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Female Reproductive Tract |
Center for Animal Biotechnology and Genomics and Department of Animal Science,3 Texas A&M University, College Station, Texas 77843-2471
Departments of Obstetrics and Gynecology,4 Division of Basic and Clinical Women's Research, Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.
developmental biology, female reproductive tract, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Development of other epitheliomesenchymal organs involves extracellular matrix (ECM) synthesis and degradation that is regulated, in part, by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) [13]. With at least 25 family members identified, MMPs are a large group of ECM-degrading enzymes that regulate cell migration, proliferation, and invasion [14, 15]. Most MMPs are secreted in a proactive form and are activated by protease cleavage. The synthesis and activity of MMPs are regulated by a variety of growth factors, ECM, and specific inhibitor found both in serum and in tissues (e.g., TIMPs). Active MMPs can degrade ECM and, in turn, liberate growth factors involved in ECM remodeling [16]. Furthermore, MMP-14 can activate MMP-2 with the aid of TIMP-2 [17], signifying that MMP activation can occur via other MMPs.
Results of available studies strongly support the idea that MMPs and TIMPs are important regulators of uterine growth and remodeling in both humans and primates during the menstrual cycle and pregnancy [18–20]. Many MMP and TIMP knockout mice do not exhibit overt reproductive phenotypes, which may be attributed to compensation and redundancy because of the size of the MMP and TIMP family as well as their overlapping activities [18]. The TIMP-1-null mice exhibit fertility problems [21]. Interestingly, TIMP-1-null mice have an increased number of uterine glands on PND 15 and PND 20 compared to wild-type mice [22], suggesting that MMPs and TIMPs regulate uterine adenogenesis. The MMP-9-null mice also have decreased fertility, although the precise cause of this fertility defect is not known [23]. Little information is available regarding expression of the MMP and TIMP system in the developing neonatal mouse uterus. Our working hypothesis is that MMPs and TIMPs are important regulators of postnatal uterine development and, in particular, of uterine gland morphogenesis. As a first step in testing this hypothesis, the objective of the present study was to determine the effects of postnatal age on expression of MMPs and TIMPs in the mouse uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult virgin CD-1 female mice were obtained from Charles River Laboratories (Wilmington, MA) and mated with fertile males of the same strain to establish pregnancy. Day of birth was designated as PND 0. All mice were housed in a temperature-controlled room (21–22°C) with a 12L: 12D photoperiod in the Kleberg Center Mouse Facility (Texas A&M University) and provided with fresh reverse-osmosis/deionized water and NIH-31 lab chow ad libitum. The University Laboratory Animal Care and Use Committee of Texas A&M University approved all experimental procedures involving these mice.
For transcriptional profiling, uteri were obtained from mice on PNDs 3, 6, 9, 12, or 15 (n 
20 per day). Uteri were snap-frozen in liquid nitrogen and stored at –80°C for RNA or protein extraction. Some uteri were frozen in Optimal Cutting Temperature compound (OCT; Ted Pella, Inc., Redding, CA). Using methods described previously by Hu et al. [11], uterine epithelia and stromal/myometrial cells were isolated by enzymatic digestion from mouse uteri sampled at PND 3, PND 6, and PND 9. As described recently [11], total RNA was extracted from either the entire uterus or separated epithelia and stroma/myometrium and then analyzed using a CodeLink UniSet Mouse I Expression Bioarray (Amersham Biosciences, Piscataway, NJ).
Real-Time Quantitative Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) was performed using an Applied Biosystems GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the SYBR Green method and procedures described previously [11]. Primer pairs for mouse genes were designed with Primer Express software, version 1.5 (Applied Biosystems). Primer sequences were checked for sequence homology against known genes using a BLAST search. To ensure specific amplification, various negative controls (i.e., water only, reaction without primers, and templates derived without reverse transcriptase) were performed. Values for cycle threshold (CT), the point at which exponential amplification of the PCR products begins, were determined using the Applied Biosystems software. The normalized CT values were analyzed by least-squares ANOVA using the general linear models procedure of the Statistical Analysis System (Cary, NC). The model included the CT value of DNA fragmentation factor 
as a covariate to correct for differences in cDNA input. As suggested by Applied Biosystems, the data from real-time PCR analyses are presented as expression level. The expression level was calculated using formula of 2x–y, where y is the normalized CT value obtained from the sample taken at PND 3 and x is the normalized CT value obtained from samples taken at the other PNDs.
