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Female Reproductive Tract |
Uterine Biology Laboratory,3 Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
Department of Physiology & Pharmacology,4 The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, female reproductive tract, menstrual cycle, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many of the molecular and cellular events of menstruation have now been identified [2, 3]. Leukocyte numbers increase dramatically immediately premenstrually and can contribute as much as 40% of the total cellular composition of the tissue at this time [4]. Other events include actions of matrix metalloproteinases (MMPs) to degrade the tissue matrix, of vasoactive substances such as prostaglandins, endothelin, and nitric oxide, and of products of a variety of leukocytes. However, as menstruation occurs naturally only in women, some Old World primates, the elephant shrew (Elephantus myuras jamesoni) and the bat (Glossophaga soricina) [5], most of the data supporting roles for such factors, is either correlative or based on in vitro experiments using tissue explants or cell cultures. Old World primates are not widely available for experimentation. There is thus a clear need for a nonprimate animal model of menstruation, in which the sequence of events leading to menstruation can be established and which can also be used for functional studies to examine the relevance of individual molecules to the processes of menstruation and endometrial repair.
During the 1980s, Finn and Pope [5] developed a model of endometrial breakdown in the mouse, in which progesterone support was withdrawn from artificially decidualized endometrium, to mimic the fall in serum progesterone that occurs in women following luteal regression. Features that occur in the endometrium of women at the time of menstruation, including influx of leukocytes and tissue degeneration, were observed in this model. However, considerable temporal variation in events was observed and the model was not further used.
The purpose of the present study was to optimize this mouse model for menstruation in an inbred strain of mouse commonly used for genetic manipulation, to establish the temporal sequence of events of tissue breakdown and restoration, and to assess the numbers of leukocytes and extent of apoptosis during the phase of tissue breakdown. Progesterone was delivered via silastic implants, providing an opportunity to induce a rapid decline in serum progesterone levels and thus reduce variability in responses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female C57BL/6 mice of 8–12 wk of age were obtained from Monash University Animal Services. Mice were housed in standard conditions with food and water provided ad libitum and a constant light cycle of 12 h (lights-on from 0800 h to 2000 h). Ethics approval for this project was granted by the Monash University/Monash Medical Centre Animal Ethics Committee B.
Induction of the Mouse Model of Menstruation
All surgeries were performed under xylazine/ketamine-induced anesthesia. Mice were ovariectomized 7 days prior to the first of three daily subcutaneous injections of 100 ng 17ß-estradiol (Sigma Chemical Co., St. Louis, MO) in arachis oil at approximately 0900 h. After resting the mice for 3 days, progesterone (P) implants were inserted subcutaneously into the back of each mouse and 5 ng of 17ß-estradiol in arachis oil was injected subcutaneously at approximately 0900 h on that and the subsequent 2 days. The progesterone implants were prepared essentially as described by Milligan and Cohen [6] by filling silastic tubes (0.062 inches inner diameter; Dow Corning, Midland, MI) with progesterone and sealing the ends with polyethylene plugs, such that the functional length of each implant was 1 cm. However, crystalline P (Sigma) was used rather than a slurry of P in oil. Prior to use, the implants were incubated overnight at 37°C in phosphate buffered saline, pH 7.35 (PBS)/1% fetal calf serum (Trace Biosciences, Sydney, Australia). At approximately 1100 h on the day of the final 17ß-estradiol injection, 20 µl of sesame oil was injected into the lumen of the right uterine horn of each mouse to induce decidualization. The left horn remained untreated as a control. The P implants were removed 49 h later. Mice were killed at the time of implant removal (0 h) and 12, 16, 20, 24, 36, and 48 h thereafter (6–8 mice per time point) and the uteri were harvested for further analysis. This sequence of events is shown diagrammatically in Figure 1. Uteri were cleaned of fat and weighed. Any mouse in which the oil-treated horn had not decidualized (as evidenced by weight 
400% of the untreated contralateral horn; approximately 30% of animals) was excluded from the study. Some uterine tissue was fixed in phosphate buffered formalin overnight and processed to wax. Other tissue was snap frozen in liquid nitrogen. Blood was taken from the mice at the 0- and 12-h time points for estimation of serum progesterone concentrations.
