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Regular Article |
a University of Kansas Medical Center, Department of Obstetrics and Gynecology and Molecular and Integrative Physiology, Kansas City, Kansas 66160
ABSTRACT
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.
female reproductive tract, uterus
INTRODUCTION
The primary function of the uterus is to house and nurture the developing fetus. Throughout the reproductive cycle, the uterus undergoes dynamic cellular changes that enhance its ability to support an implanted conceptus. These changes involve active tissue remodeling as well as periods of cell proliferation, arrest, cell death, and differentiation. It is well established that the extent of tissue remodeling that occurs is controlled by a balance between proteolytic factors (such as matrix metalloproteinases or MMPs) and their inhibitors (such as tissue inhibitors of metalloproteinases or TIMPs) [1, 2]. To date, four distinct TIMPs have been identified; TIMP-1, TIMP-2, and TIMP-4 that are secreted proteins, and TIMP-3 that is bound to the extracellular matrix.
The patterns of expression and regulation for both uterine MMPs and TIMPs are well characterized in many species. Numerous studies have elegantly mapped the pattern of expression of TIMPs in the cycling endometrium of humans [3–6] and nonhuman primates [7] as well as in sheep [8, 9]. In addition, studies have examined uterine TIMP expression during the pre- to peri-implantation period in human [10], mouse [11–14], pig [15], and sheep [8, 9] in an attempt to understand the mechanisms by which uterine TIMPs may act in a coordinated fashion with embryo/trophoblast-derived MMPs to allow successful implantation.
Uterine TIMP expression appears to be influenced by the steroidal milieu. In the sheep, cyclic uterine expression of TIMP-1 appears to be downregulated by estradiol-17ß, while that of TIMP-2 may be upregulated by progesterone [8]. In the human, TIMP-3 expression is upregulated by progesterone in endometrial stromal cells [3, 5]. In both species it appears that stromal cells express the majority of TIMP-1, TIMP-2, and TIMP-3, with epithelial cells expressing variant levels of TIMP-1 and TIMP-2 [3, 5, 6, 8]. Detailed analysis of TIMP-4 expression, regulation, and localization is, to our knowledge, unexplored in uterine tissue of any species. Leco and colleagues [16] were unable to detect TIMP-4 transcript expression in mouse uterine tissue; however, it is unclear from this study the reproductive cycle stage from which these uteri were obtained.
The role of TIMPs in uterine physiology is uncertain, but due to the fact that these proteins are multifunctional, TIMPs may regulate a variety of physiologic events within the uterus. The TIMPs were first identified based upon their ability to regulate MMP activity. However, recent evidence suggests that TIMPs can act as growth factors and antiangiogenic agents as well as modulators of steroidogenesis and apoptosis. Both TIMP-1 and TIMP-2 exert erythroid-potentiating activities that stimulate the growth of erythroid precursors in vitro [17, 18] and in vivo [19]. In addition, TIMP-1 and TIMP-2 have been shown to be serum-derived growth factors for a wide array of cell types in vitro [20, 21] as well as a growth promoter of cultured human keratinocytes [22]. Furthermore, TIMP-1 and TIMP-2 exhibit antiangiogenic activity in vitro [23, 24], while TIMP-1 has also been suggested as a modulator of steroidogenesis [25, 26].
Like TIMP-1 and TIMP-2, TIMP-3 has been reported to exhibit growth-promoting activity [27]. In contrast, recent evidence indicates that TIMP-3 may exert antiproliferative or apoptotic effects [28–30]. Taken together, it appears that TIMPs are capable of eliciting a wide array of functions that might suggest that the role of TIMPs in uterine physiology is multifaceted. Despite a thorough knowledge of the expression and regulation of uterine TIMPs in many species, there is little to no in vivo information on the function of TIMPs in uterine physiology. To begin to examine the role of TIMPs in uterine physiology in vivo, reproductive cyclicity, systemic hormone levels, and uterine morphology were assessed using TIMP-1-deficient and wild-type mice.
