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BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029181
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BIOLOGY OF REPRODUCTION 71, 534–539 (2004)
DOI: 10.1095/biolreprod.104.029181
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Disruption of the TIMP-1 Gene Product Is Associated with Accelerated Endometrial Gland Formation During Early Postnatal Uterine Development1

Han-E Zhou3, Xuan Zhang3, and Warren B. Nothnick2,4

Department of Obstetrics and Gynecology, Division of Basic and Clinical Women's Research3 Department of Molecular and Integrative Physiology,4 University of Kansas School of Medicine, Kansas City, Kansas 66160


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.

developmental biology, female reproductive tract, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All mammalian uteri contain endometrial glands, which are essential for normal uterine function. Endometrial gland development (termed adenogenesis) involves the same basic morphogenetic events in all female species, including bud formation and tubulogenesis with coiling, branching, and branching morphogenesis more evident in primate species and ungulates [1]. In rodents, bud formation occurs between Postnatal Days (PNDs) 5 and 7, with onset of gland development occurring between PNDs 7 and 9. Tubulogenesis proceeds from PNDs 9 to 15, culminating in histological maturity by PND 15. In rodents, compared with ungulates and primates, endometrial glands are simple and lack a tightly coiled, highly branched structure.

The mechanisms that regulate endometrial gland development are complex and involve epithelial-mesenchymal interactions, steroid-steroid receptor interactions, and endometrial remodeling. Tissue recombination studies have demonstrated that, in the mouse, interactions between germinal epithelium and stroma are required for endometrial morphogenesis [2, 3]. The role of steroids and their respective receptors appears to be more involved in branching morphogenesis than in differentiation or budding of germinal epithelium from luminal epithelium. In the rat, early postnatal events in uterine development and endometrial adenogenesis appear to be ovary [4] and adrenal independent [5]. Between PNDs 9 and 11, circulating estrogen levels increase and are associated with postnatal endometrial remodeling and tubulogenesis [6]. While the process of tissue remodeling during adenogenesis is apparent, the specific proteases and their regulation during this period of uterine development are not.

Matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) are proposed to play a vital role within the endometrium [7]. Previous studies from this laboratory indicate that regulation of MMP action by TIMP-1 is a critical event during the reproductive cycle [8–10]. Using TIMP-1-deficient mice, we have demonstrated that TIMP-1 may play a role in regulating the tissue remodeling that occurs during the reproductive cycle and may also be required for normal fertility. As such, similar regulation of tissue remodeling may be vital to allow for proper endometrial growth and differentiation during the postnatal period. As the pattern of expression of uterine TIMPs and their potential function within the uterus during this time period is largely unknown, the purpose of the current study was to characterize the pattern of uterine TIMP transcript expression during postnatal development and to assess the functional role of TIMP-1 during this time period. To meet this objective, TIMP-1 wild-type and null mice were killed at various stages of postnatal development and TIMP steady-state transcript expression, MMP activity, and uterine morphology were assessed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Wild-type and TIMP-1 null mice were used throughout this study. The TIMP-1-deficient animals (SVTER 129 background) were generated by homologous recombination of a neo-containing gene-targeting vector in mouse embryonic stem cells. Transmission of the mutant allele and the genotype of mice were determined by polymerase chain reaction analysis of the neo sequences in genomic tail DNA. The TIMP-1 deficiency was confirmed at the transcript and protein levels by Northern analysis and protease inhibitor assays, respectively [11].

A breeding colony of both genotypes was generated at the University of Kansas Medical Center. Mice were housed within environmentally controlled conditions under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Mice were maintained on a 14L:10D cycle and provided water and mice chow ad libitum.