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Progesterone Assay
The concentration of progesterone in serum was determined by the Biochemistry Unit, Southern Cross Pathology (Clayton, Australia) using a microparticle enzyme immunoassay (Abbott AxSYM System, Abbott Australasia Pty. Ltd., Doncaster, Victoria, Australia). Concentrations were expressed as nmol/L (mean ± SEM).
Histology
Uterine cross-sections of formalin-fixed tissue were deparaffinized and hydrated by processing sections through Histosol (Sigma) and a graded series of ethanol to distilled (d)H2O. The hydrated sections were stained with hematoxylin and eosin using standard staining procedures. Stained sections were dehydrated and mounted under coverslips using DPX mounting medium (BDH Laboratory Supplies, Poole, England).
Morphological Analysis of Murine Endometrium
Hematoxylin and eosin-stained sections of murine uterus were scored blind using an arbitrary scoring system, ranging from 0 to 4, such that 0 = no, 1 = minimal, 2 = moderate, 3 = extensive, 4 = profound signs of destruction of the target cellular structure (including loss of adherence between cells, rounding of nuclei, large interstitial spaces). Sections were scored blind, with at least five sections from different mice within each group analyzed. Semiquantitative analysis of the destruction of the decidualized stromal tissue and the luminal epithelium was performed.
Immunohistochemical Analysis of Leukocytes
Transverse sections of formalin-fixed, paraffin-embedded mouse uteri were deparaffinized in Histosol and hydrated via immersion in baths of absolute ethanol, 70% ethanol, and dH2O. They were then immersed in 0.1 M citrate buffer and heated for 5 min in a 700-W microwave set to medium. Once the slides had returned to room temperature, they were rinsed in dH2O and immersed in 0.6% H2O2 in dH2O for 30 min at room temperature. After further rinsing in dH2O, the sections were bathed in a blocking solution of Tris buffered saline (TBA; pH 7.5)/10% normal mouse serum for 30 min at room temperature, after which time the blocking solution was replaced with 100 µl of either rat anti-mouse CD45 (Cat. No. 553076; Pharmingen, BD Biosciences, Lexington, KY), 5 µg/ml in TBS/10% normal mouse serum, or an equivalent amount of nonspecific rat IgG for an incubation overnight at 4°C. Sections were washed thrice in TBS with an additional wash step of TBS/0.5% Tween 20 between the first and second washes in TBS. A secondary antibody solution consisting of biotinylated rabbit anti-rat Ig (DAKO [Australia] Pty. Ltd., Botany, NSW, Australia) in TBS/10% normal mouse serum was applied to each section for 1 h at room temperature, followed by four washes as above. The StrepABC kit and DAB solution (DAKO) were used in accordance with the manufacturer's specifications to reveal the CD45 staining. Sections were lightly counterstained with Harris hematoxylin (Accustain; Sigma Diagnostics, Castle Hill, NSW, Australia), dehydrated, and mounted using DPX mounting medium.
Stereological Analysis
Stereology was used to determine the average number of leukocytes per unit area (2729 µm2) [7]. Leukocytes were identified by staining for CD45, and the DH Castgrid, version 1.6, software package (Olympus, Denmark) was used to randomly move the counting frame (area of 2729 µm2) throughout the designated region of interest. At least 100 cells in each category (stained and unstained) were counted. Leukocyte numbers were analyzed separately in basal and decidualized areas of the tissues. Cell counting was performed by the same observer and sections were scored blind. Leukocyte numbers were also assessed in control tissues (no oil injection, no decidualization).
