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.