MATERIALS AND METHODS
Animals
Wild-type and TIMP-1 null mice were utilized for all studies. 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 level by Northern analysis and protease inhibitor assays, respectively [25].
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. Eight- to 12-wk-old female mice of both genotypes were monitored for reproductive cyclicity by daily vaginal lavage at 0800 h for a period of at least 14 consecutive days. Animals were sacrificed in each of the four stages of the estrous cycle.
Ten to 20 animals of each genotype were monitored in each trial, and four separate trials were performed over the course of a 6-mo period (n = 45 wild-type and n = 75 TIMP-1 null mice monitored in all, over the 6-mo period). All animals were sacrificed by decapitation. Trunk blood was collected, and serum was obtained and stored at -70°C until analyzed for steroid content. Uteri were removed, trimmed of fat and connexion, weighed, and either snap-frozen in liquid nitrogen until utilized for RNA extraction (left uterine horn) or prepared for histological assessment (right uterine horn). The duration spent in each stage of the cycle (expressed as days) was calculated by determining the average duration of each stage of the cycle using the last two consecutive cycles. Mice of either genotype that displayed constant diestrus or estrus were excluded from analysis.
Establishment of the Estrous Cycle Stages in Wild-Type Mice
Because there can be a great degree of variability in the cellular composition of vaginal lavages across the estrous cycle between as well as within animals from cycle to cycle, the first goal was to establish a normal pattern of vaginal cytology for each of the stages of the estrous cycle in the TIMP-1 wild-type mice. To accomplish this goal, 2–3-mo-old female wild-type mice (n = 20) were monitored daily by vaginal lavage for 2 wk, and the cytology of the smears was recorded. Four months later, this experiment was repeated with 20 separate 2- to 3-mo-old wild-type females. All 40 observations were then pooled, and an average cellular composition of rounded epithelial, cornified epithelial, and leukocytes were determined for each stage (proestrus, estrus, metestrus, and diestrus). Stages of the estrous cycle were then defined based upon the following criteria:
Proestrus was defined as a vaginal smear that contained primarily (>80%) rounded epithelial cells (RE) with or without some (0–20%) cornified epithelial cells (CE) and/or leukocytes/white blood cells (WBC; 
5%). Estrus was defined as a smear that contained primarily (>80%) cornified epithelial cells with or without rounded epithelial cells (0–20%). A metestrus smear was considered one that contained cornified epithelial cells and leukocytes (WBC 
CE or CE > WBC), while a diestrus smear was characterized as containing predominantly (>80%) leukocytes with some rounded (0–20%) or cornified epithelial cells (>5% CE).
RNA Isolation and Northern Analysis
Total RNA was isolated from the left uterine horn of each animal by separately homogenizing the tissue in 1 ml of TRIZOL reagent (Life Technologies/GIBCO-BRL, Gaithersburg, MD) per 100 mg of tissue wet weight. The RNA was then extracted with chloroform and precipitated with isopropyl alcohol according to the recommendations of the manufacturer. Total RNA samples (10 or 20 µg/lane) 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, TIMP-2, TIMP-3, and TIMP-4 cDNA probes (kindly provided by Dr. Dylan Edwards, University of East Anglia) were excised from their respective plasmids with the appropriate restriction endonucleases [25], and the resulting inserts were labeled using a random primer kit (Life Technologies/GIBCO-BRL). Probes were labeled to a specific activity of 5 x 108 to 1 x 109 dpm/µg of DNA using [
-32P]dCTP (NEN-DuPont, Boston, MA). Filters were hybridized overnight, washed, and exposed to Kodak-XAR-5 film for 24 h at -75°C. Hybridization signals were allowed to decay and filters were subsequently hybridized for the 18S transcript using a rat cDNA probe (kindly provided by Dr. Michael Melner, Vanderbilt University) that cross-hybridizes with the mouse transcript. Membranes were then exposed to Kodak XAR-5 film for 1 h at -75°C. In all experiments, TIMP data were normalized to the relative expression of the 18S transcript for each of the study groups.
