Hum. Reprod. Advance Access published online on October 4, 2007
Human Reproduction, doi:10.1093/humrep/dem312
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Menstrual-like changes in mice are provoked through the pharmacologic withdrawal of progesterone using mifepristone following induction of decidualization
1 Department of Cell Biology, National Research Institute for Family Planning, Beijing 100081, People's Republic of China 2 Graduate School, Peking Union Medical College, Beijing 100730, People's Republic of China
3 Correspondence address. E-mail: jdwangmail-hr{at}yahoo.com.cn
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Abstract |
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BACKGROUND: Cyclic shedding of the endometrium is unique to menstruating species, and mouse menstruation models by physiologic progesterone withdrawal have been previously reported. Since progesterone action ablated pharmacologically may provide more insight into the mechanism of action, a mouse menstruation model using mifepristone was established.
METHODS: Mifepristone was administered following oil-induced decidualization in ovarectomized mice primed with hormones. Morphology, hormone levels, leukocytes and apoptosis were evaluated over a period of 48 h after treatment. Vaginal smears were used to monitor bleedings.
RESULTS: Mifepristone induced menstrual-like changes. Tissue breakdown was drastic by 16 h, and the decidual zone was shed by 24 h while the mice bled. The endometrium regenerated from 24 h onwards and became completely restored by 48 h. These results are consistent with previous reports. However, although progesterone levels remained constant, estradiol levels increased after the treatment. The CD45+ cells showed two peaks of increase at 16 h (breakdown phase) and 32 h (regeneration phase) (Leukocyte levels also increased in the unstimulated horns, but no breakdown changes occurred there). Moreover, apoptosis drastically increased by 16 h concurrent with tissue destruction. These results differed from those of the physiologic withdrawal models.
CONCLUSIONS: The pharmacologic withdrawal of progesterone by mifepristone successfully provoked a menstrual-like process in mice after artificial decidualization.
Key words: decidualization/menstruation/mifepristone/mice/pharmacologic withdrawal of progesterone
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Introduction |
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The lining of the uterus in human is unique because it sheds every cycle if no fertilized ovum is implanted. It is one of the mysteries of reproductive physiology and has great relevance to clinical medicine. However, the underlying mechanism of menstruation has not been well understood yet, partly due to the lack of suitable model systems.
The best candidate for menstruation model should be the non-human primates, but many factors limit their use, including limited sources, difficulties in practice, ethical considerations, etc. In the 1980s, Finn and Pope (1984)
established a model for menstruation in mouse, a mammalian species that does not menstruate naturally. The model is very valuable as it provides a convenient means to study menstruation. In their study, the prevention of progesterone supply led to endometrial bleeding after inducing decidualization by oil in ovarectomized mice, following a sequential administration of estrogen and progesterone. The menstrual-like changes were similar to those of menstruating endometrium in primate females. Recently, Brasted et al. (2003) refined Finn's model with progesterone implants. They also optimized the starting time for progesterone withdrawal after induction of decidualization and attained more reproducible results.
In menstruation research, there are two methods to induce progesterone withdrawal. One is to stop the supply of progesterone, as described in the previous mouse models, either by stopping progesterone injection or by removing progesterone implants, which mimics the fall of progesterone levels at the end of the menstrual cycle as caused by the demise of the corpus luteum in primates (Finn and Pope, 1984; Brasted et al., 2003). This method is defined as the physiologic withdrawal of progesterone. Another method is the ablation of progesterone action by mifepristone, a progesterone receptor (PR) antagonist. This method is defined as the pharmacologic withdrawal of progesterone and is a valuable tool in the studies of pregnancy and uterine physiology (Baulieu et al., 1987; Baulieu, 1991; Critchley et al., 2003). Thus, a comparison between models induced by physiologic and pharmacologic withdrawals of progesterone will provide more insight into the mechanism of menstruation.
In this report, a pharmacologic withdrawal model of mouse menstruation, in which mifepristone was administered after artificial decidualization, is presented. Indeed, we observed similar events as those in the physiologic progesterone withdrawal model. At the same time, the discrepancies in the uterine changes and hormone levels caused by different withdrawal models are also discussed.
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Materials and Methods |
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Animals
Female virgin NIH mice (8–12 weeks old) were obtained from the Animal Services of the National Research Institute for Family Planning. Mice were maintained under controlled conditions of light (lights were on from 0600 to 1800 h) and temperature (21 ± 1°C) and were allowed free access to food and water. Experimental and surgical procedures were likewise approved by the Animal Ethics Committee of the same institute mentioned above.
