furthest from the stroma into the granulosa cell layer. Single arrowheads indicate stromal vessels. Double arrowheads indicate capillary luminal spaces with red blood cells in the control-IgG injected follicle only. (C) shows representative measurements of granulosa cell layer thickness (brown lines) and endothelial cell invasion (green lines). (A–C) are oriented as shown in (A), with stroma (st) at bottom, granulosa cell (gc) layer central, and follicle antrum (an) at top. Images in (A–C) are at the same magnification; bar in (B) is 100 μm. Granulosa cell layer thickness (D), endothelial cell invasion (E), and the percent of the granulosa cell layer penetrated by endothelial cells (F) after intrafollicular injection with control IgG (IgG) or THBS1 antibody. Groups are different by two-tailed unpaired t-test as indicated by *p < 0.05. Data are expressed as mean + SEM, n = 4/group. (H,J) 3D modeling of endothelial cells (white on black background) is shown alongside (G,I) representative VWF immunostained ovarian sections after intrafollicular injection with control IgG (G,H) or THBS1 antibody (I,J). Green arrows indicate stromal vessels, green arrowheads indicate capillary-like structures that connect to a stromal vessel, and yellow arrowheads indicate endothelial cells that lack connect to a stromal vessel. (G–J) are oriented with antrum at the top, granulosa cells central, and stroma at the bottom of each image/model; (G,I) at the same magnification.