Luteinized human granulosa cells are associated with endogenous basement membrane-like components in culture

Abstract

Human granulosa cells (GC), prepared from follicular aspirates using a non-enzymic method, were maintained in culture on chamber slides in a defined medium without additional attachment factors or extracellular matrix (ECM). In this system, GC clustered to a limited extent and attached only loosely to the substratum necessitating medium replacement through repeated partial changes to avoid cell loss. Using this new culture system, cell size and progesterone production per cell increased, consistent with continuing luteinization. These processes were associated with maintenance and deposition of endogenous ECM components. Thus, pericellular heparan sulphate proteoglycan (HSPG) was clearly visible by immunocytochemistry around the luteinized GC after culture. Also progressive accumulation of laminin (particularly alpha(2)-, beta(1)- and gamma(1)-subunits) during culture was shown by Western blotting of GC extracts. Small patches of collagen IV, shown to be already present between freshly prepared GC, were maintained in culture. A clear effect of gonadotrophin on the maintenance of progesterone production in culture was paralleled by an apparent increased pericellular deposition of HSPG. To conclude, luteinization and maintenance of the GC-derived layer of the corpus luteum is likely to involve deposition and conservation of pericellular ECM components.

Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells

“…By immunohistochemistry using antibodies raised against LN-1, we have confirmed the presence of LN in basement membrane, granulosa and theca cells of ovine antral follicles, as shown previously in various animal species (Bagavandoss et al 1983, Wordinger et al 1983, Bortolussi et al 1989, Leardkamolkarn & Abrahamson 1992, Zhao & Luck 1995, Lee et al 1996, Huet et al 1997, van Wezel et al 1998, Alexopoulos et al 2000. Laminins are heterotrimeric proteins containing , and subunits, and the association of structural isoforms of these subunits gives rise to different LN (Mercurio 1995).…”

“…This basement membrane is believed to play a role in influencing proliferation and differentiation of GC (Amsterdam et al 1989, Luck 1994 and in protecting them from entering apoptosis in early atretic follicles (Monniaux et al 1999). Laminin (LN) has previously been shown to be one of the most abundant ECM components within the basement membrane (Wordinger et al 1983, Leardkamolkarn & Abrahamson 1992, Zhao & Luck 1995, Lee et al 1996, Huet et al 1997, van Wezel et al 1998, Alexopoulos et al 2000. In vitro, purified LN used as a culture substratum improves GC survival in rat (Aharoni et al 1996) and sheep (Huet et al 2001) and stimulates their proliferation in sheep (Huet et al 2001).…”

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle

“…Laminin subunits (Alexopoulos et al, 2000) and collagen type IV (Yamada et al, 1999;Alexopoulos et al, 2000) have previously been detected between granulosa cells isolated from human pre-ovulatory follicles and also in bovine cumulus cells (Sutovsky et al, 1995). However, focimatrix as described here first appears in less mature follicles.…”

Studies of granulosa cell maturation in dominant and subordinate bovine follicles: novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1

“…1) is deposited as plaques or aggregates of basal lamina material between the epithelial granulosa cells in follicles O5 mm in diameter in bovine and its abundance increases as follicles enlarge. Although discovered in the bovine, focimatrix also exists in sheep (Huet et al 1997) and humans (Yamada et al 1999, Alexopoulos et al 2000. The novel aspect of this matrix is its conformation.…”