Induction of the mouse menstruation model
The manipulation of the animals roughly followed the procedure described by Brasted et al. (2003), with modifications as outlined in Fig. 1. Animals were ovarectomized under anesthesia and allowed to recover for two weeks. They were then sequentially administered with steroid hormones wherein on Days 1, 2 and 3, all mice were subcutaneously (s.c.) injected daily with 100 ng of 17
-estradiol (E2) (Alfa Aesar Inc., Heysham, UK) in arachis oil at ~0930 h. On Day 7, progesterone implants were inserted s.c. into the back of the mouse at ~0930 h and 50 ng of progesterone (Sigma-Aldrich Inc., St. Louis, MO, USA) and 5 ng of 17-E2 in arachis oil were also instantaneously injected s.c. The implants were prepared as described previously (Milligan and Cohen, 1994; Brasted et al., 2003). On Days 8 and 9, 5 ng of 17-E2 in arachis oil was also injected s.c. at 0930 h, while on Day 9, 20 µl of arachis oil was injected into the lumen of the left uterine horn of each mouse to induce decidualization through dorsal incision at 1130. However, the right horn was not treated with arachis oil to serve as a negative control. Forty-eight hours later (i.e. on Day 11), mifepristone (120 mg/kg, Beijing Zizhu Pharmaceutical Co., Ltd, Beijing, China) was given to the mouse through intragastric administration at 1130 h (regarded as 0 h). In our pilot study, the dosages of 30–200 mg/kg mifepristone were tested and an optimal dose of 120 mg/kg was chosen. Mice were sacrificed by cervical dislocation at 0, 8, 16, 24, 32, 40 and 48 h after mifepristone administration and uterine horns were harvested at each time interval. After being weighed, one-half of each horn was fixed in Bouin's solution and the other half was frozen immediately in liquid nitrogen, before being stored at –70°C.
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Meanwhile, blood was collected from the orbital sinus of the sacrificed mice to estimate the serum concentrations of progesterone and E2.
Vaginal smear examination
Vaginal smear examinations were performed on all the mice at the time just before sacrificing them to monitor the endometrial bleedings. For those mice sacrificed at 32, 40 and 48 h after mifepristone administration, a vaginal smear examination was also performed in advance at 24 h, a time whereby most animals bled as shown in our pilot study. The slides were put into 95% ethanol for ~10 min and then stained with hematoxylin and eosin, using standard staining procedures.
Progesterone and E2 assays
Hormonal serum levels were measured using the enzyme-linked immunosorbent assay (ELISA) kit (Sun Biomedical Technology, Inc., Beijing, China), according to the manufacturer's instructions. The limit of detection was 5 pg/ml for E2 and 0.1 ng/ml for progesterone, respectively. The intra-assay coefficients of variation for E2 and progesterone were 7 and 6%, respectively. The inter-assay coefficients of variation for E2 and progesterone were 12 and 14%, respectively.
Reticular fiber staining
Gordon and Sweets' staining method was used to observe the reticular structure of the endometrium (Gordon and Sweets, 1936).
Immunohistochemical analysis of leukocytes
Immunohistochemistry was performed using Histostain Plus kit (Zymed Laboratories, San Francisco, CA, USA). Briefly, uterine cross-sections were deparaffinized and rehydrated, followed by an antigen retrieval procedure. After being soaked in 3% of H2O2 in methanol for 15 min and bathed in a blocking solution of rabbit serum for 20 min, both of which were conducted at room temperature, every section was added with rat anti-mouse primary antibody CD45 (1:150, BD, Biosciences, Inc., Franklin Lakes, N.J., USA) and incubated overnight at 4°C. Meanwhile, non-immunized rat serum was applied in the adjacent section to replace the anti-CD45 antibody in the staining procedures to serve as a negative control. After that, the sections were added with biotinylated rabbit anti-rat IgG secondary antibody (Zymed Laboratories) in blocking buffer and incubated for 1 h at room temperature. The reaction was visualized by 3,3'-Diaminobenzidine tetrahydrochloride (DAB) solution counterstained with hematoxylin. Each treatment step mentioned above was followed by three 5-min washes in PBS, unless specifically described.
