Distribution of the alpha 1 to alpha 6 chains of type IV collagen in bovine follicles

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV col lagen is an important component of many basal laminae, Each molecule is com posed of three alpha chains; however, six different type IV collagen chains have been identified, It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition o f the basal lamina changes during follicular development. Therefore, we imm unolocalized each of the six chains in bovine ovaries using antibodies dire cted to the nonconserved non-collagenous (NC) domains. Additionally, dissec ted follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot, methods . The follicular basal lamina of almost all primordial and preantral follic les was positive for all type IV collagen alpha chains, Colocalization of t ype IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alph a 1, alpha 2, alpha 3, alpha 4, and alpha 5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining wa s less intense than in the preantral follicles, Staining for the alpha 1 an d alpha 2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immun ostaining for alpha 1 and alpha 2 chains of type IV collagen was confirmed by Western immunoblots, As well as being detected in the basal lamina of ap proximately half of the antral follicles examined, type IV collagen alpha 4 also colocalized with 3 beta-hydroxysteroid dehydrogenase-immunopositive c ells in the theca interna, Type IV collagen alpha 6 was detected in the bas al lamina of only one of the 16 antral follicles examined. Thus, the follic ular basal lamina changes in composition during follicular development, wit h immunostaining levels being reduced for all type IV collagen chains and i mmunoreactivity for IV collagen alpha 6 being lost as follicle size increas es. Additionally, immunoreactivity for alpha 1 and alpha 2 appears in the e xtracellular matrix of the theca as it develops.