Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function

Abstract

The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis.When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I-and/or FSH-stimulated progesterone secretions were totally abolished.Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.

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

“…It is well established that the loss of cell attachment to ECM causes apoptosis, according to a process named anoikis (Ruoslahti & Reed 1994, Giancotti & Ruoslahti 1999. The present result is consistent with previous data showing that LN enhances cell survival in rat (Aharoni et al 1996) and ovine GC (Huet et al 2001). In vitro, histological observations have established that GC adjacent to the follicular basement membrane are protected from entering apoptosis in early atretic follicles (Monniaux et al 1999).…”

“…Accordingly, we have shown previously that LN, compared with non-coated substratum, enhances proliferation of sheep GC (Huet et al 2001). In other cell types, the effect of LN on cell proliferation is the object of controversy.…”

“…cells with condensed or fragmented nuclear chromatin (Aharoni et al 1995), and non-pyknotic cells in 20 different microscopic fields (objective 100). Previous results established that the presence of pyknotic cells was associated with DNA fragmentation characteristic of the apoptotic process in cultured GC (Huet et al 2001).…”

“…5C,D). The apoptosis rate of GC, estimated by the pyknotic index (Huet et al 2001), was higher in cells from small compared with large follicles (Fig. 5E,F).…”

“…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). However, the effects of LN on GC steroidogenesis are still the object of controversy since LN enhances P4 secretion in rat (Aten et al 1995, Aharoni et al 1996 and pig (Sites et al 1996), but inhibits it in human .…”