The positive cells in the basal and the functional zones of the endometrium were counted by two independent observers, using a Leica microscope LMD 6000 (Leica, Wetzlar, Germany), and the average of the two observers' counts was taken as the value of the specimen. The areas of the basal and functional zones were determined by the image processing software of Leica LMD version 6.3.1. Both the left and right horns were assessed, and the results were then expressed as the number of positive cells per 5000 µm2.
Detection of apoptosis
TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed to detect apoptosis of the endometrium. The In Situ Cell Death Detection Kit, POD (Roche Applied Science Inc., Mannheim, Germany) was used according to the manufacturer's instructions. Briefly, cryopreserved tissue sections were fixed in 4% paraformaldehyde for 20 min at 15–25°C and then incubated with 3% of H2O2 in methanol for 10 min at 15–25°C. After incubation in a permeabilization solution on ice for 2 min, the sections were incubated with 50 µl TUNEL reaction mixture (containing 5 µl of terminal transferase solution and 45 µl label solution) for 60 min at 37°C and counterstained with propidium iodide to determine the cell count. The negative control sections were treated with the label solution alone. Each treatment step mentioned above was followed by repeated washes in PBS. The sections were observed under a laser-scanning confocal microscope (Leica TCS SP2, Leica). The number of endometrial cells undergoing apoptosis at different time points was independently determined by two observers, and the final result was the average of the two observers' counts.
Statistical analysis
A comparison between the weights of the left and right uterine horns was performed using the Student paired t-test, while a comparison among different groups was performed using one-way ANOVA followed by a Tukey–Kramer multiple comparison test. All statistical tests were two-sided, and a P < 0.05 was considered statistically significant.
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Results |
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Morphologic observation of the uterus after mifepristone treatment
The weights of the uterine horns were significantly different between the two sides from 0 to 24 h (Fig. 2, P < 0.05). At 0 h following mifepristone treatment, namely, 48 h after arachis oil infusion, the stimulated horn was grossly congested and enlarged as compared with its unstimulated counterpart. This indicates that decidual reaction was successfully induced, a fact which was further confirmed by histologic examination (Fig. 3A). There was a variation in this reaction along the length of the horn, as well as among animals. Little change was exhibited at 8 h as compared with the situation at 0 h. After 16 h, the stimulated horn showed various degrees of darkening in color that evidenced a process of tissue breakdown (Fig. 3I). Furthermore, after 24 h, the stimulated horn displayed apparent congestion and a dark purple color, implying that bleeding had taken place. From 32 h onwards, such differences between the two horns diminished with time and the weights between two sides of the horns became insignificantly different (Fig. 2). At 48 h, the stimulated horn macroscopically appeared to be the same as the unstimulated one, and the occurrence of bleeding was only verified through the presence of red blood cells (RBCs) in the vaginal smear examined in advance at 24 h (Table I). This implies that regeneration of the broken down tissue in the horn had been attained.
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Meanwhile, histologic examination revealed detailed changes after mifepristone administration. At 8 h (Fig. 3B), the boundary between the basal and functional layers became clear and could be easily identified, and the epithelial cells showed focal breakdown and shedding. At 16 h (Fig. 3C), the lining of the epithelium began to develop necrosis and desquamation, and a large area of the stroma exhibited necrosis with hemorrhage. The stromal reticular fiber likewise underwent fragmentation and disaggregation (Fig. 4A). Leukocyte infiltration became apparent as well. After 24 h (Fig. 3D) following mifepristone treatment, most of the decidualized tissue were sloughed into the uterine lumen. Meanwhile, tissue reconstruction was initiated as luminal epithelia recovered rapidly. The regeneration became more obvious at 32 and 40 h (Figs 3E and F) as the endometrium began to thicken due to the proliferation of the stromal cells. Furthermore, the number of leukocytes increased significantly in both the basal and functional zones of the endometrium. Reticular fibers were recovering in the endometrium at this time point (Fig. 4B). The endometrium regenerated completely by 48 h, as it began to morphologically look the same as the unstimulated horn (Fig. 3G). In contrast, the abovementioned changes were not seen in the unstimulated horns during the entire course of the experiment (Fig. 3a–g).
