Role for Inhibitor of Differentiation/Deoxyribonucleic Acid-Binding (Id) Proteins in Granulosa Cell Differentiation

Abstract

Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual inhibitor of differentiation (Id) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared with levels of Id1, Id3, and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3, and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, because factors that promote inhibitory MAPK signaling (TGF-alpha and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3, and Id4. Furthermore, overexpression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3, and Id4 mRNA but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knockdown studies using small interfering RNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side-chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.

Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells

“…These effects on ID expression are completely blocked by treatment with the selective MAP kinase/ERK inhibitor, U0126. We have recently provided evidence that changes in ID protein expression can modulate FSHR mRNA expression in undifferentiated granulosa cells (Johnson et al 2008a, Johnson & Woods 2009. In the present studies, BMP2 treatment was found to increase ID1, ID3, and ID4 expression (Fig.…”

“…3). This is noteworthy because treatment with BTC or TGFA was reported to increase ID1, ID3, and ID4 expression (and decrease levels of ID2) in undifferentiated granulosa cells (Johnson et al 2008a). These effects on ID expression are completely blocked by treatment with the selective MAP kinase/ERK inhibitor, U0126.…”

“…Forward and reverse primers specific for Gallus EGF, BTC, FSHR, ID protein isoforms (ID1, ID2, ID3, and ID4), and 18S rRNA have previously been described , Johnson et al 2008a. Random-primed, reverse-transcribed cDNA synthesis reactions were performed using the Promega RT System (Promega), according to the conditions described by the manufacturer.…”

“…Western blot analysis was conducted as described previously (Johnson et al 2008a). Briefly, cells were homogenized in a protein lysis buffer containing a cocktail of enzyme (including phosphatase) inhibitors (Sigma-Aldrich).…”

“…Importantly, it has been reported that activation of the rat FSHR promoter is dependent upon a functional E-box element (Heckert 2001). More recently, levels of ID proteins (ID1, ID3, ID4, and ID2) in the hen granulosa cells were found to be regulated by BTC (acting in a paracrine/autocrine fashion) and TGFA (acting in a paracrine fashion), and reflective of FSHR mRNA expression (Johnson et al 2008a). Specifically, elevated levels of ID1, ID3, and ID4 in granulosa from prehierarchal follicles are associated with decreased levels of FSHR, whereas enhanced expression of ID2 is associated with enhanced FSHR expression and FSH responsiveness.…”