As in another mouse study and in human endometrium, the data point tendentially towards cyclic Il18 expression. A similar scenario with ambiguous results from human endometrial biopsies was found for CXCL12. Interestingly, Laird et al. describe a CXCL12 mRNA expression pattern in human endometrium that appears to be comparable to the one seen in the overtly bleeding mice. Concerning the Tgfb cytokine family, which is important for the induction of wound healing, we observed partial correlation of our data with human data: TGFB1 mRNA is increased during the menstrual phase in humans, corresponding to the expression pattern observed in this study. In contrast, average TGFB3 mRNA levels were reported to be increased 3-fold from the human secretory to the menstrual phase, but were fairly constant in our study. In principle, when translating gene expression, histology, and bleeding data from mice to women or vice versa one must consider that human endometrial biopsies are taken over a period of days and not at a clearly defined point in time as was the case in this study. Our data demonstrate that the decidualized endometrium is a highly dynamic tissue in which processes such as tissue destruction and repair proceed in parallel. Accordingly, it must be expected that changes occur within a very short time frame in humans as well, especially during menstruation. The exact time at which tissue is collected will therefore determine the results obtained and is decisive for the transferability of data from mouse to man. In conclusion, the comparison of Loganin overtly bleeding mice and human data for menstruation indicates a very strong correlation, not only in terms of morphology and hormones but also regarding the common regulation of marker genes, thus indicating comparable underlying processes. Since intact pseudopregnant mice convincingly mimic human menstruation, it is expected that the further use of these mice will contribute to a better understanding of the cellular and molecular mechanisms accompanying menstruation. Moreover, they will be a valuable tool for pharmacologic testing. A diverse set of interventions, in addition to drugs, is known to have antidepressant action, including cognitive and electroconvulsive therapies, sleep deprivation, and exercise. Mechanistic understanding of what is common to environmental and drug treatments may provide valuable clues about the mode and site of action of antidepressants. Recent studies demonstrate Cinnamaldehyde the importance of adult hippocampal neurogenesis for the action of antidepressants. Both exercise and enriched environment have also been found to increase hippocampal neurogenesis and cause antidepressant-like behavioral change. Chronic exercise reduces depressive-like behavior in rats and mice, as measured in standard models of depression such as the forced swim test. Environmental enrichment has similar consequences and both produce notably similar effects on the brain in stimulating cell proliferation and recruitment of new neurons into the dentate gyrus of the hippocampus. Currently no molecular pathway is known that is common to these treatments. The finding that gene expression data show structured correlation, together with the development of weighted gene co-expression network analysis, provide a system-level approach for using gene expression to detect the common mechanisms of different interventions. WGCNA organizes genes into modules that are co-regulated and therefore are more likely to be functionally related and to participate in similar cellular processes. WGCNA also alleviates the multiple testing problem inherent in testing tens of thousands of transcripts, a problem that otherwise substantially reduces the power of standard differential expression analysis.

This observation argues against the hypothesis put forward by Profet that menstruation is a mechanism for pathogen defense. The induction of menstruation in a species that does not normally menstruate and in the absence of any modification of the number of sexual contacts refutes the assumption that the copiousness of menstruation increases with the promiscuity of the mating system. Rather, our data indicate that it is a matter of costs and benefits: where there are large quantities of decidualized endometrial tissue, as was found in the mice in this study, it seems to be metabolically more economical to repel tissue rather than to absorb it. Taking both into account – the morphological uterine changes demonstrated here and the variations in hormone levels – we propose a functional correlation between overtly menstruating mice and human menstruation as shown in Figure 6. The proestrus in mice might represent the proliferative phase in women, since estrogen is the dominant hormone at this time. After induction of vitexicarpin pseudopregnancy, however, progesterone levels are greatly increased, indicating the similarities to the human secretory phase. The bleeding between day 7 and day 10 which is triggered by the drop in progesterone mimics menses, and day 12 corresponds to tissue repair in the early proliferative phase. Indeed, similar regulation of marker genes also supports the proposed link between overtly bleeding mice and human menstruation. Fn1 mRNA was increased in the ‘‘secretory-like’’ phase on day 4 of pseudopregnancy in line with human progestin-stimulated FN1 expression. Increased expression of stromal cell-derived vimentin during the human secretory phase and on day 4 of mouse pseudopregnancy might account for the cyclic variations in tissue composition in both species. The significant down-regulation and subsequent up-regulation of the epithelium-derived keratin 19 mRNA is thought to be a molecular sign of ongoing epithelial breakdown and repair as suggested by immunohistological investigations. In addition, the expression of Pecam1 was increased during bleeding in mice, and PECAM1 is intensely stained in stroma and endothelial cells during human menstruation. Correspondingly, the mRNA of the pro-angiogenic factor VEGF-A,Praeruptorin-B which is highly up-regulated in human menstruation, reached its highest level of expression during bleeding in mice. The growth activity of stroma cells is elevated in both the proliferative phase and the late secretory phase of the menstrual cycle, whereby the latter is induced by progesterone in a second wave of proliferation. By investigating the expression of Mki-67 mRNA which is expressed by cycling cells, we demonstrated progesteroneinduced proliferation on day 4 of pseudopregnancy in mice. Moreover, humans display cyclic mRNA expression of the prosurvival factor BCL-2. A contribution of apoptosis was likewise demonstrated in menstruation-like processes in mice. The Bcl2/Bax ratio was exclusively decreased during bleeding as an indicator of the regulation of survival and apoptosis. Whether Hmox1 displays cycle-dependent expression during human menstruation is not completely understood. However, Hmox1 mRNA is typically induced after erythrocyte lysis and in hypoxia and inflammation, suggesting increased expression during tissue breakdown and bleeding which was confirmed by our data. Menstrual breakdown is triggered by prostaglandins, and both rate-limiting enzymes of prostaglandin synthesis, PTGS1 and PTGS2, display cyclic expression. The high expression of PTGS1 on day 4 of pseudopregnancy and increased expression of PTGS2 during bleeding are consistent with human data. It is widely accepted that immunological mediators play a role in menstruation and, correspondingly, we detected regulated gene expression for pro-inflammatory cytokines like Il6 and Il15, as has been described in humans.