In Situ Hybridization Analysis
Mouse MMP-2, MMP-7, MMP-10, MMP-11, TIMP-1, TIMP-2, and TIMP-3 cDNAs were kindly provided by Dr. Lynn Matrisian (Department of Cancer Biology, Vanderbilt University, Nashville, TN). Partial mouse MMP-9, MMP-14, and MMP-23 cDNAs were generated by reverse transcription-PCR and confirmed by sequencing. In situ hybridization was conducted on uterine tissues using methods described previously [24]. Antisense and sense radiolabeled cRNA probes were generated using appropriate polymerases by in vitro transcription with -[35S]UTP. Transcripts protected from RNase digestion were visualized by liquid emulsion autoradiography. Slides were stored at 4°C for 1–2 wk as judged from autoradiographs and then developed and counterstained with hematoxylin for imaging.
Gelatin and Casein Zymography
Zymography was conducted using previously described methods with a few modifications [25, 26]. All zymographic studies were performed using three pools of uteri from for each PND (n = 3). Frozen mouse uteri were homogenized (1:5, w/v) in homogenization buffer (0.5 M Tris-HCl [pH 7.6], 0.2 M NaCl, 0.01 M CaCl2, and 1.0% [w/v] Triton X-100). Uterine homogenates were placed on ice for 5 min, followed by centrifugation at 12 000 x g for 30 min at 4°C. Supernatants were then collected, and an aliquot was subjected to the DC Protein Assay (Bio-Rad Laboratories, Richmond, CA). For gelatinolytic and caseinolytic activity analysis, uterine extracts (30 µg of total protein) were immediately subjected to zymographic analysis by 10% PAGE containing 1 mg/ml of gelatin (gelatin zymography) or in precast, 4%–16% PAGE containing 1 mg/ml of blue casein (casein zymography; InVitrogen, Carlsbad, CA). Following electrophoresis, gels were washed twice with 2.5% (v/v) Triton X-100 for a total of 60 min at room temperature to remove SDS. The gels were then placed in incubation buffer (50 mM Tris [pH 8.0] and 5 mM CaCl2) for 30 min at room temperature. Finally, gels were incubated in fresh incubation buffer for 18–24 h for gelatin gels or 48 h for casein gels at 37°C to allow proteolysis of the substrates in the gels. Gelatin-impregnated gels were then rinsed with water and stained with Brilliant Blue R-250 dye (2.5 mg/ml) and destained until clear bands became evident. Blue casein-impregnated gels required no staining to allow detection of the caseinolytic activity. Prestained molecular weight markers (InVitrogen) were coelectrophoresed in all gels to determine the masses of the lysis bands.
To verify that the bands of proteolytic activity were MMPs, inhibition studies were conducted. The MMP inhibitors, 1,10-phenanthroline (20 mM; Sigma) and EDTA (5 mM; Sigma), were added separately to the incubation buffer, and gels were incubated overnight as described for zymography. Additionally, the serine protease inhibitor, PMSF (5 mM; Sigma), was also used as a control to verify MMP activity.
Western Blot Analysis
Western blot analysis was done essentially as described previously [27]. Briefly, proteins (50 µg) extracted from the entire uterus were denatured and separated by 10% SDS-PAGE, and Western blot analysis was conducted using enhanced chemiluminescence detection. Immunoreactive MMP-2 and MMP-9 proteins were detected using either galectin-15 or polyclonal rabbit anti-human MMP-2 or anti-mouse MMP-9 antibodies (Triple Point Biologics, Forest Grove, OR) at a 1:1000 final dilution. Negative control blots were performed in which primary antibody was replaced by rabbit immunoglobulin (Ig) G at the same concentration.
Immunofluorescence Analysis
Frozen sections (thickness, 4–8 µm) of the uterus were mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA), fixed in –20°C methanol for 10 min, and permeabilized with 0.3% Tween-20 in 0.02 M PBS. After blocked with 10% normal goat serum in antibody dilution buffer (two parts 0.02 M PBS, 1.0% BSA, and 0.3% Tween-20, [pH 8.0], and one part glycerol) for 1 h at room temperature, slides were incubated overnight at 4°C with rabbit polyclonal anti-human MMP-2 or anti-mouse MMP-9 antibodies (Triple Point Biologics) at a 1:200 final dilution. Immunoreactive protein was detected using a fluorescein-conjugated goat anti-rabbit IgG from Zymed (San Francisco, CA). Images of representative fields were recorded using a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) fitted with a Hamamatsu C-5810 chilled three-color CCD camera (Hamamatsu Corporation, Bridgewater, NJ).
