Distribution of the α1 to α6 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 collagen is an important component of many basal laminae, Each molecule is composed 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 of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected 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 follicles was positive for all type IV collagen alpha chains, Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha 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 was less intense than in the preantral follicles, Staining for the alpha 1 and alpha 2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining 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 approximately half of the antral follicles examined, type IV collagen alpha 4 also colocalized with 3 beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna, Type IV collagen alpha 6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for IV collagen alpha 6 being lost as follicle size increases. Additionally, immunoreactivity for alpha 1 and alpha 2 appears in the extracellular matrix of the theca as it develops.