The first was characterized by a peak in endometrial mRNA expression on day 4 of pseudopregnancy. This was shown for the stromal cell-derived vimentin, fibronectin 1, insulin growth factor 1, the proliferation marker Mki67, and the prostaglandin-endoperoxide synthase 1. The second expression pattern had a typical maximum of expression on day 9; the day of heaviest visible bleeding. By this day significantly increased expression was detected for Pecam1, which accounts for a large proportion of intercellular junctions of endothelial cells, and for the growth factor Vegfa. In addition, prostaglandin-endoperoxide synthase 2 was up-regulated 10-fold during bleeding compared to day 4. To sum up, the results imply that genes important for various cell- and tissue-specific processes were differentially regulated within the decidualized endometrium of pseudopregnant mice. In general, three expression patterns were represented. The pattern most frequently detected here was the up-regulation of genes during bleeding on day 9. Experimentally induced overt menstruation has not been reported for any species so far. Mouse models mimicking menstruation are known,Madecassoside but overt bleeding has never been described. The data presented in this study show that menstruation is not just species-specific. It is more likely that previous experimental approaches have not fulfilled the prerequisites for overt menstruation. Our data confirmed that pseudopregnant mice with decidualized endometrium display endometrial tissue destruction and repair. Moreover, for the first time, active flushing of shed tissue and blood from the vagina as a feature of overt menstruation was observed in mice. Uterine bleeding was triggered by the natural drop of previously increased Sanggenone-D endogenous progesterone, which occurred between day 7 and day 8 or day 8 and day 9 of pseudopregnancy. This temporal variation may reflect the individuality of mice with respect to the degradation of their corpora lutea in the ovary. Withdrawal of endogenous progesterone coincided with the onset of bleeding. The causal relationship of both events was confirmed by experimental antagonizing endogenous progesterone signaling through mifepristone administration that apparently caused almost immediate bleeding. The induction of overt bleeding in a naturally non-menstruating species like the mouse support the hypothesis that the amount of tissue and blood determines whether expulsion or reabsorption takes place. It was possible to achieve differentiation of a large amount of tissue by increasing the stimulus for decidualization in the form of a 5-fold larger volume of oil than that used by other authors. This led to decidualization of both uterine horns, which further increased the amount of tissue and blood to be flushed out. In order to obtain overt bleeding, we profited from the intrinsic ovarian regulation of intact pseudopregnant female mice compared to ovariectomized mice supplied by exogenous hormones used in former studies. In comparison to the established model, we prolonged the duration of the processes underlying bleeding: changes in uterus size and morphology occurred very rapidly within hours after progesterone withdrawal in ovariectomized mice, whereas these processes took place over a few days in our study. This extension of time, together with the determination of heavy bleeding by scoring bleeding intensity, will increase the feasibility of analyzing the kinetics of bleeding or its intensity. Hence, overtly bleeding mice are expected to be valuable for studying the molecular pathways affecting menstrual disorders such as menorrhagia. Our data demonstrate that the conversion of non-menstruating mice to mice displaying overt bleeding as a characteristic of menstruation is feasible.