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Using vaginal smears to monitor bleeding
The appearance of RBCs in the vaginal smear is an indicator of endometrial bleeding and can thus be used to monitor bleedings in live animals. In this study, we performed vaginal smear examinations throughout the course of the mifepristone treatment. In the pilot study, most animals bled at 24 h, thus for those animals sacrificed from 24 h onwards, smears at 16 and/or 24 h were examined in advance. As summarized in Table I, no RBCs were detected among the mice sacrificed before 16 h (i.e. 0 and 8 h). Percentages of RBC-positive cases against the total inspected at 16, 24, 32, 40 and 48 h were 24.0, 96.6, 100, 57.1 and 16.7%, respectively. For a total of 29 smears examined at 24 h, RBCs were found in 96.6% of the cases. This result indicated that most of the animals had been bleeding at 24 h after mifepristone treatment (Table I and Fig. 3H). In addition, RBCs were found in all eight animals examined at 32 h, but the RBC count was much less than that at 24 h. These eight animals were also positive at 24 h examination, signifying that RBCs persisted in the vagina for at least 8 h after the initiation of bleedings.
Assessment of leukocytes
Leukocytes were identified by immunostaining for CD45 (Fig. 4C and D). The changes in the number of CD45 positive cells at various time points after mifepristone treatment were found to be complicated. These are shown in a diagram in Fig. 5.
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During the first 24 h, a remarkable change took place in the basal zone of the stimulated horn. At 16 h, a dramatic rise in CD45 positive cells was noticed in the basal zone (P < 0.05; 16 versus either 8 or 24 h, Fig. 5A). However, there was also an increase in the number of CD45 positive cells in both the basal and functional zones of the unstimulated horn at 16 h after mifepristone treatment (P < 0.05; 8 or 16 versus 0 h, Fig. 5B for the basal zone; P < 0.05; 16 versus 8 or 24 h, Fig. 5D for the functional zone). The parallel rise in leukocyte influx in both horns, with breakdown changes only in the stimulated horns, suggested that decidual status determined the response of the horn to leukocyte influx.
The evaluation of the number of leukocytes in the functional zone of the stimulated horn was complicated by the striking change in decidual destruction during the first 24 h following mifepristone treatment. As seen in Fig. 5C, the CD45 positive cell number in the decidual zone was much lower than that in the basal zone (Fig. 5A) of the stimulated horn during the first 24 h of treatment.
Meanwhile, the leukocyte profile in the second 24 h was different from that in the first 24 h. Interestingly, there was a peak of CD45 positive cells at 32 h in both the basal and functional zones of the stimulated horn after mifepristone was administered (P < 0.05; 32 versus either 24 or 40 h, Fig. 5A and C). On the other hand, in the unstimulated horn, no significant fluctuation was observed in both its basal and functional zones during the period from 24 to 40 h (P > 0.05; Fig. 5B and D).
Analysis of apoptosis
The mifepristone treatment resulted in a remarkable increase of apoptosis in the stimulated horn. At 0 h, the number of apoptotic cells was very low, and there was no apparent difference between the stimulated and unstimulated horns. However, the number of apoptotic cells increased slightly at 8 h, following mifepristone administration. Afterwards, the number of apoptotic cells sharply increased at 16 h, and then declined at 24 and 32 h, before rapidly dropping down from 40 h onwards. In contrast, the level of apoptosis remained low in the unstimulated horns. The trend in differences in apoptosis between the two uterine horns was obvious, but statistical evaluation was not performed due to the limited number (n = 3) of specimens available at the time of assay (Fig. 6).
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Serum levels of E2 and progesterone
High levels of progesterone remained constant during the course of the mifepristone treatment, and no significant difference was found at different time points (P > 0.05; Fig. 7). The levels of progesterone were in the range of 41–52 ng/ml, which were within the normal range of those in pregnant mice. The E2 levels were 26 ±6 pg/ml at 0 h and were progressively raised to 46 ± 18 pg/ml at 16 h, which was the highest during the entire mifepristone treatment, before they gradually decreased to 13 ± 3 pg/ml by 48 h. The serum E2 levels at 8 and 16 h were significantly higher than those at 0, 24, 32, 40 and 48 h (P < 0.05 for either 8 or 16 h against any other time points; Fig. 7).
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Discussion |
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Finn and Pope (1984)
established a model for menstruation in mice. This model defines fundamental requirements for the occurrence of menstrual-like changes in non-menstruating species of mammals. These requirements state that (i) the endometrium should be well prepared by a natural endocrine milieu or by a sequential administration of estrogen and progesterone in ovarectomized animals; (ii) a differentiated status, as predecidual reaction in human or decidual reaction in mouse, should be evoked in the stroma of the endometrium and (iii) the withdrawal of progesterone following decidual-like differentiation should be achieved.