Assessment of Serum Steroid Content
Serum progesterone and estradiol were quantitated as previously described [25] with the exception that a double antibody assay (Diagnostic Products Corp., Los Angeles, CA) was used for estradiol determinations. Samples (n = 6–9/genotype/stage of cycle) were analyzed singly or in duplicate (50–100 µl) when sufficient serum was available for the latter. The detection limits of the assays were approximately 0.02 ng/ml (progesterone) and 1.4 pg/ml (estradiol). The inter- and intraassay coefficients of variation for the progesterone assays were 7.9% and 4.6%, respectively, and the inter- and intraassay coefficients of variation for estradiol were 8.0% and 6.0%, respectively.
Histological Assessment of Uterine Tissue
The middle three fourths of the right uterine horn was removed from animals of both genotype in all stages of the estrous cycle (n = 4/genotype/stage of cycle). Tissues were fixed in toto in 10% neutral buffered formalin. Tissues were embedded in paraffin, serially sectioned at 5 µM, and counterstained with hematoxylin and eosin as previously described for ovarian tissue [25]. Every 10th section (approximately 50-µm intervals) was analyzed for each uterine specimen to assess the structure/morphology of the uterine lumen, height of the luminal epithelial cells, the number of endometrial epithelial glands, and the diameter of each gland. Height of luminal epithelial cells was assessed with the aid of an ocular micrometer, while the diameter of each epithelial gland was determined using the mean of two right angle determinations through the gland. Average height of luminal epithelium was assessed by measuring the luminal epithelial at five random locations on each section and then taking the average height/section. The number of epithelial glands was counted on every 10th section (approximately 50-µm intervals), and an overall average number of glands was calculated. For each gland counted, the diameter was also determined. An overall average was then obtained for each parameter for uterine sections of each stage of the cycle for mice of both genotypes.
Statistical Analysis
All data were analyzed across stage of the estrus cycle by one-way ANOVA. When an F-test indicated statistical significance, posthoc analysis was made using the Student-Newman-Keuls procedure. Planned comparisons between genotypes within each stage of the estrous cycle were made using unpaired t-tests. Significance was set at P < 0.05 for all comparisons.
RESULTS
The TIMP-1 Null Mice Exhibit Altered Reproductive Cyclicity
Disruption of the TIMP-1 gene product was associated with an alteration in the normal reproductive cycle of TIMP-1 null mice. The majority (82.2% ± 2.0%; or 37/45 mice) of wild-type female mice exhibited normal 4- to 5-day estrous cycles, with 5 of the 8 (62.5% ± 31.0%) and 3 of the 8 (37.5% ± 25.0%) of the noncycling wild-type mice exhibiting vaginal smears indicative of constant diestrus and estrus, respectively. Compared to wild-type counterparts, significantly fewer (P < 0.001) TIMP-1-deficient mice (41.3% ± 4.2%; or 31/75 mice) exhibited normal cycles (Fig. 1A). Of those TIMP-1 null mice that did not cycle (44 mice total), three types of abnormal vaginal cytology/smears were detected. Constant diestrus (22.7% ± 2.4%; or 10/44 mice) and estrus smears (13.6% ± 3.6%; or 6/44 mice) were detected in a fraction of the null mice that did not cycle. The majority (63.6% ± 3.0%; or 28/44) of those TIMP-1 null mice that did not exhibit normal cycles failed to display a period of estrus as determined by vaginal cytology. More specifically, the rounded epithelial cells characteristic of the period of proestrus dominated until the influx of white blood cells occurred during the period of metestrus. Very few (<5%) if any cornified epithelial cells indicative of estrus were ever detected in the vaginal lavages of those noncycling TIMP-1 null mice. The vaginal cytology of those null mice that did exhibit a period of estrus consisted of primarily rounded epithelial cells with some degree of cornified epithelial cells present (ranging from approximately 25 to 100% cornified epithelial cells present). To assess quantitatively the duration of each stage of the estrous cycle, length of each cycle stage was compared between genotypes. The TIMP-1 null mice exhibited significantly longer periods of proestrus (1.5 ± 0.15 days vs. 0.9 ± 0.2 days) and shorter periods of estrus (0.6 ± 0.2 days vs. 1.1 ± 0.25 days) compared to wild-type counterparts (Fig. 1B). No significant differences in the duration of either metestrus or diestrus were noted between genotypes (Fig. 1B).