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RESULTS |
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Microarray analysis found that MMP-2, MMP-10, MMP-11, MMP-14, and MMP-23 were the most abundant MMP mRNAs expressed during postnatal development of the mouse uterus (Table 1). The MMP-2 mRNA was predominantly expressed in uterine stroma/myometrium, whereas the MMP-10 mRNA was predominantly expressed in epithelium (Table 2). All other MMPs were expressed in all uterine epithelium and stroma/myometrium. The expression level of MMP-10 decreased after PND 9, whereas the expression level of all other MMPs remained relatively constant from PND 0 to PND 15. We found that TIMP-1, TIMP-2, and TIMP-3, but not TIMP-4, were expressed in the developing mouse uterus (Table 1) and in both the uterine epithelium and stroma/myometrium (Table 2). Expression of TIMP-1, TIMP-2, and TIMP-3 genes did not change between PND 0 and PND 15.
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Semiquantitative, real-time PCR analyses were conducted for eight MMPs and TIMPs to verify and validate results of the microarray analyses (Fig. 1). With the exception of TIMP-2 and TIMP-3, the expression level of the selected MMPs and TIMPs were coordinate between microarray and real-time PCR analyses. After the PCR reaction, amplification plots were obtained to confirm specific amplification, and all PCR products were analyzed by agarose gel electrophoresis to confirm a single amplicon (data not shown).
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As predicted from the array analysis, MMP-2 mRNA was expressed in the uterine stroma and myometrium, but not in epithelia (Fig. 2A). In contrast, low levels of MMP-10 mRNA were detected exclusively in the epithelium (Fig. 2B). Expression of MMP-11, MMP-14, MMP-23, TIMP-1, TIMP-2, and TIMP-3 mRNAs were detected in all uterine cell types (Fig. 2C). As expected from the microarray analysis, expression of all other MMPs (MMP-3, MMP-9, MMP-13, MMP-15, MMP-19, and MMP-24) and TIMP-4 mRNAs were less than the detection limit of the in situ hybridization analysis (data not shown).
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Zymography and Western Blot Analysis of MMPs in the Postnatal Mouse Uterus
Gelatin and casein zymography of whole uterine extracts were performed to determine the presence of active MMPs. Gelatinase zymography detected five major gelatin-degrading bands of approximately 82, 78, 66, 62, and 50 kDa (Fig. 3). The 82- and 78-kDa bands of gelatin-degrading activity were consistent with the active forms of MMP-9 (gelatinase B), whereas the 66-, 62-, and 50-kDa bands of activity agreed with the reported active forms of MMP-2 (gelatinase A). These bands of activity were all inhibited by the MMP inhibitors, 1,10-phenanthroline and EDTA, but not by the serine protease inhibitor, PMSF.
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Casein zymography revealed three major bands of casein-degrading enzymes in the uterus (Fig. 4). The apparent molecular masses of these bands of activity were approximately 80, 62, and 50 kDa. Surprisingly, these apparent molecular sizes were not consistent for the stromelysin family (52–43 kDa). The possibility that these bands of activity could be caused by non-MMP proteases was confirmed by the observation that their activity could not be blocked by the MMP inhibitors, EDTA or 1,10-phenanthroline. However, these bands of activity were inhibited by the broad-spectrum serine protease inhibitor, PMSF, suggesting that they are serine proteases.
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As observed by gelatinase zymography, Western blot analyses detected immunoreactive, 62/66-kDa MMP-2 in the mouse uterus (Fig. 5). Several other immunoreactive, high-molecular-mass proteins were observed in the Western blot analyses that were not detected by zymography. The 105-kDa pro-form of MMP-9 was also detected in the mouse uterus (Fig. 5). The 92-kDa active form of MMP-9 was detected only in the uterus from PND 3, whereas the 82/78-kDa forms of active MM-9 were detected in uteri from all PNDs.
