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Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital development

Lori M. Roberts^1,2, Jenny A. Visser^1, and Holly A. Ingraham^1,2,3,*

¹ Department of Physiology,
² Graduate Programs in Biomedical Sciences and
³ Developmental Biology, University of California San Francisco, Box 0444, San Francisco, CA 94143-0444, USA
Present address: Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, The Netherlands

View larger version (72K): [in a new window]   Fig. 1. The regressing Müllerian duct expresses MMP2. (A) Gelatin zymography was carried out on E13 mouse genital ridge extracts and the corresponding MMP activity is shown in the second lane (E13 GR). Lane 1 shows zymography of purified recombinant MMP2 (1 ng), as a control marker. Zymography reveals both the inactive proenzyme and mature metalloproteinase; most of the activity detected in cell extracts of genital ridges represents the intracellular pro-form of MMP2. This zymogram is shown as a negative image. (B) Whole-mount in situ hybridization of MMP2 profiles showed prominent expression in male but not in female E13 Müllerian ducts (M, arrow). Control experiments using an MMP2 sense probe showed no signal in the Müllerian duct (data not shown). The prominent signal observed in the ovary and testis was observed with both sense and antisense MMP2 probes. O, ovary; T, testis. (C) In situ hybridization of MMP2 in E13 genital ridge sections shows marked expression in the Müllerian mesenchyme, which encapsulates the visible epithelial Müllerian duct (M) in male tissue. The MMP2 staining observed in the coelomic epithelium (CE) in female tissue is indicated with an arrowhead.

View larger version (81K): [in a new window]   Fig. 2. MIS regulates MMP2 expression in the Müllerian duct. (A) In situ hybridization shows both MMP2 and MISIIR expression in Müllerian mesenchyme, which surrounds the Müllerian duct (M) in E13 male genital ridges. At E14, MMP2 and MISIIR are detected in a narrow ring surrounding the regressing Müllerian duct (M). (B) MMP2 message is detected in the Müllerian mesenchyme of male Amh+/– ridges at E13 and E14, but not in male Amh–/– genital ridges. MMP2 expression is still detected in the coelomic epithelium (CE) of these male Amh–/– ridges. In the E14 Amh–/– panel, owing to a difference in the plane of section, the Müllerian duct appears further away from the coelomic epithelium than in the other micrographs. The absence of MMP2 staining in the E14 Amh–/– Müllerian mesenchyme and its persistence in the coelomic epithelium illustrates that MMP2 expression is independently regulated in these two tissues; the Wolffian duct is out of the frame of the photomicrograph. The dotted circles indicate the position of the Müllerian ducts.

View larger version (44K): [in a new window]   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).

View larger version (44K): [in a new window]   Fig. 4. MMP2 maturation correlates with Müllerian duct regression. (A) A schematic of MMP2 activation by MMP14 is shown. (B) Gelatin zymograms of conditioned media obtained from treated male and female genital ridges are shown for both the latent (pro-MMP2) and mature forms of MMP2 (mature MMP2). The first lane shows the control marker for purified recombinant (MMP2). The treatment is indicated above each lane and all zymograms are shown as negative images. (C) Female genital ridges treated with concanavalin A (Con A) and control analog undergo regression in the absence of MIS (Con A + control). GM6001 blocks con A-induced regression (Con A + GM6001). The Müllerian duct is not as clearly visible in con A + GM6001 ridges as in the untreated ridges, possibly because of incomplete inhibition of con A-stimulated MMP2 maturation, and mild toxicity of these compounds in organ culture. M, Müllerian duct; W, Wolffian duct. (D) Con A causes Müllerian duct regression independent of MIS receptor signaling. The relative luciferase activity of the Tlx2 promoter fused to luciferase reporter in P19 cells over a wide concentration range of con A (10-200 µg/ml) is shown. No enhancement of the MIS receptor signaling is observed in the absence of MIS, despite high doses of con A. In genital ridge organ cultures 50 µg/ml of con A is sufficient to cause full Müllerian duct regression in the absence of MIS (see 4C).

View larger version (67K): [in a new window]   Fig. 5. Exogenous MMP2 causes genital ridge apoptosis and loss of MMP2 impairs regression. (A) Hoechst staining (blue) reveals that exogenous MMP2 causes widespread death in the genital ridge compared to control cultures. Arrowheads indicate condensed apoptotic nuclei. Pax2 staining (red) shows the absence of the Müllerian duct after MMP2 treatment. GM6001 attenuates the effect of exogenous MMP2, indicated by the preservation of the Müllerian duct. (B) Antisense oligos against MMP2 (anti-MMP2) block Müllerian duct regression compared to control MMP2 oligos containing four mismatched bases (Mismatch). (Left panel) Full Müllerian duct regression is defined as the absence of any visible duct remnants. (Right panel) The percentage of Müllerian duct remnants present in genital ridge sections is shown for both anti-MMP2 and the control MMP2 oligonucleotide treatments (Mismatch). The number of each independent experiment or the number of sections examined is indicated in parentheses above the bars. *P=0.03; ** P=0.01. Müllerian duct remnants were judged by Pax2 staining as shown in C. (C) Representative examples of Hoechst (blue) and Pax2 (red) stained sections counted in B, show full regression of the Müllerian duct in control cultures treated with MIS, whereas both Müllerian and Wolffian ducts are often present in genital ridges treated with anti-MMP2 oligos. M, Müllerian duct; W, Wolffian duct.

View larger version (32K): [in a new window]   Fig. 6. Potential roles of MMP2 in mediating Müllerian duct regression. Activation of the MIS signaling cascade induces expression of MMP2 in the Müllerian duct mesenchyme. Latent or pro-MMP2 is secreted into the extracellular space, where it is activated to its mature form by membrane bound metalloproteinases such as MMMP14. Cell death of the Müllerian duct epithelium may occur by MMP2 cleavage of a factor secreted by the mesenchyme. MMP2 activity may result in the degradation of a survival factor or activation of a death factor. Alternatively, MMP2 could cause apoptosis by cleaving substrates on the epithelial cell, such as the epithelial cell basement membrane.