For experimentation, whole uteri were collected from female pups born from natural matings at PNDs 5, 10, 15, 20, and 25. Animals were killed by either CO2 asphyxiation or cervical dislocation and uteri were removed with the aid of a dissecting microscope. Removed tissues were placed immediately into either RNAlater (Ambion, Midland, TX) and stored at 4°C for subsequent RNA isolation, fixation solution (HistoPrep Buffered 10% formalin; Fisher Scientific, Pittsburgh, PA) at room temperature for histological assessment, or snap-frozen in liquid nitrogen and stored at –75°C for subsequent protein isolation. Extracted protein fractions were then used for assessment of total uterine matrix metalloproteinase activity as described below.

RNA Isolation and Northern Analysis

Total RNA was isolated from uterine tissue by pooling tissue from 2 to 10 mice per PND of age for each genotype and homogenizing the pooled tissue in 1 ml of TRIZOL reagent (Life Technologies/GIBCO-BRL, Gaithersburg, MD). Using this amount of pooled tissue allowed us to obtain sufficient RNA to perform all Northern analysis using the same RNA pool for each of the study groups. RNA was extracted with chloroform and precipitated with isopropyl alcohol according to the recommendations of the manufacturer. Total RNA samples (20 µg/lane for TIMP-1 and TIMP-4 and 10 µg/lane for TIMP-2 and TIMP-3) were then electrophoresed through 1.0% agarose gels containing 2.2 M formaldehyde and were transferred to nylon membranes (Nytran; Schleicher and Schuell, Keene, NH) as recommended by the manufacturer. The murine TIMP-1, -2, -3, and -4 cDNA probes (kindly provided by Dr. Dylan Edwards, University of East Anglia, Norwich, U.K.) were excised from their respective plasmids with the appropriate restriction endonucleases and the resulting inserts were labeled using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Probes were labeled to a specific activity of 5 x 108 to 1 x 1010 dpm/µg of DNA using [{alpha}-32P] dCTP (Perkin Elmer Life Sciences, Boston, MA). Filters were hybridized overnight, washed, and exposed to Blue Sensitive autoradiography film (Midwest Scientific, St. Louis, MO) for up to 24 h at –75°C. Membranes were then stripped of probe by washing the membranes in stripping solution (75% formamide, 0.2x SSC, and 0.5% SDS) at 65°C for 1 h and subsequently hybridized for the 18S transcript using a rat cDNA probe (kindly provided by Dr. Michael Melner, Vanderbilt University), which shows greater than 90% sequence homology and cross-hybridizes with the mouse transcript. In all experiments, TIMP data were normalized to the relative expression of the 18S transcript for each of the study groups. Data were expressed as a ratio of each TIMP to 18S, and data are reported as a fold change in this ratio from the lowest level of expression for each TIMP. All data were digitized and quantitated using the GDS-8000 System (Ultra Violet Products, Upland, CA).

Extraction of Uterine Proteases and Assessment of Total Matrix Metalloproteinase Activity

To obtain total protein, uteri were pooled from two to six mice per PND of age for each genotype (separate pools from those used for RNA isolation). Uterine proteinases were extracted from pooled tissues (1:5, w/ v) by homogenizing uteri 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). Homogenized samples were then placed on ice for 5 min, followed by centrifugation at 12 000 x g for 30 min at 4°C. Supernatants were then removed, and an aliquot was subjected to the DC Protein Assay (Bio-Rad Laboratories, Richmond, CA) to determine protein concentration. The remaining sample was aliquoted and stored at –75°C until analyzed for protease activity.