In two published models (Finn and Pope, 1984; Brasted et al., 2003), the cessation of progesterone supply was followed by tissue breakdown and uterine bleeding. However, it is uncertain whether or not such events could be induced by the blockage of progesterone at the receptor level. To address this issue, we established a menstruation model of mice through pharmacologic withdrawal of progesterone. In our protocol, instead of the removal of progesterone implants to stop progesterone supply (Brasted et al., 2003), a single dose of mifepristone was administered following the induction of decidualization.
In the present study, the weights of the uterine horns were significantly different between the two sides of these horns at 0 h (i.e. 48 h following oil infusion), indicating that the decidual reaction was properly prepared. Meanwhile, mifepristone administration evoked progressive tissue breakdown changes within 24 h in the functional zone of the decidualized horn. At 8 h, the epithelial cells began a focal breakdown as shedding and dead cell debris were seen in the uterine lumen, while some stromal cells were undergoing degenerative changes. These changes were most apparent with necrosis at 16 h, which then spread to the whole decidual zone of the horn. By 24 h, the whole zone was sloughed into the uterine lumen. At the same time, the functional zone began to regenerate from the basal zone of the organ and the endometrium completely recovered by 48 h after mifepristone treatment. This sequence of events is consistent with that seen in the previous models, indicating that both the blockage of progesterone at the receptor level and the fall of progesterone level were equally effective in inducing a menstrual-like process (Finn and Pope, 1984; Brasted et al., 2003).
We used vaginal smears to monitor endometrial bleedings during the course of the mifepristone treatment. It was revealed by vaginal smear inspections conducted throughout the course of the treatment that endometrial bleeding appeared in most animals at 24 h. By 32 h, the RBCs were still detectable, though the amount was greatly reduced, signifying that the bleeding lasted for at least 8 h. This is a significant parameter, particularly for those animals sacrificed at time points beyond 24 h, to which the occurrence of endometrial bleeding can only be verified by this monitoring method.
Nevertheless, although the mifepristone-induced changes were quite similar to those previously described (Finn and Pope, 1984; Brasted et al., 2003), some differences were noted. First, the serum levels of E2 and progesterone in the present model were significantly different from those in the previous study (Brasted et al., 2003). This is due to the fact that the implant was not removed and that the level of progesterone was maintained, although its action was ablated by mifepristone. On the other hand, the level of E2 was elevated after mifepristone treatment. The mechanism of the increase is unclear. It may be related to metabolism pathways in adrenal glands, uterus or other organs since the ovary of the animal was removed. Because the enzymes for estrogen metabolism were not examined in our experiments, the exact pathway led to the elevation of E2 remains to be determined. However, this increase in estrogen did not prevent the occurrence of menstrual-like changes. This is similar to a study conducted on rhesus monkey, in which mifepristone-induced menstruation when it was administered together with E2 and progesterone (Slayden et al., 1993).
We also observed an increase in the number of CD45 positive cells in both sides of the horns, which peaked at 16 h after mifepristone treatment. Although both sides of uterine horns were infiltrated by leukocytes, only the decidualized tissue broke down, suggesting that the effect of leukocyte infiltration in the endometrium depends on the decidual status of the tissue.
In the previous report, the rise of CD45 positive cells was only seen in the stimulated horn, and no change was found in the unstimulated counterpart (Brasted et al., 2003). As mentioned above, mifepristone treatment, in addition to the ablation of progesterone, led to an increased level of the serum E2 in our model (Fig. 7), which was different from the report of Brasted et al. (2003). This discrepancy in endocrine milieu may explain the different patterns of leukocyte influx between the two models. As documented in the studies of PR knockout, the uterus of deficient mice showed a strong inflammatory reaction, with marked infiltration of polymorphonuclear leukocytes, in response to E2 and progesterone (Lydon et al., 1996; Tibbetts et al., 1999;). Further analysis demonstrated that estrogen is responsible for the recruitment of leukocytes and that progesterone antagonizes this effect. Meanwhile, PR deficiency led to loss of the antagonistic action, which resulted in the increased level of leukocyte number (Tibbetts et al., 1999). Thus, it was most probably the pharmacodynamic effects of mifepristone (i.e. the elevation of E2 level and the loss of progesterone antagonism), which were responsible for the leukocyte pattern in the present study.
