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 alpha1, alpha2, alpha3, alpha4, and alpha5 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 alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 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 alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 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 type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.

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Within the lumbar sympathetic ganglia of guinea pigs, the endings of different populations of neuropeptide-containing preganglionic neurons form well-defined pericellular baskets of boutons around target neurons in specific functional pathways. We have used multiple-labelling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to investigate synaptic organisation within pericellular baskets labelled for immunoreactivity to calcitonin gene-related peptide (CGRP), substance P (SP), or the pro-enkephalin-derived peptide, met-enkephalin-arg-gly-leu (MERGL) in relation to their target neurons. Different functional populations of neurons, identified by their neurochemical profile, showed a significant degree of spatial clustering and predicted well the distribution of specific classes of pericellular baskets. Most of the boutons in a basket were completely surrounded by Schwann cell processes and did not form synapses. The synapses that were present were made mostly onto dendrites enclosed by the Schwann cell sheath surrounding the neuron within the basket. These dendrites probably originated from neurochemically similar neighbouring neurons. Nevertheless, some of the boutons in the baskets did form synapses with the cell body or proximal dendrites of the neuron they surrounded. Occasionally, cell bodies received a relatively high number of synapses and close appositions from boutons in a pericellular basket. Synaptic convergence of two immunohistochemically distinct types of preganglionic inputs was found in baskets of SP-immunoreactive or MERGL-immunoreactive, but not CGRP-immunoreactive, boutons. Taken together, our results show that the appearance of pericellular baskets is primarily due to the packing of the target neurons. The grouping of functionally similar classes of neurons with their pathway-specific projections of peptide-containing preganglionic neurons suggests that peptides could exert their effects in relatively well-defined zones within the ganglia.

The composition of a basal lamina markedly affects its ability to filter material and affects the fate of adjacent epithelial cells. Therefore, basal laminae differ in composition with tissue development, and between different tissues in the body. Laminins are a component of basal laminae and consist of one alpha, one beta and one gamma chain, of which there are at least five, three and two isoforms, respectively. This is the first study to immunolocalize a range of these individual laminin chains (alpha 1, alpha 2, beta 1, beta 2, gamma 1) in ovarian follicles. Frozen sections of bovine ovaries (n = 6) were immunostained using specific antisera to laminin chains and factor VIII-related antigen (to identify endothelial cells). Secondary antisera were labelled with one of two different fluorochromes (DTAF and Cy3), and dual localization of laminin chains and factor VIII-related antigen was performed. The alpha 1, beta 2 and gamma 1 chains were consistently localized to the follicular basal lamina in all healthy follicles. Staining was less intense in the atretic antral follicles. Conversely, alpha 2 and beta 1 were rarely present in the follicular basal laminae of healthy antral follicles. Two of nine healthy antral follicles observed stained weakly for alpha 2 in their basal lamina, and beta 1 was present at low concentrations in growing preantral follicles. In atretic antral follicles, the follicular basal lamina stained positively for alpha 1, alpha 2, and beta 2 but no beta 1 was detected and the gamma 1 staining was less intense than in healthy follicles. Antisera to Englebreth Holm-Swarm tumour laminin stained basal laminae of all follicles. In the theca of antral follicles, beta 1 and beta 2 chains were both present in the vasculature. Staining for the gamma 1 chain was present in the thecal vasculature and generally throughout the theca of healthy and atretic antral follicles. Therefore, the composition of the follicular basal lamina alters during development and atresia, and potentially plays a role in the changing identity of the granulosa cells and the accumulation of antral follicular fluid.

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