Total matrix metalloproteinase activity was determined in uterine extracts as previously described, with minor modifications [9]. Briefly, uterine proteinase activity was determined by adding a volume that contained 100 µg of total protein into 0.5 ml of ACA buffer (0.05 M Tris, 0.01 M CaCl2, 0.2 M NaCl, 0.05% Brij 35, and 0.02% NaN3, pH 7.5). Samples were preincubated for 1 h at 37°C in the presence or absence of chelating agents known to inhibit MMP activity (5 mM EDTA or 5 mM 1,10-phenanthroline). After the preincubation period, an additional 0.5 ml of ACA buffer containing 2 mg/ml of the colorimetric substrate Azocoll (Calbiochem, Richmond, CA) was added to each sample (final Azocoll concentration = 1 mg/ml), and samples were incubated for 18 h at 37°C with continuous shaking. Protease activity (reflected as a breakdown of the Azocoll substrate and liberation of the colored dye into the buffer) was quantitated using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at a wavelength of 520 nm. Data were expressed as arbitrary units, which reflect the change in optical density (OD) units per milligram of protein compared with a blank buffer sample. Total MMP activity was calculated using the following formula: OD520 control – OD520 EDTA = MMP activity and was based on the rationale that control samples contain all proteases capable of cleaving substrate while those samples treated with EDTA contain all non-MMP proteases capable of cleaving substrate. As such, the amount of MMP activity could be obtained from the differences between these two treatments. For this report, data obtained from EDTA-treated samples were used, but 1,10-phenanthroline-treated samples gave similar results with respect to the proportion of total MMP activity in each sample.

Histological Assessment of Uterine Tissue

The middle two thirds of each right uterine horn were used for histological analysis (N = 6 mice/PND/genotype. Tissues were embedded in paraffin, serial sectioned at 5 µM, and stained with hematoxylin-eosin. Sections were analyzed for each uterine specimen to assess the structure/ morphology of the uterine lumen and the number of endometrium epithelial glands per cross-section. Endometrial gland number was determined by counting the total number of uterine glands in a complete cross-section of the uterine horn portion. The observation of a gland cross-section with a visible open lumen was counted as a single gland. The number of glands was counted on every 10th section (approximately 50-µm intervals) using a minimum of 10 different sections, and an overall average number of glands was calculated for each specimen. Histological assessment was conducted on tissue from N = 6 mice PND–1 genotype–1, three of which were used for RNA analysis and three of which were used for assessment of MMP activity.

Statistical Analysis

All data were analyzed using SPSS version 11.5 software (SPSS Inc., Chicago, IL). Data were analyzed across time points (PNDs 5–25) within genotype by one-way ANOVA. When an F-test indicated statistical significance, post hoc analysis was made using the Tukey procedure on data that displayed homogeneity of variance. Data that did not display homogeneity of variance were analyzed by the Games-Howell procedure. Unpaired t-tests were used for planned comparisons between genotypes within each postnatal day of age. Significance was set at P < 0.05 for all comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Uterine wet weights increased with postnatal age in mice of both genotypes (Fig. 1). Compared with wild-type mice, significantly greater uterine wet weights were detected in TIMP-1 null mice at PNDs 5, 10, and 25 (Fig. 1). Gross uterine morphology was similar between genotypes across PNDs 5–25, with the exception that TIMP-1 null mice had a significantly greater number of endometrial glands at PNDs 15 and 20 compared with wild-type mice (Fig. 2). More specifically, there was an approximately twofold increase in the number of glands per uterine cross-section in the null mice at PNDs 15 and 20 (Figs. 2 and 3), but by PND 25, the number of glands were similar in mice of both genotypes.



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  		FIG. 1. Uterine wet weights during postnatal uterine development in TIMP-1 wild-type and null mice. Mice of both genotypes were sacrificed at PNDs 5, 10, 15, 20, and 25, and uterine wet weights were determined. Data are displayed as the mean ± standard error of the mean (SEM). Data points were obtained from 50 PND 5, 30 PND 10, 20 PND 15, 15 PND 20, and 15 PND 25 mice of each genotype. Different letters indicate statistical significance (P < 0.05) among PND of age as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice while bold letters indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within PND of age by planned comparisons using unpaired t-test. For all statistical analysis, P < 0.05 was considered statistically significant



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  		FIG. 2. Number of endometrial glands during postnatal uterine development in TIMP-1 wild-type and null mice. The number of endometrial glands per cross-section were calculated as described in Materials and Methods and were compiled from 10 different cross-sections per specimen with PND of age/genotype, N = 6. Different letters indicate statistical significance (P < 0.05) among PND of age as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice while bold letters indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within PND of age by planned comparisons using unpaired t-test (* = P < 0.005; ** = P < 0.01)



