Spatial differences within the membrana granulosa in the expression of focimatrix and steroidogenic capacity

Abstract

In the ovarian follicular membrana granulosa there are morphological and functional differences between cells adjacent to the follicular fluid lumen, or aligning the basal lamina. Amongst the observed functional differences are steroidogenic capacity and expression levels of a novel basal lamina, focimatrix; both of which increase in the later stages of antral follicle growth. A number of different studies have produced apparently inconsistent results as to which cell layers are more steroidogenic. To examine this systematically, individual bovine follicles, confirmed as healthy by post hoc histological examination, were used to isolate populations of apical and basal granulosa cells. Cell counts revealed that the respective groups did not differ in the numbers of cells, thus confirming the separation of these populations. We measured gene expression (quantitative RT-PCR, n=8-10, follicle diameter 14.0±0.5 mm) and protein levels (Western immunoblotting, n=14, follicle diameter 11.9±0.5 mm) and hormone production from granulosa cells (2.5×10(5) viable cells/well in serum-free conditions for 24 h, n=15, diameter 12±0.5 mm). Levels of mRNA of HSD3B1 and CYP19A1 and three focimatrix genes COL4A1, HSPG2 and LAMB2 and LHCGR were significantly lower in apical granulosa cells (P<0.05), whereas, expression of CYP11A1 and HSD17B1 were not different (P>0.05). The protein levels of steroidogenic enzymes P450scc and P450arom were significantly higher in apical cells (P<0.05), whereas those of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase type 1 were not different (P>0.05). Progesterone production was significantly lower and oestradiol production was significantly higher in apical granulosa cells (P<0.05). These results confirm that apical and basal cells are functionally different, and the differences might be explained by the location of cells of different ages and maturity within the membrana granulosa. Discrepancies in the literature on their steroidogenic capacity may reflect differences in the steroidogenic parameters measured.