Fig. 3. Metalloproteinase inhibitor GM6001 blocks MMP2 processing and Müllerian duct regression. (A) Both the Müllerian (M) and Wolffian (W) ducts remains intact in control female genital ridges cultured without MIS (Untreated). Female ridges cultured with bioactive recombinant MIS ligand for 72 hours undergo Müllerian duct regression, leaving only the Wolffian duct intact. GM6001 negative control does not inhibit Müllerian duct regression (MIS + control analog), while GM6001 prevents Müllerian duct regression (MIS + GM6001). GM6001 was prepared as described in the Materials and Methods and added at a concentration of 10 µM. Male genital ridges cultured for 72 hour undergo regression (Control). The caspase inhibitor Boc-D-FMK and metalloproteinase inhibitor GM6001 block regression in male genital ridges. The presence of the Müllerian duct is indicated in all experimental conditions by an arrowhead. O, ovary; T, testis. (B) The percentage of apoptotic cells in the Müllerian duct epithelium of female genital ridge sections was determined by TUNEL staining as described in the Materials and Methods. Untreated rat female ridges show low levels of apoptosis in the Müllerian duct epithelium (-), compared to MIS (+) or MIS plus the GM6001 control analog treated ridges (+ Control). Addition of GM6001 to MIS treated ridges lowers significantly the number of TUNEL-positive cells (+ GM6001). (C) Metalloproteinase inhibitor GM6001 blocks regression at a step downstream of MIS receptor signaling and target gene activation. Luciferase activity of Tlx2 promoter activity is shown without MIS (-), with MIS and the control analog of GM6001 (+ Control) and with MIS and GM6001 (+GM6001) at a concentration of 10 µM. This concentration potently blocks Müllerian duct regression (refer to A and Table 1).