TUNEL Staining to Detect Apoptosis
Hydrated cross-sections of formalin-fixed mouse uteri were deparaffinized in Histosol and processed through a gradient of ethanol into dH2O. Sections were immersed in methanol/3% H2O2 for 30 min at room temperature. Following two washes of 5 min in 0.01 M PBS (pH 7.4), slides were placed on an ice-cold tray. DNA labeling mixture (50 µl) was added to each section; this solution contained 10 µl 5x terminal deoxynucleotidyl transferase (TdT) buffer, 1 µl DIG DNA labeling mix, 2 µl 2.5 mM CoCl2, 0.2 µl TdT (25U/µl) (all from Roche Diagnostics Australia Pty. Ltd., Castle Hill, NSW, Australia) and 37 µl MilliQ H2O. A glass coverslip was used to contain the DNA labeling mix solution over the section during the 30-min incubation at 37°C in a humid chamber. Control assays replaced TdT with an equal volume of MilliQ H2O. Sections were washed twice in PBS for 5-min duration and then bathed in a blocking solution of 20% normal rabbit serum in PBS for 10 min at room temperature in a humid chamber. After this blocking step, approximately 100 µl of sheep anti DIG solution (Roche, 1.5 mU/µl in 10% normal rabbit serum/PBS) was applied to each section and incubated for 30 min at room temperature in a humid chamber. Sections were washed twice in 0.05 M PBS with gentle shaking, bathed in a solution of biotinylated rabbit anti-sheep Ig 1:500 (DAKO) in TBS/5% normal rabbit serum for 30 min at room temperature and then washed twice in TBS. The StrepABC kit and DAB solution (both from DAKO) were used in accordance with the manufacturer's specifications to reveal the TUNEL staining. Sections were lightly counterstained with hematoxylin, dehydrated, and mounted using DPX mounting medium. The relative abundance of stromal cells that were stained during TUNEL processing were compared by scoring each tissue with an arbitrary scoring system such that 0 indicated no stained cells in the tissue, through 4, where more than half of the cells were stained. All sections were scored blind by the same observer.
Statistical Analysis
All statistical analysis was performed using Prism 3.0. Comparison between progesterone levels at 0 and 12 h and between weights of unstimulated and stimulated uterine horns was made by paired t-test. Where a number of groups were compared, analysis of variance was performed following Bartlett test for equal variance. Individual differences were analyzed by Dunnett multiple comparison test. In all cases, significance was taken as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following the injection of oil into the uterine lumen of a sensitized mouse, there was an increase in the weight of the stimulated horn harvested 49 h later. The weights of the stimulated horns greatly exceeded those of their nonstimulated counterparts (66.6 ± 7.5 vs. 16.0 ± 1.5 mg, respectively, P < 0.0001, n = 8 animals). Following the withdrawal of progesterone, the mean weight of the stimulated uterine horns continued to increase until the 20-h time point, at which time it was significantly greater than the weight at 0 h (P < 0.05), decreasing thereafter (Fig. 2). The murine endometrium undergoes extensive remodeling during the process of decidualization, and at 49 h following the injection of the deciduogenic stimulus into the uterine lumen (0 h after progesterone withdrawal: Fig. 3A), expansion of the stromal cell population and their differentiation into decidual cells was apparent. Glands were absent from the decidual zone, but small glands could be found within the basal zone, proximal to the myometrium. It has been previously reported [8] that extensive neovascularization of the tissue supports the increase in tissue size, although blood vessels are largely absent from the primary decidual zone. By this time, there is closure of the lumen, the surface of which is generally mostly intact through re-epithelialization of the lumen damaged by the application of oil. These features were also apparent in the present study (Fig. 3A) and contrast with the control horn, to which a decidual stimulus was not applied (not shown).
