Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus

Aims —To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor β1 (TGF-β1) in the mouse uterus. Methods —In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) and TGF-β1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or (2) ovariectomised (OVX) and administered estradiol-17β (E 2 ) or progesterone (P 4 ), either alone or in combination. Uteri collected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-β1. Results —On days 0.5–2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-β1 were principally colocalised to uterine epithelial cells, with much smaller amounts in the stroma. On days 3.5–4.5, there was a reduction of CCN2 (CTGF) and TGF-β1 in the epithelium but an increase in stromal and endothelial cells, corresponding to a period of extracellular matrix remodelling and neovascularisation within the endometrium. In OVX mice, epithelial cells were weakly positive for both CCN2 (CTGF) and TGF-β1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently stimulated in OVX mice cells by E 2 . These effects were antagonised by P 4 , which itself transiently stimulated epithelial CCN2 (CTGF) production, although less robustly than E 2 . CTGF and TGF-β1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the stroma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-β1 were lower after co-administration of E 2 and P 4 than in response to each hormone individually. Although ccn2 (ctgf) is a TGF-β1 inducible gene in other systems, and both growth factors were often co-localised in uterine tissues in these studies, several treatment regimens resulted in high amounts of TGF-β1 protein in stromal cells without the concomitant production of ccn2 (ctgf) mRNA. Conclusions— Maternal factors are principal cues for CCN2 (CTGF) and TGF-β1 production in the uterus because (1) their expression during pseudopregnancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-β dependent and independent mechanisms of ccn2 (ctgf) gene transcription exist in the uterus that are variably regulated by steroid hormones. Collectively, the data support a role for CCN2 (CTGF) in mediating the effects of steroid hormones and TGF-β on endometrial function.