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  		FIG. 3. Histological assessment of endometrial gland density at PND 15 in TIMP-1 wild-type and null mice. Uterine tissue was processed and morphology was assessed as described in Materials and Methods. A representative photomicrograph of uterine cross-sections from PND 15 wild-type (+/+) and TIMP-1 null (–/–) mice is shown. Arrows indicate endometrial glands and magnification is x220. Similar histological results were obtained from PND 20 specimens (increased gland number; data not shown; N = 6 specimens/PND of age/genotype

To begin to examine the mechanisms that may contribute to this altered rate of adenogenesis, we examined uterine TIMP expression in mice of both genotypes. In wild-type mice, TIMP-1 steady-state mRNA expression was greatest at PND 5 and decreased between PNDs 10 and 15 and then again between PNDs 20 and 25 (Fig. 4). As expected, TIMP-1 transcript was not detected in TIMP-1 null mice (Fig. 4). TIMP-2 transcript was detected in uteri of mice of both genotypes across all days of postnatal development (Fig. 5). In wild-type mice, neither the 3.5- nor the 1.0-kilobase (kb) transcripts of TIMP-2 showed significant changes across PNDs 5–25 (Fig. 5). In contrast, the steady-state levels of the 3.5-kb transcript of TIMP-2 significantly increased between PNDs 10 and 15 in TIMP-1 null mice (Fig. 5). The most striking difference in TIMP-2 steady-state mRNA levels was detected between genotypes within PND 10, as TIMP-1 null mice expressed significantly lower TIMP-2 transcript expression of both the 3.5 and kb transcripts compared with wild-type mice (Fig. 5).



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  		FIG. 4. Expression of steady-state levels of TIMP-1 mRNA during postnatal uterine development in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for TIMP-1 expression as described in Materials and Methods using 20 µg of total RNA/lane. TIMP-1 mRNA was detected as a single transcript of approximately 0.9 kb, consistent with previous reports. TIMP-1 mRNA expression is expressed as the fold change in the mean ratio of TIMP-1/18S transcript from PND 25 wild-type values, which was set at 1.0. Data are representative of four separate observations (N = 4 tissue pools/treatment group) and are reported as the fold change in ratio ± SEM. Autoradiographic exposures for TIMP-1 were for 24 h at –75°C, while that of 18S rRNA was for 1 h at –75°C. Different letters indicate statistical significance (P < 0.05) among PND of age as determined by one-way ANOVA



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  		FIG. 5. Expression of steady-state levels of TIMP-2 mRNA during postnatal uterine development in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for TIMP-2 expression as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-2 mRNA was detected as two transcripts of approximately 3.5 kb (upper band) and 1.0 kb (lower band), which is consistent with previous reports. TIMP-2 mRNA expression is expressed as the fold change in the mean ratio of TIMP-2/18S transcript from PND 5 null values, which was set at 1.0 for each transcript, and both transcripts were analyzed separately. Data are representative of four separate observations (N = 4 tissue pools/treatment group) and are reported as the fold change in ratio ± SEM. Autoradiographic exposures for TIMP-2 were for 24 h at –75°C, while that of 18S rRNA was for 1 h at –75°C. Different letters indicate statistical significance among different PND of age as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice while bold letters indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within PND of age by planned comparisons using unpaired t-tests

Examination of uterine TIMP-3 steady-state mRNA levels revealed a similar pattern to that of TIMP-2. In wild-type mice, uterine TIMP-3 steady-state mRNA levels did not significantly change across PNDs 5–25. In contrast, TIMP-3 steady-state mRNA levels were significantly greater in the null mice between PNDs 15, 20, and 25 compared with PND 5 levels (Fig. 6). Comparison within PND between genotypes revealed that TIMP-1 null mice expressed significantly lower levels of TIMP-3 steady-state mRNA levels at PND 10 (Fig. 6). Last, TIMP-4 transcript expression was not detected using Northern analysis, and this observation is in accord with our previous findings conducted in uteri of reproductive-age intact and ovariectomized, steroid-reconstituted mice [8].



