Immunolocalization of BRG1–SWI/SNF protein during folliculogenesis in the porcine ovary

Abstract

Dynamic changes in chromatin structure and gene expression occur during follicular and oocyte growth. Epigenetic mechanisms regulate these changes through biochemical reactions that modify the nucleosome structure, and consequently affect transcription. Chromatin remodellers that alter DNA-histone interactions can influence transcriptional activity by facilitating or repressing DNA access. The SWItch/Sucrose NonFermentable (SWI/SNF) complex represents an important chromatin remodelling family, which comprises many protein subunits including the BRG1 (brahma-related gene 1). Our aim in this study was to analyse BRG1 expression patterns in different stages of follicular development. Ovaries (n = 10) were collected from prepubertal gilts and then rinsed in phosphate-buffered saline (PBS). Ovarian fragments of 8 × 8 × 8 mm were cut and placed into a 4% paraformaldehyde solution. For immunofluorescence analysis, samples were incubated with primary antibodies: polyclonal rabbit anti-BRG1 (1/200) or control rabbit IgG at the same concentration, overnight at 4°C. Primary antibodies were detected using Alexa Fluor 594-anti-rabbit 1/1000 diluted secondary antibody. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Positive fluorescence signal for BRG1 was detected in all analysed samples. In primary follicles, the protein was detected only in the oocyte nucleus. However, in growing follicles, BRG1 was identified in granulosa and theca cells in a well defined pattern, according to the proximity of the cells from the oocyte. These results suggest an important role for BRG1 in the regulation of follicular growth, probably modulating granulosa and theca cell proliferation, as well as oocyte growth and maturation.