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The TIMP-1 Null Mice Have Altered Uterine Morphology
Of those TIMP-1 null mice that did exhibit normal 4- to 5-day cycles, there was an alteration in uterine wet weight gain during the course of the estrous cycle in these mice. Specifically, uteri obtained from null mice during the estrus stage of the estrous cycle weighted significantly (P < 0.05) more compared to their wild-type counterparts (Fig. 2). Histological assessment of uteri of TIMP-1 null mice across the estrous cycle revealed dramatic changes in the structure of the uterine lumen. These changes were detected during all stages of the cycle but were most pronounced during the period of estrus and were characterized by precocious luminal folds that exhibited profound branching often extending to and following the periphery of the circular smooth muscle (indicated by arrows in Fig. 3, B and F). Furthermore, the uterine lumen could not be clearly identified in the uteri of the TIMP-1 null mice during the period of estrus (compare Fig. 3F to 3E). Also during the period of estrus, the stroma appeared more edematous in TIMP-1 null mice, which may have contributed to the increase in uterine wet weight detected during this stage of the estrous cycle. Lastly, no significant differences were detected during any stage of the estrous cycle between genotypes in either the number of endometrial glands/section, the average diameter of endometrial glands or height of the luminal epithelial cells (data not shown).
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The TIMP-1 Null Mice Have Reduced Serum Steroid Content
As TIMP-1 has been reported to be a steroidogenic factor and to ascertain if the differences in uterine wet weight, morphology, and/or vaginal cytology could be due to alterations in endogenous steroid levels, serum steroid concentrations were assessed in mice of both genotypes. Systemic progesterone concentrations did not differ between genotype across the estrous cycle, except during the period of estrus. During estrus, TIMP-1 null mice had significantly lower serum progesterone levels (Fig. 4A) compared to wild-type counterparts. Coupled with the decrease in serum progesterone levels was a significant (P < 0.05) increase in serum estradiol concentrations during the period of estrus in TIMP-1 null mice (Fig. 4B). A significant increase in serum estradiol was also detected in the TIMP-1 null mice during the period of diestrus (Fig. 4B). The finding that during the period of estrus there is a significant decrease in serum progesterone coupled with a significant increase in estradiol levels may aid in explaining the increase in uterine wet weight in the null mice during the period of estrus as well as the altered uterine morphology and vaginal cytology.
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Disruption of the TIMP-1 Gene Product Is Associated with Reduced Uterine TIMP-3 Expression
To verify that disruption of the TIMP-1 gene did not induce compensatory increases in expression of other TIMP family members and to characterize the pattern of uterine TIMP expression during the murine estrous cycle, Northern analysis for TIMP-1, TIMP-2, TIMP-3 and TIMP-4 was performed. In wild-type mice, TIMP-1 transcript expression was lowest during diestrus, significantly increased during the periods of proestrus and estrus, and then declined again during metestrus to levels similar to those detected during diestrus (Fig. 5). As expected, TIMP-1 transcript was not detected in uterine tissue of TIMP-1 null mice (data not shown). In contrast to TIMP-1, TIMP-2 displayed relatively constant levels of expression across the estrous cycle showing little change in expression of either the 3.5- or 1.0-kilobase (kb) transcripts (data not shown). No significant differences in TIMP-2 mRNA expression (3.5- or 1.0-kb transcripts) were noted between genotypes during any stage of the cycle (data not shown). The TIMP-3 mRNA was the most abundant of the uterine TIMPs. In the wild-type mice, uterine TIMP-3 expression was lowest during the period of diestrus and increased during the periods of proestrus through metestrus (Fig. 6). In the TIMP-1 null mice, TIMP-3 expression was significantly (P < 0.01) reduced during the periods of estrus and metestrus but increased during diestrus compared to their wild-type counterparts (Fig. 6). A similar decrease in uterine TIMP-3 mRNA expression was also detected in those null mice that did not show a normal period of estrus (data not shown). These animals were sacrificed in what appeared to be the metestrus stage of the cycle (RE WBC) and displayed similar levels of TIMP-3 mRNA expression compared to normally cycling TIMP-1 null mice in the period of metestrus, i.e., significantly lower TIMP-3 mRNA expression. Uterine TIMP-4 mRNA expression was not detectable by Northern analysis using up to 20 µg of total RNA per lane during any stage of the estrous cycle in mice of either genotype (data not shown).