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  		FIG. 6. Expression of steady-state levels of TIMP-3 mRNA during postnatal uterine development in TIMP-1 wild-type and null mice. Total mRNA was extracted and subsequently analyzed for TIMP-3 expression as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-3 mRNA was detected as a major transcript of approximately 4.5 kb, which is consistent with previous reports. TIMP-3 mRNA expression is expressed as the fold change in the mean ratio of TIMP-3/18S transcript from PND 5 null values, which was set at 1.0. Data are representative of four separate observations (N = 4 tissue pools/treatment group) and are reported as the fold change in ratio ± SEM. Autoradiographic exposures for TIMP-3 were for 8–12 h at –75°C, while that of 18S rRNA was for 1 h at –75°C. Different letters indicate statistical significance among PND of age as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice while bold letters indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within PND of age by planned comparisons using unpaired t-tests

To begin to examine if disruption of the TIMP-1 gene product was associated with increases in uterine matrix metalloproteinase (MMP) activity that might influence endometrial gland density, total MMP activity was assessed. MMP activity was lowest in PND 5 wild-type samples, and MMP activity for all other samples was expressed as a percentage of this amount of activity. In wild-type mice, total MMP activity increased from PND 10 and reached statistically significant greater levels at PND 15 (Fig. 7; P < 0.05 PND 5 vs. PND 15). MMP activity then decreased at PNDs 20 and 25 (Fig. 7). In TIMP-1 null mice, a similar pattern of total MMP activity was detected, but compared with wild-type counterparts, total MMP activity was greater in the null mice (Fig. 7). Compared with PND 5, MMP activity was significantly (P < 0.05) greater in the null mice at PNDs 10, 15, and 20 (Fig. 7). Comparison between genotypes within PND of age revealed that TIMP-1 null mice had significantly greater total MMP activity at PNDs 10, 15, and 20 (Fig. 7), a time that just preceded and included the time period of increased gland density in the null mice (Fig. 2). Collectively, these data demonstrate, in TIMP-1 null mice, increased uterine total MMP activity is associated with the increase in endometrial gland density.



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  		FIG. 7. Uterine total MMP activity in TIMP-1 wild-type and null mice during postnatal uterine development. Total MMP activity was determined as described in Materials and Methods and data are expressed as percent change from PND 5 wild-type values ± SEM for four separate experiments (N = 4 tissue pools/genotype/PND of age. Different letters indicate statistical significance among the PNDs as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice while bold letters indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within PND by planned comparisons using unpaired t-tests


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of proper endometrial gland formation and function is evident in a variety of species, as endometrial gland secretions are critical for establishment of early pregnancy (reviewed in [1]). Despite the well-characterized physiology and overall necessity of these glands in the reproductive process, little information exists on the mechanisms by which they develop. It is evident that, for adenogenesis to occur, tissue remodeling that allows for the budding and branching of the luminal epithelium into the surrounding stroma must occur. Despite a general understanding of this process, virtually no information exists on the proteases and their inhibitors that dictate the events necessary for adenogenesis to occur. Because MMPs and TIMPs are key tissue remodeling factors required in the branching morphogenesis of various organs [12–14], it would not be surprising that this tissue remodeling system may also play a role within the uterus during similar events associated with uterine development. It is evident that MMPs and TIMPs play an important role within the uterus of a number of species (reviewed in [7]). Anatomical support for this has previously been described by our laboratory using TIMP-1-deficient mice [8]. In this study, it was demonstrated that TIMP-1 dictates the uterine histoarchitecture within the uterus of reproductively mature female mice. Thus, a similar role of not only TIMP-1, but other TIMP family members as well, may be required for endometrial gland formation during the postnatal developmental period. This postulate of a functional role for TIMP-1 during uterine adenogenesis is supported by the observation in the current study that, in the absence of TIMP-1, endometrial gland density is increased. The mechanism for this increase may be associated with an imbalance in the TIMP: MMP ratio that favors net MMP activity. In mice of both genotypes, total MMP activity increased from PND 5 to PND 20 and coincided with the period of gland formation and development. The observation that total MMP activity is elevated in TIMP-1 null mice and that these mice have an increased number of endometrial glands suggests an active role for MMPs in uterine adenogenesis. MMPs may play an active role in directing the tissue remodeling necessary for adenogenesis to occur and TIMP-1 might dictate the extent to which this occurs. The exact MMPs involved and their cellular origin (stromal, epithelial, or both) are currently under investigation and may include epithelial cell-derived MMPs such as MMP-7 [8, 15, 16] and MMP-26 [17, 18].