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The withdrawal of progesterone support by the removal of the subcutaneous progesterone implants led to a rapid fall in serum levels of circulating progesterone from 180 ± 33 nmol/L at 0 h to 6 ± 2 nmol/L at 12 h (P < 0.0001, n = 3 mice per time point). However, the influence of this change was not evident by 12 h, as indicated by the morphology of the decidualized endometrium (Fig. 3B). At this time, the morphology of the uterus was similar to that observed at the 0 time point (49 h following the deciduogenic stimulus, Fig. 3A), including the infiltration by red blood cells throughout the tissue, presumably from the highly permeable blood vessels that are a feature of the decidual response. However, by 16 h following the withdrawal of progesterone (Fig. 3C), changes in the structural integrity of the decidualized endometrium became apparent. Large spaces between decidual cells were evident, and fewer blood vessels in the basal zone remained intact. This reduction in cell-cell contact was even more marked by 20 h following progesterone withdrawal (Fig. 3D), when large regions of necrotic decidual tissue were frequently observed. The loosening of connective tissue in the basal zone presumably contributed to the disassociation of the endometrium from the myometrium, which was further exaggerated by 24 h following withdrawal of progesterone (Fig. 3E). Morphology during the subsequent 24 h showed extensive remodeling of the tissue with isolation of the dead tissue (evidenced by loss of adherence between cells, rounding of nuclei, and large interstitial spaces) within the lumen apparent by 36 h after the withdrawal of progesterone (Fig. 3F). This cellular debris was isolated within the lumen following re-epithelialization, which is initiated antimesometrially from glands in the basal zone. By 48 h, there was a relatively small quantity of debris remaining within the lumen and the endometrium had undergone extensive restoration toward a predecidualized state (Fig. 3G).
Semiquantitative analysis of the morphological features of the decidualized endometrium following the withdrawal of progesterone was performed and representative sections with a score of 0 (representing no evidence of tissue destruction), and 4 (indicating complete tissue destruction) are seen in Figure 3, A and E, respectively. Such analysis further emphasized the progressive destruction of the decidualized zone of stromal cells (Fig. 4A) and luminal epithelium (Fig. 4B). The extent of destruction of the stromal tissue did not differ at the early time points (0, 12, and 16 h following the withdrawal of progesterone) but was significantly greater at the 20- and 24-h time points (P < 0.0001). The extent of destruction of the luminal epithelium varied considerably within groups and occurred even in tissues harvested at the early time points. Thus, no statistically significant differences were recorded. Nonetheless, intact epithelium was observed infrequently in the uteri harvested at 24 h after the withdrawal of progesterone compared with the earlier times.
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Transverse sections taken through control uterine horns (not decidualized) across the range of time points following progesterone withdrawal did not show signs of tissue breakdown.
Immunohistochemical Analysis of Leukocytes
The importance of leukocytes in the remodeling of the human endometrium throughout the menstrual cycle [4] and in particular, the dramatic increase in their numbers just prior to menstruation, heralded these cells as targets for analysis in this model. Leukocytes, identified by immunostaining for CD45, a marker of hematopoietic cells, were present throughout the endometrium in both decidualized and nondecidualized tissue, often in close association with the luminal epithelium and throughout the basal zone (Fig. 3, H–J). They were also closely associated with the newly generated luminal epithelium at the later time points (Fig. 3I). Leukocytes were also localized around glands, which are located in the basal zone of the decidualized endometrium. At the time when breakdown of the decidual zone was well progressed (24 and 36 h), very large numbers of leukocytes, most of which were identified morphologically as macrophages, were present within the breakdown area (Fig. 3J): given the variability between tissues at these time points, leukocyte numbers were not assessed.
Stereological analysis of leukocyte abundance in the basal zone in decidualized horns (Fig. 5A) revealed an increase in cells per unit area within 12 h of the removal of the implant (P = 0.016 compared with 0-h control). This increase in leukocyte abundance was maintained until 24 h after progesterone withdrawal (Fig. 5A), at which time analysis was impaired by the lack of structural integrity of the tissue (as seen in Fig. 3E). Analysis of leukocyte abundance throughout the entire tissue and decidual zone did not indicate changes in the leukocyte populations (other than those in the basal zone), although the changes in tissue weight and size could potentially mask any such changes (data not shown). Leukocytes were also counted in the basal zone in control horns (no induction of decidualization). Although the mean numbers of leukocytes per field was higher than in the decidualized tissue, no changes in numbers were detected following progesterone withdrawal (Fig. 5B).