The increased level of leukocytes may not be a direct effect of antiprogestin. However, mifepristone treatment can up-regulate potential chemokines that mediate leukocyte traffic. Critchley et al. (1996) reported that interleukin-8 and MCP-1 increased in response to mifepristone treatment and may be responsible for leukocyte influx in human decidua. Although there is no homolog of interleukin-8 in the mouse, a similar signaling pathway may exist. This deserves further exploration in the present model.
Interestingly, there was a second increase in the number of leukocytes in the basal and functional zones of the decidualized horn at 32 h after the administration of mifepristone, a phenomenon which was not seen in the control horn during the same period. However, this event may not be induced by mifepristone. As the regeneration of the broken down tissue had been initiated at 24 h, the event is more likely related to the process of tissue reconstruction, which would be completed 16 h later (i.e. 48 h after mifepristone administration). It is not certain whether or not similar phenomena took place in the previous mouse model, since data were not available in the report (Brasted et al., 2003). Nevertheless, a recent study from this group has shown that the depletion of neutrophils effectively suppressed the reconstruction process of menstruated uterus in the model (Kaitu'u-Lino et al., 2006). Moreover, in women, leukocytes were observed to be involved in the tissue-repair process 4–5 days from the start of menstruation (Ludwig et al., 1990; Ludwig and Spornitz, 1991) and have been postulated to have an important role in the tissue-repair process (Salamonsen, 2003).
The role of apoptosis has been implicated in the extensive observation of human menstruation. Dahmoun et al. (1999) observed increased apoptosis in both epithelial and stromal cells at the end of the cycle and during the menstrual period. In the report of Brasted et al. (2003), apoptosis declined 16 h upon removal of the progesterone implants. However, in the present study, we observed a significant increase of apoptosis at 16, 24 and 32 h after mifepristone treatment, with a peak at 16 h during the breakdown period in the stimulated horn. This is consistent with Slayden's observation in rhesus monkeys that both the physiologic and pharmacologic withdrawals of progesterone led to an increase in apoptotic cells (Slayden et al., 1993).
In our experiment, as the stromal apoptosis in the tissue was abruptly raised at 16 h, a time when tissue necrotic changes of the decidual zone were most drastic, it seemed that the rise of apoptosis was a concurrent event rather than a causal one for tissue breakdown. A study on endometrial explants has shown that tissue breakdown took place in a culture from biopsies taken during the secretory phase of the cycle, and that E2 and progesterone treatment reduced or eliminated menstrual-like breakdown, but did not affect the degree of apoptosis observed (Li et al., 2005). Thus, the stromal apoptosis may not be critical in the initiation of breakdown, but may serve a role in the subsequent process of endometrial repair following menstruation (Dahmoun et al., 1999; Li et al., 2005). In a normal menstrual cycle, stromal apoptosis increased on the onset of menstruation, paralleled with a highest proliferate index of Ki-67 (Dahmoun et al., 1999). This active cell turnover supports the role of stroma in the renewal process of the endometrium. The increase of apoptosis, which spans from the breakdown to the regeneration phases (from 16 to 32 h), in our observation thus provides additional support for the role of apoptosis in tissue remodeling.
The consistency of our model with that proposed in Finn's original and recently refined work in the sequential events of tissue breakdown and regeneration in decidualized endometrium, through the pharmacologic withdrawal of progesterone, confirms that menstrual-like changes could be achieved by the blockage of PR in mice. Furthermore, the differences between the two methods of progesterone withdrawal provide more insight into the detailed mechanism of menstrual bleeding and shedding. In particular, mifepristone possesses potential clinical applications relevant to menstrual disorders, and its possibility as an agent for menstrual induction has been proposed (Bygdeman, 2003; Xiao et al., 2003). The model presented in this report will thus favor research conducted in this field.
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Funding |
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Natural Science Foundation of China (30371490, to J.D.W.).
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Acknowledgement |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementReference |
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We would like to express our gratitude to Mr. C.Y. Zhang for his aid in animal experiments, as well as to Mr. R.H. Xu and Ms. Cheng Jie for their technical assistance. We would also like to thank the Beijing Zizhu Pharmaceutical Co. Ltd, China for the donation of the mifepristone used in the study.
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Footnotes |
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These authors contributed equally to this work as first authors. 
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Reference |
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Submitted on March 21, 2007; resubmitted on July 3, 2007; accepted on September 4, 2007.
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