In addition to TIMP-1 regulation of MMP activity, we also observed significantly lower levels of TIMP-2 and TIMP-3 steady-state mRNA levels in the TIMP-1 null mice. This is an interesting observation in that one might anticipate a compensatory increase in the expression of other TIMP family members to overcome the TIMP-1 deficiency, yet a decrease in levels were detected. It is also plausible that these lower levels of TIMP-2 and TIMP-3 may also contribute to the increased gland density in the TIMP-1 null mice as these lower levels of MMP inhibitory action/increased MMP activity precede the increase in uterine gland numbers/rate of gland formation. Like TIMP-1, TIMP-2 and TIMP-3 can regulate many of the MMPs expressed within the uterus (reviewed in [7]) and these TIMPs may also functionally contribute to regulating the tissue remodeling that occurs during the process of adenogenesis. While we did not assess TIMP activity in the current report, we did assess total MMP activity. We elected to assess MMP activity to determine the net effect of disruption of the TIMP-1 gene product on the overall balance of the MMP: TIMP ratio. From this study, it was determined that, in the wild-type mice, net MMP activity is highest during the period of gland development and loss of the TIMP-1 gene product is associated with a further increase toward net MMP activity.

Another novel aspect of the current study was the characterization of TIMP expression during the period of postnatal uterine development and the reported differences in the pattern of mRNA steady-state levels between genotypes. In wild-type mice, TIMP-2 (both the 3.5- and 1.0-kb transcripts) and TIMP-3 steady-state mRNA levels were unchanged during the time points examined. In contrast, both TIMP-2 and TIMP-3 steady-state mRNA levels were lower in the TIMP-1 null mice at PND 10. This observation may suggest that TIMP-1 either directly or indirectly controls expression of these other TIMP family members. Possible mechanisms for this regulation by TIMP-1 may be MMP-dependent or MMP-independent. It is well established that MMPs can regulate growth factor/cytokine signaling pathways, which in turn may influence TIMP-2 and/or TIMP-3 expression [19–21]. If this mechanism is a MMP-dependent process, elucidation of those MMPs whose activity is increased in the null mice will be the first step in the dissection of the pathway responsible for the reduction in uterine TIMP-2 and TIMP-3 expression in the TIMP-1 deficient mice.

In summary, this is the first report, to our knowledge, to describe uterine expression of the TIMPs during the period of adenogenesis and assess the role of TIMP-1 in this process. The results from the current study indicate that, in the absence of TIMP-1, endometrial gland density is increased and this is associated with decreases in steady-state mRNA levels of TIMP-2 and TIMP-3 as well as an increase in total MMP activity within the uterus. It can be concluded from this study that TIMP-1 regulates uterine MMP activity, which in turn dictates the rate of endometrial gland formation and gland density. Identification and cellular localization of those MMPs involved in the process of adenogenesis will provide additional insight into the complex mechanism that allows for endometrial gland formation.


   FOOTNOTES
 
1 Supported by NIH grant award HD39765 to W.B.N. Back

2 Correspondence: Warren B. Nothnick, University of Kansas Medical Center, Department of Obstetrics and Gynecology, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 6271; wnothnic{at}kumc.edu Back

Received: 3 March 2004.

First decision: 17 March 2004.

Accepted: 30 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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