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Assessment of Apoptosis
TUNEL staining was used to assess the extent of cell death in the decidualized endometrium of mice and following the withdrawal of progesterone. Quantitative scoring of these tissues revealed that stromal cells containing nicked DNA (indicative of cells undergoing the initial stages of cell death) were present immediately before progesterone withdrawal (49 h after the decidualizing stimulus) and decreased significantly in abundance by 16 h (P < 0.05 at each of 16, 20, and 24 h compared with 0 and 12 h; Fig. 6). The lack of a significant difference between the extent of TUNEL staining at the time of withdrawal of the progesterone implant compared with 12 h later indicates that the increase in leukocyte abundance observed at this 12-h time point does not occur in response to increased cell death following the withdrawal of progesterone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In humans, leukocyte numbers increase dramatically late in the secretory phase of the cycle [4]. In the present model, the number of leukocytes per unit area increased significantly in the basal zone by 12 h after progesterone withdrawal, and this increase in numbers preceded the destruction of the stroma that was first evident at 16 h but significantly increased by 20 h. Given that the decidualized stroma appears to separate from the basal zone, the increased numbers of leukocytes at this site supports the contention that leukocytes play an important role in initiation of tissue breakdown via their production of MMPs, serine proteases, and other bioactive factors and is in agreement with previous findings in the human [10]. Leukocyte numbers per unit area did not increase in the decidual zone following progesterone withdrawal. The higher number of leukocytes per unit area in the control horns was not surprising given the relatively small stromal area compared with the area in stimulated horns. However, the constant numbers found in these control horns following progesterone withdrawal indicates that the signals recruiting leukocytes must arise from the decidual cells or from cells such as the displaced glands, requiring cross-talk with the decidual cells.
In the human uterus, there is clear demarcation between the basal layer of the endometrium and the inner functional layer and it is largely the latter that takes part in menstruation. Although there is no such clear demarcation in the mouse endometrium, it does appear that there is a basal layer of stroma that remains undifferentiated during the decidual reaction in both pregnant and artificially stimulated uteri [5]. Indeed, it is likely that, in the mouse as in the human, once the stroma has embarked on differentiation, it cannot revert to its former state if progesterone support is removed and must be shed.
Apoptosis has been shown to increase in the human endometrium prior to and during menstruation, predominantly in the epithelium. Little apoptosis is seen in the stroma until menstruation has commenced [11, 12]. TUNEL staining was performed to establish whether the tissue loss seen in the present study was primarily due to increased apoptosis. Although apoptosis was detected in the decidual zone immediately before progesterone withdrawal, it was clearly not induced by this withdrawal. Indeed, by 16 h, there were significantly less apoptotic cells in the decidual zone than at the time of progesterone withdrawal. Apoptosis in the epithelium was not assessed due to the variation in epithelial integrity in sections within each group. This pattern of apoptosis resembles that seen in artificial menstrual cycles in spayed macaques [13]. In these animals, menstruation began 2–3 days after removal of progesterone implants. However, 12 h after progesterone withdrawal, there was a consistent, dramatic increase in cell death by apoptosis, especially in the basalis. Leukocyte invasion into the stroma occurred 1–2 days after progesterone withdrawal. This sequence of events is similar to that seen in our mouse model and leads to the conclusion that the increase in leukocytes in the basal zone relies on quite different signals from those that regulate apoptosis.
No differences were seen between time points when destruction of the luminal epithelium was assessed, although most was lost by 24 h. Although there was considerable variability between animals, it was unusual to observe tissues in which there was a complete absence of luminal epithelium: this reflects the situation at menstruation in women in whom re-epithelialization is thought to occur at least in part from residual luminal epithelium [14, 15]. The very rapid restoration of the luminal epithelium (within 48 h) in this model is also in agreement with postmenstrual repair in the human and the rhesus monkey [16, 17]. Thus, our mouse model provides also a useful model for studying the events of endometrial repair.
Silastic implants containing progesterone have been used to provide long-acting treatment in a variety of species, including mice: serum hormone levels in treated mice remain steady over 21 days compared with the marked fluctuations seen with daily subcutaneous injections [6]. Furthermore, withdrawal of such implants results in a very rapid fall in progesterone concentrations (95% in 24 h [6]), more resembling the rate of fall in women. This was seen also in the present study in which progesterone levels were minimal 12 h after implant withdrawal. As substantial variation in morphology was seen at each time point following cessation of progesterone injections, both in published work [5] and in preliminary studies in our laboratory (data not shown), we chose to use implants to better enable examination of the kinetics of events following progesterone withdrawal. The very rapid sequence of events described here is likely to be a consequence of the rapid fall in serum P.