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DISCUSSION
Tissue inhibitor of metalloproteinase-1 is a multifunctional protein [1] capable of exerting growth factor [18–22], antiangiogenic [23, 24], and steroidogenic effects [25, 26]. The majority of these functions of TIMP-1 have been determined using in vitro methodologies, with little to no in vivo data to support these potential functions. In the current study, TIMP-1 null mice incapable of expressing the TIMP-1 gene product were used to assess the effect of this protein on the uterine events that occur during the course of the murine reproductive cycle; an event characterized by marked periods of cellular proliferation, differentiation, and angiogenesis. The findings from the current study lend the first in vivo supportive evidence for a role of TIMP-1 in modulating uterine events that occur during the reproductive cycle.
In the current study, it was demonstrated that uterine TIMP-1 and TIMP-3 mRNA expression fluctuates during the reproductive cycle, TIMP-2 mRNA expression remains constant, and TIMP-4 mRNA expression is expressed at undetectable (by Northern analysis) levels. These findings are in accord with those of others that have demonstrated that uterine TIMP expression in humans [3–6], monkeys [7], and sheep [8] exhibit specific patterns of expression during the course of the reproductive cycle. Based upon the detected pattern of uterine expression in the mouse (current study), it could be implied that murine uterine TIMP-1 and TIMP-3 expression may be under steroidal control similar to uterine TIMP expression in these other species. Murine uterine TIMP-1 mRNA expression is expressed throughout the reproductive cycle and peaks during the stages of proestrus and estrus, while TIMP-3 mRNA is expressed during the course of the reproductive cycle. As both TIMP-1 and TIMP-3 are capable of controlling cell proliferation, differentiation, and cell death [1, 28–30], these TIMPs may play a role in controlling these cellular events that occur during the course of the reproductive cycle.
Disruption of the TIMP-1 gene was associated with an altered uterine reproductive cycle and uterine phenotype. The uteri of TIMP-1 null mice were characterized by profound branching of the uterine lumen that appeared to invade the uterine stroma in an uncontrolled fashion. This abnormal lumen structure was most evident during the estrus stage of the estrous cycle. Potential factors that might contribute to this aberrant uterine morphogenesis may be due to perturbations in mechanisms resulting from deletion of the TIMP-1 gene product. It is well established that estradiol influences uterine epithelial cell proliferation [31]. In the current study the significantly higher serum estradiol levels, coupled with the significantly lower progesterone levels during the period of estrus in the TIMP-1 null mice, may have altered luminal epithelial cell growth/proliferation, which in turn could contribute to the abnormal structure of the uterine lumen detected in the null mice. Alternatively and/or equally plausible, the integrity of the uterine extracellular matrix (ECM) that surrounds the luminal epithelial cells may be compromised. Tissue inhibitor of metalloproteinase-1 regulates ECM integrity by controlling proteases capable of degrading the components that compose the ECM [1, 2]. Although not determined in the current study, the absence of TIMP-1 may be associated with an increase in uterine protease activity during the period of estrus. The uterus expresses several MMPs whose expression peaks during the period of estrus [32, 33]. Left unopposed, MMPs could conceivably breakdown the uterine ECM. A weakened ECM would provide less resistance to proliferating cells allowing for the invasion of the stroma by the luminal epithelial cells in an uncontrolled fashion.
