Immunostaining of the Thecal Vasculature
The endothelial cell marker, factor VIII-related antigen, was colocalized with each of the type IV collagen a chains. In antral follicles, the arterioles and venules were located mainly at the extremities of the theca interna and in the externa, and capillaries were common in the theca interna. Intense fluorescence was observed in the subendothelial basal lamina of arterioles, venules, and capillaries with antibodies to a1 and a2, and the basal laminae of smooth muscle cells of arterioles also contained a1 and a2. In the thecal layers, no fluorescence was observed in the subendothelial cell basal laminae or the basal laminae of the vascular smooth muscle cells using antibodies to a3, a4, a5, or a6. buy birth control online
In basal laminae, type IV collagen molecules assemble into a meshwork via disulfide bonds and by other nonreducible covalent cross-linking bonds; therefore we digested the collagenous domains of type IV collagen with collagenase, releasing the NC1 domains. Each NC1 domain is a trimer of three chains, and each NC1 domain is bonded to the NC1 domain of another molecule of type IV collagen. Upon reduction, these hexameric structures yield monomers (approximately 25 kDa) and dimers (approximately 50-60 kDa) of single peptide chains from the NC1 domains. The dimers are due to nonreducible, covalent crosslinks. Bovine kidney was chosen as a positive control as this tissue had previously been reported to contain four type IV collagen chains: a1, a2, a3, and a4.
FIG. 4. Western blot analyses of type IV collagen a1 and a2. Samples were digested with collagenase, and monomers (small arrowhead) and dimers (large arrowhead) were detected. Lane 1, bovine kidney; lane 2, follicles < 3 mm; lane 3, follicles 3-5 mm; lane 4, follicles 5-10 mm; lane 5, follicles > 10 mm.