Variability in the extent of decidualization between mice was a problem in these and similar studies and is also seen in women during the late secretory phase of the menstrual cycle. In the present study, this was minimized as far as possible by our choice of 49 h of decidualization as the starting point. When establishing the model, we started with 24 and 36 h of decidualization as Time 0, but found considerable variability in the extent of decidualization between mice. Indeed, it was not clear in some mice whether there was complete failure to decidualize or whether decidualization was merely retarded (data not shown). When 49 h was chosen as Time 0, there was still variability between the extent of decidualization as reflected in the weights of the stimulated horns, but any mice that failed to decidualize could be clearly identified at the time of sacrifice and could thus be excluded from the study.
One important difference between the mouse model and menstruation in the human is the extent of decidualization. During the normal human cycle, decidualization is initiated close to the spiral arterioles during the mid to late secretory phase and then spreads throughout the upper two thirds of the endometrium [18]. This predecidual cell expansion is not only by cellular hypertrophy but also by mitosis [1], as for decidualization in the mouse. Once pregnancy is established, decidualization continues throughout the first trimester [19]. Highly decidualized endometrium is not commonly found in the absence of pregnancy but is often seen in women using progestin-only contraceptives, particularly the levonorgestrel-releasing intrauterine system [20]. By contrast with this slow progression in women, once decidualization is induced in mice (only in the presence of a blastocyst or by artificial stimulation), it progresses rapidly from the site of initiation immediately below the uterine epithelium and means have not yet been found to restrain the process following induction. This is one limitation of the mouse model of menstruation. There may be fundamental differences between stromal cells from rodents and primates, as mouse and rat endometrial stromal cells decidualize spontaneously when put in culture unless they are derived from ovariectomized animals that have not been treated with progestin [21]. In contrast, human endometrial stromal cells derived from cycling women in either the proliferative or secretory phase of the cycle, decidualize in culture only when treated with progesterone for >6 days. This can be accelerated by addition of decidualizing stimuli such as interleukin-11 [22], activin A [23], and prostaglandin E2 [24] or most commonly by cAMP.
It appears that decidualization or at least initiation of a predecidual state in the endometrial stroma is a prerequisite for menstruation. Although the stroma of rhesus and cynomolgus macaques do not decidualize to the extent seen in women during the nonfertile menstrual cycle, some enlargement of the stromal cells is observed, especially around the spiral arteries. This is likely to be similar in kind but not in degree to the response of stromal cells to progesterone in the human [18]. Indeed, it is in the area around the spiral arterioles that decidualization is initiated in the human. In the menstruating bat (Glossophaga soricina) during the late luteal phase, a swollen polyp containing large decidual-like cells is seen in the endometrium, and this breaks down and bleeds at the end of the cycle [25]. It is important to note that, in the model described here, tissue breakdown was not observed in the nondecidualized horn, supporting the contention that at least some progress toward stromal differentiation is required for initiation of tissue breakdown. Given the major phenotypic changes that accompany decidualization [26], it is likely that new secretory products from these cells initiate or promote the tissue breakdown.
This mouse model for menstruation thus demonstrates many features in common with spontaneous menstruation in higher primates. Given the wide availability of genetically modified mice and of inhibitors of many of the factors thought to be involved in both menstruation and in tissue repair, the model will allow functional studies to determine which factors are critical to these processes and closer examination of the sequence of events. Such studies are essential if we are to progress toward rational treatment of disorders of menstruation and endometrial repair.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: L.A. Salamonsen, Prince Henry's Institute of Medical Research, Level 4, Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. FAX: 61 3 9594 6125; lois.salamonsen{at}med.monash.edu.au
Received: 19 February 2003.
First decision: 20 March 2003.
Accepted: 16 May 2003.
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