In addition to providing structural support, the ECM also influences the development, migration, proliferation, shape, and metabolic function of those cells that come in contact with it [34, 35]. Upon ECM degradation/turnover, protein-bound growth factors may be released that may directly impact cell proliferation. Tissue inhibitor of metalloproteinase-1 can regulate the bioavailability of growth factors via protease-dependent mechanisms [36–40] and subsequent cell/tissue proliferation [41, 42]. Thus, it is tempting to speculate that the absence of TIMP-1 in the TIMP-1 null mice may contribute to an increase in bioavailability of uterine growth factors that could increase uterine cell proliferation resulting in the detected uterine phenotype.
Disruption of the TIMP-1 gene was also associated with a significant reduction in uterine TIMP-3 expression during the period of estrus. Coupled with the absence of TIMP-1 in the null mice, reduction in activity of both of these TIMPs could further increase the susceptibility of the uterine stroma to penetration by the luminal epithelial cells. It is of interest that this alteration in TIMP-3 expression occurred only during the periods of estrus and metestrus and not during other stages of the estrous cycle. This finding may imply that some factor or factors specific to the estrus/metestrus stage of the reproductive cycle may be responsible for this altered expression. A progesterone surge occurs just prior to the period of estrus. Progesterone is a known regulator of uterine TIMP-3 expression [5]. In the current study, progesterone levels were significantly lower during the period of estrus in the TIMP-1 null mice that coincided with the period of reduced uterine TIMP-3 expression. Thus, the reduced levels of serum progesterone may be at least one mechanism that might contribute to the reduced levels of TIMP-3 mRNA expression.
Tissue inhibitor of metalloproteinase-1 has been reported to be a steroidogenic factor in vitro [25, 26]. Previous in vivo studies have examined the effect of TIMP-1 deletion on the ovarian events that occur during the periovulatory period and found no significant changes in either systemic estradiol or progesterone content [25]. These findings are in conflict with those obtained in the current study that demonstrate significant alterations in the levels of estradiol and progesterone during the course of the murine reproductive cycle. While both of these studies used the TIMP-1 null mice as a model, the study by Nothnick and colleagues [25] used an immature, gonadotropin-primed model compared to mature cycling female mice (current study). It is plausible that the gonadotropins may have overridden potential differences in the mechanisms responsible for steroid production by superstimulating the system. The current study provides in vivo evidence that TIMP-1 regulates steroidogenesis under a more physiological milieu. Clearly, further studies are needed that incorporate a combination of in vitro and in vivo experimental approaches to ascertain the true role of TIMP-1 in ovarian steroidogenesis.
The impact of the altered reproductive cyclicity and abnormal uterine morphology upon gestation or fecundity has not been quantitatively assessed. We do know that less than 50% of normally cycling TIMP-1 null mice ever produce offspring compared to approximately 70% to 75% of cycling wild-type mice when exposed to proven males (Nothnick, unpublished observation). Tissue inhibitor of metalloproteinase-1 has been postulated to play a role in embryo maturation [43], while a coordinated action between MMPs and TIMPs (primarily TIMP-3) has been implicated to play a pivotal role in the implantation process [8–15]. Thus, the absence of TIMP-1 could impact several steps in embryo development and implantation that may influence the ability to achieve a successful pregnancy.
In summary, the current study provides the first in vivo evidence that TIMP-1 plays a role in the uterine events that occur during the murine reproductive cycle. Disruption of the TIMP-1 gene was associated with abnormal morphogenesis of the uterine lumen and reduced uterine TIMP-3 expression as well as with altered systemic steroid levels. The mechanisms by which TIMP-1 induces this uterine phenotype may be multifaceted and could include alterations in the physical resistance imposed by the ECM, increased bioavailability of growth factors, altered ovarian steroid production, or a combination of these mechanisms acting in synergy.
FOOTNOTES
First decision: 17 April 2000.
1 This research was supported by a grant award (HD37941) from the Office of Research on Women's Health in conjunction with the NICHD and core support from NICHD center grant HD33994. 
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
Accepted: May 2, 2000.
Received: March 29, 2000.
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