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. 2012;7(3):e32922.
doi: 10.1371/journal.pone.0032922. Epub 2012 Mar 7.

Induction of overt menstruation in intact mice

Marion Rudolph  1 , Wolf-Dietrich Döcke, Andrea Müller, Astrid Menning, Lars Röse, Thomas Matthias Zollner, Isabella Gashaw
Affiliations

Induction of overt menstruation in intact mice

Marion Rudolph et al. PLoS One. 2012.

Abstract

The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply.

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Conflict of interest statement

Competing Interests: All authors are employed by Bayer Healthcare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Scoring system for the determination of bleeding intensity on the basis of hematoxylin and eosin (H&E) staining of vaginal lavage fluid.
The increase in erythrocytes correlates with the increase in the indicated score (from 0 = no erythrocytes, no bleeding to 5 = large amounts of erythrocytes, heavy bleeding). Black arrows indicate eosin-stained squamous epithelial cells and white arrows eosin-stained erythrocytes.
Figure 2
Figure 2. A drop in endogenous progesterone levels induces overt, extravaginally visible bleeding.
A: Experimental setup: Female mice were mated with vasectomized male mice (day 0). Pseudopregnant female mice were identified by vaginal plugs the next day. On day 4, endometrial decidualization was triggered by intra-uterine injection of sesame oil. The bleeding was monitored visually (detection of extravaginal blood) and microscopically (in H&E stained vaginal lavage fluid) from day 6 until necropsy. B: Systemic progesterone concentrations (mean) were determined by ELISA in sera from mice treated according to Figure 2A. The Figure shows pooled data from two independent experiments (N≥10). C: Representative photograph of a mouse bleeding visibly extravaginally 4 days after decidualization on day 8 of pseudopregnancy. D: Visual determination of bleeding intensity of pseudopregnant mice with decidualized endometrium over 3 days from day 7 to day 9 (N = 16). E: Bleeding intensity (H&E bleeding scores; left axis) and pattern of serum progesterone levels (right axis) over time (open square = result for one individual mouse; filled squares = mean score for all mice in the group; N≥7; red = H&E bleeding score; gray = progesterone concentration). Data were collected in a single experiment. F: Bleeding scores of mifepristone-treated, pseudopregnant mice from day 7 to day 9 (treatment started on the evening of day 6; 10 mg/kg sc; open/filled squares see Figure 2E; N = 7). The unpaired two-tailed Student's test was used for statistical analysis of the progesterone data (B, E), the two-tailed Mann-Whitney test for the ordinal H&E scores (*p≤0.05, **p≤0.01, ***p≤0.001).
Figure 3
Figure 3. Bleeding pattern and uterine weight mirror changes such as tissue construction, breakdown, and repair.
Pseudopregnant BALB/c mice were treated according to Figure 2A and sacrificed before (day 4) or after decidualization (days 6, 7, 8, 9, 10, 12). A: Macroscopic analysis of uteri at indicated points in time. B: Microscopic analysis of H&E-stained uteri (S, stromal cells; E, epithelium; L, lumen; B, extravasated blood). C: Analysis of uterus weights (left axis; open square = individual data; bar = mean) and bleeding intensity determined by H&E scores according to Figure 1 (right axis; filled square and bar analogous to the preceding; N≥7). The two-tailed Spearman test indicates a significant correlation (p = 0.0167, Spearman r = 0.9429) between increased uterus weight and H&E score. Data were assigned to the respective time (day 4 to day 12) and to the dominant processes at that time (construction - blue, breakdown - red, repair - green).
Figure 4
Figure 4. Tissue breakdown and repair are dynamic processes which take place in parallel.
Representative H&E-stained sections (right) from the same decidualized uterine horn (left) on day 9 of pseudopregnancy displaying: A: Decidualization and first signs of extravasation of blood (black arrow). B: Clear signs of destruction in the form of detachment of tissue structures (blue arrow) and extravasation of blood. C: Pronounced tissue destruction with marked extravasation of blood combined with signs of re-epithelialization (orange arrow; repair).
Figure 5
Figure 5. Regulation of different cellular processes in the endometrium is time point-specific.
Endometrial mRNA levels of the indicated genes were determined by quantitative real-time PCR. All values were normalized to HPRT mRNA levels. Boxplots summarize the results of 5 to 9 mice per group (unpaired two-tailed Student's t-test, *p≤0.05, **p≤0.01, ***p≤0.001).
Figure 6
Figure 6. Overt bleeding in mice mimics the human menstrual cycle.
Comparison of the overtly bleeding mice presented here and the human menstrual cycle. The proestrus, with high levels of estrogen, might mimic the proliferative phase in humans. Induction of pseudopregnancy causes progesterone levels to increase in the same way as in the human secretory phase. The drop of progesterone induces bleeding between day 7 and day 10, which is comparable to menses. By day 12 tissue repair is well advanced, something which is also seen in the early proliferative phase. These processes are governed by differential expression of various genes. Genes that were expressed at a maximum at certain time points in overtly bleeding mice are listed. The vast majority of them are regulated similarly in mice and humans (for details see text).
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