Cellular Mechanisms and Modulation of Activin A- and Transforming Growth Factor β-Mediated Differentiation in Cultured Hen Granulosa Cells1

Abstract

Undifferentiated granulosa cells from prehierarchal (6- to 8-mm-diameter) hen follicles express very low to undetectable levels of LH receptor (LH-R) mRNA, P450 cholesterol side chain cleavage (P450scc) enzyme activity, and steroidogenic acute regulatory (StAR) protein, and produce negligible progesterone, in vitro, following an acute (3-h) challenge with either FSH or LH. It has previously been established that culturing such cells with FSH for 18-20 h induces LH-R, P450scc, and StAR expression, which enables the initiation of progesterone production. The present studies were conducted to characterize the ability of activin and transforming growth factor (TGF) beta, both alone and in combination with FSH, to promote hen granulosa cell differentiation, in vitro. A 20-h culture of prehierarchal follicle granulosa cells with activin A or transforming growth factor beta (TGFbeta)1 increased LH-R mRNA levels compared with control cultured cells. Activin A and TGFbeta1 also promoted FSH-receptor (FSH-R) mRNA expression when combined with FSH treatment. Neither activin A nor TGFbeta1 alone stimulated progesterone production after 20 h culture. However, preculture with either factor for 20 h (to induce gonadotropin receptor mRNA expression) followed by a 3-h challenge with FSH or LH potentiated StAR expression and progesterone production compared with cells challenged with gonadotropin in the absence of activin A or TGFbeta1 preculture. Significantly, activation of the mitogen-activated protein (MAP) kinase pathway with transforming growth factor alpha (TGFalpha) (monitored by Erk phosphorylation) blocked TGFbeta1-induced LH-R expression, and this effect was associated with the inhibition of Smad2 phosphorylation. We conclude that a primary differentiation-inducing action of activin A and TGFbeta1 on hen granulosa cells from prehierarchal follicles is directed toward LH-R expression. Enhanced LH-R levels subsequently sensitize granulosa cells to LH, which in turn promotes StAR plus P450scc expression and subsequently an increase in P4 production. Significantly, the finding that TGFbeta signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro

“…Our results show that treatment of cultured secondary follicles with activin-A does increase the follicular level of FSH-R mRNA, which is in accordance with the previously demonstrated activin-induced FSH-R formation in rat granulosa cells (XIAO et al, 1992). When combined with FSH, activin-A similarly also stimulates expression of FSH-R mRNA in granulosa cells from 6-8 mm hen follicles (JOHNSON et al, 2004). These studies suggest that effects of activin-A on the synthesis of FSH-R appear simultaneous with its role in the amelioration of the sensitivity of follicular cells to this gonadotropin.…”

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

“…As it has been shown that ActA increased LH-R mRNA expression in 6-8 mmGC (Johnson et al 2004), the present and previous data lead us to propose that GC of prehierarchical follicles approaching selection, which are primarily FSH-driven, have reduced expression of key ActRs (ActRI and ActRIIB) and therefore a diminished responsiveness to Act. In turn, as follicles are selected and become more responsive to LH, ActRI and ActRIIB mRNA expression is upregulated leading to greater co-stimulation by Act.…”

“…ActA also enhanced FSH-R and LH-R mRNA expression in GC from preovulatory follicles (Johnson et al 2006). An ActA-induced increase in FSH-R mRNA expression has also been observed in GC from preheirarchical hen follicles (Johnson et al 2004, 2006, Woods & Johnson 2005.…”

“…In support of this, ActA was recently shown to enhance the expression of both FSH-R and LH-R in GC from preovulatory follicles (Johnson et al 2006). There are other reports (Johnson et al 2004, 2006, Woods & Johnson 2005) that ActA augments gonadotrophin receptor mRNA expression in granuolsa cells from prehierarchical (small yolky) hen follicles.…”

“…Inh and Act have been implicated as direct (paracrine/autocrine) and indirect (endocrine) regulators of ovarian development in birds (Davis et al 2001, Johnson et al 2004review: Knight et al 2005) and mammals (reviews: Knight & Glister 2003, Philips 2005.…”