Oestrogen formation and connective tissue growth factor expression in rat granulosa cells

Abstract

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450 aromatase (P450 arom )) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450 arom mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450 arom mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450 arom . Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450 arom . Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSHstimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10 K7 -10 K5 M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450 arom diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.

Expression and regulation of oestrogen receptors in the human corpus luteum

“…One major role of these cells is steroidogenesis, but we could not detect acute effects of oestradiol on the major regulators of luteal steroidogenesis LHCGR or STAR. Both VEGF and CTGF are major regulators of tissue and vascular remodelling in the corpus luteum (Duncan et al 2005b), but oestradiol had no effect on their mRNA expression over 24 h. This was surprising as it has been demonstrated that VEGF has a functional oestrogen response element in its promoter (Hyder et al 2000) and oestradiol has been shown to up-regulate Ctgf mRNA in rat granulosa cells (Harlow et al 2007). Others have demonstrated a very rapid increase in VEGF expression after stimulating isolated human endometrial cells (Shifren et al 1996) or human MCF-7 breast carcinoma cells (Ruohola et al 1999) as rapidly as 1 h. It may be that such rapid effects do occur or that in these cells the major hormonal regulator is LH.…”

Glial-derived neurotrophic factor promotes ovarian primordial follicle development and cell–cell interactions during folliculogenesis

“…CTGF is a matrix-associated protein with many possible roles in development and differentiation processes (Leask & Abraham 2003). CTGF is regulated by gonadotropins and is expressed in antral follicles and corpus lutea (Landis 2002, Harlow et al 2007. Early follicle development is a vital process for female fertility, and GDNF participates in a network of compensatory factors that act as checks and balances.…”

Age-associated changes in bovine oocytes and granulosa cell complexes collected from early antral follicles

“…Of the apoptosis related genes, CCDC80, COL1A1, CTGF, DNAJB1, LGALS9, LTF, LNM, MT2A, MTH11, SOX4, and TIMP2 showed significant differences in expression levels between the two age groups (Supplementary Table 2). Table 3 lists the marker genes for the largest healthy follicles and the subordinate follicles in cows [31][32][33][34][35][36][37], and shows the expression levels of these genes in aged and young cows. All genes associated with subordinate follicles were expressed at lower levels in young cows.…”