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Immunofluorescence Localization of MMP-2 and MMP-9 Proteins in the Postnatal Mouse Uterus
Immunoreactive MMP-2 protein was detected in both the uterine epithelium and stroma, but this protein was most abundant in the endometrial luminal epithelium (Fig. 6A). In contrast, MMP-9 protein was observed almost exclusively in the uterine stroma (Fig. 6B). The Postpartum Day 0 uterus was included as a positive control.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gelatinases are important enzymes that degrade collagen and are highly expressed in invading cells [19]. In the present study, abundant MMP-2 mRNA and protein were observed in the developing neonatal mouse uterus. Although MMP-2 mRNA was expressed only in the uterine stroma, MMP-2 protein was present in both the uterine epithelium and stroma. These results support the idea that MMP-2 protein is produced by the stroma and then either diffuses or is transported to the uterine epithelium. Interestingly, a functional feedback loop between epidermal growth factor-membrane type MMP-14 and MMP-2 is critical in branching morphogenesis of fetal lung [28]. Furthermore, MMP-2-null mice exhibit impaired branching morphogenesis of the mammary gland. Therefore, interactions between MMP-2 and MMP-14 may be an important regulatory system governing endometrial adenogenesis in the neonatal mouse uterus.
In mice and humans, MMP-9 is expressed in the invading trophoblast during implantation and is thought to regulate implantation [29, 30]. The MMP-9-null mice of various genetic backgrounds are subfertile for unknown reasons [23]. Microarray analysis showed that MMP-9 mRNA was expressed predominately in the uterine stroma/myometrium. Active MMP-9 protein was detected in the neonatal mouse uterus by gelatinase zymography, and MMP-9 protein was observed in the uterine stroma. Balanced interactions between MMP-2 and MMP-9 as well as other MMPs may modulate uterine development in the neonatal mouse.
Three major forms of stromelysins (MMP-3, MMP-10, and MMP-11) were expressed in the developing neonatal mouse uterus. Stromelysins are involved in tissue development, remodeling, and wound healing [31, 32]. The MMP-3 is a critical mediator of growth factor-induced mammary gland branching morphogenesis [16], and premature development of the mammary gland is seen in virgin mice overexpressing the MMP-3 gene [33]. Although MMP-11-null mice are fertile, MMP-11 can contribute in a paracrine manner to epithelial malignancy [34]. In the cyclic human and primate uterus, MMP-10 is observed in the uterine stroma and regulated by ovarian steroid hormones [20, 35]. In the present study, MMP-10 mRNA was detected predominantly in the uterine epithelium and decreased after PND 9. Expression of MMP-10 is correlated to the size and invasiveness of various tumors [36, 37], and MMP-10 appears to be involved in the growth of cancer cells [37]. Furthermore, an increase in MMP-10 mRNA and protein in the epithelium of diabetic corneas was associated with increased expression of insulin-like growth factor-I, a regulator of postnatal mouse uterine development [38–40]. The effects of null mutation of the MMP-10 gene have not been reported in mice. However, the potential regulatory role of MMP-10 in postnatal uterine development warrants further investigation.
Although expression of MMP-10 and MMP-11 mRNAs were relatively high in neonatal mouse uterus according to the microarray data, casein zymography failed to detect active MMP-10 and MMP-11 proteins in the uterine extract. One explanation is that the activity of those MMPs may be low relative to the overall serine protease activity in the uterus, because MMPs constitute only approximately 10%– 20% of the total protease activity in the postnatal uterus [22]. Alternatively, TIMPs or other MMP inhibitors could be released during tissue homogenization and inhibit the activity of MMP-10 and MMP-11. Nevertheless, the present results show that several functional serine proteases are in the uterus during postnatal development, but the identity and function of these serine proteases remain to be investigated.
In summary, temporal and spatial alterations in the expression of specific MMPs and TIMPs were discovered in the developing neonatal uterus. Because MMPs and TIMPs regulate morphogenesis of other epitheliomesenchymal organs, we speculate that they also play important regulatory roles in postnatal development of the mouse uterus. Future studies will address the role of these MMPs and TIMPs, as well as that of the ECM, in neonatal mouse uterine development and, in particular, endometrial gland morphogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979-862-2662; tspencer{at}tamu.edu
Received: 1 May 2004.
First decision: 19 May 2004.
Accepted: 14 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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