This triggers the breakdown of endometrial tissue and the flushing of shed endometrium and blood in human overt menstruation, or the reabsorption of the endometrial lining in the estrous cycle. Consequently, common laboratory animals such as mice or rats cannot be used to directly study the mechanisms of overt menstruation as it occurs in humans. In this regard, Finn and Pope described in 1984 a mouse model that mimics menstruationlike processes: ovariectomized mice treated with a special hormone schedule showed decidualization after intrauterine oil injection, endometrial breakdown after progesterone withdrawal, and repair thereafter. However, it has to be considered that utilizing ovariectomized mice combined with an artificial hormone supply excludes any natural impact of ovarian hormones, estrogen and progesterone, which are essential to govern the endometrial functions in human menstruation. Surprisingly, even though the intrinsic drawbacks of this model have been well recognized, very little work has been done to develop it further. In recent studies, the artificial exogenous hormone supply was still necessary and visible bleeding and shed tissue comparable to that seen in women has never been observed in mice. There is therefore still a great need for a model that mimics human menstruation. Against this background it first has to be clarified Procyanidin-B1 why certain species repel rather than reabsorb endometrial tissue and which mechanisms turn a non-menstruating species into an overtly menstruating species. Among other theories, menstruation has been proposed as a mechanism to protect the uterus from sperm-borne pathogens. Another hypothesis suggests that repelling or reabsorbing the endometrial lining might be less costly than keeping it in an active metabolic state by luteal maintenance. Whether expulsion or absorption takes place might be determined by the amounts of tissue and blood: for large quantities of blood and tissue shedding and flushing might be more economical. To prove whether overt bleeding is determined by the quantity of differentiated endometrial tissue, or whether it is restricted to Procyanidin-B2 a few species the feasibility of inducing overt bleeding in mice was tested. Intact pseudopregnant mice were used to exploit intrinsic hormonal changes largely comparable to the human situation. The transferability to human menstruation was verified by investigating bleeding intensity and endometrial gene expression and by histological examination of the uteri. Importantly, in mice with decidualized endometrium the spontaneous drop of endogenous progesterone levels was sufficient to induce menstruationlike processes including the extravaginally visible bleeding typically seen in overt menstruation. Moreover, within this time course the color of the uteri changed from pink to dark red and back to pink. Overall, decidualized endometrium of pseudopregnant mice displays immense morphological changes: tissue construction, breakdown, extravasation of blood, and repair, accompanied by gain and loss of uterine weight and size, and, most importantly, by overt vaginal bleeding. Coordination of the various menstrual events is achieved by regulation of a number of genes involved in processes such as decidualization, angiogenesis, proliferation, apoptosis, and inflammation. In order to discover how the menstruation-like bleeding seen in mice correlates with the endometrial expression of genes known to be regulated in human menstruation, we analyzed mRNA levels at different points in time as shown in Figure 5. Endometrial samples were collected from mice in proestrus, on day 4 of pseudopregnancy, and after decidualization on day 9 of pseudopregnancy and on day 14. In general, we identified three different mRNA expression profiles.

We could then wonder why the major regulator of glucocorticoid metabolism, 11bHSD2, is not expressed? Could this lack of expression represent a temporal window necessary for glucocorticoid activity? The combined lack of renal 11bHSD2 activity and the absence of MR expression in the neonatal kidney leave glucocorticoids free to access and activate GR, which might have an important role in kidney development and maturation. It has been suggested that glucocorticoids are implicated in developmental programming. This hypothesis could be sustained by the fact that, at variance with the kidney, 11bHSD2 activity is detected in other organs in the newborn. It is particularly interesting to note that while it is barely expressed in the adult brain, a high 11bHSD2 activity is detected in the developping central nervous system in rats, mice and humans until the end of gestation. It has been postulated that 11bHSD2 during fetal and neonatal life is essential to protect the developing nervous system from deleterious consequences of glucocorticoid exposure. Therefore 11bHSD2 could have an organ specific pattern of expression in the neonatal period, protecting against or facilitating glucocorticoid actions. This developmental process could contribute to the increased short-term adverse outcome rate observed in extremely low birth weight infants with high cortisol concentrations. Moreover, this specific temporal window where the kidney appears to be particularly sensitive to glucocorticoid action could explain the deleterious effects of prenatal glucocorticoid overexposure on renal development or epigenetic modifications leading to the predisposition for adult hypertension. Finally, our study demonstrates the existence of Benzoylpaeoniflorin a physiological, temporal 11bHSD2 expression window specific to the kidney, which appears to be necessary for optimal fetal and neonatal development, but as a result could also represent a breach in the protective mechanisms against excess glucocorticoid exposure responsible for adverse short-term and long-term effects through fetal programming, with a higher predisposition to specific diseases in later life. Although menstruation is at least as old as the human species, knowledge of the underlying mechanisms is limited. Its physiology has rarely been studied, and this is partly due to a lack of appropriate animal models. Apart from humans very few species of Old World monkeys and apes experience a menstrual cycle. This includes the flushing out of endometrial tissue and blood from the uterus to the vagina, clearly visible as overt menstruation. Two menstruating species of non-primates have been described the elephant shrew and the bat. The estrous cycle is much more common in placental mammals than the menstrual cycle. The former is characterized by Ursolic-acid complete reabsorption of the endometrial lining, which is not externally visible. Both reproduction cycles involve proliferation of stromal cells and ovulation, followed by the formation of the progesteroneproducing corpus luteum in the ovary. However, in the menstrual cycle endometrial stromal cells differentiate into decidual cells in response to the rapidly increasing progesterone level despite the absence of a blastocyst. In contrast, in the estrous cycle decidualization occurs only after conception, e.g. in mice it starts on day 4 post coitum, when endometrial stromal cells surround the implanting blastocysts. The decidua provides a vascular network for nutrition and gas exchange for the developing embryo if implantation occurs before a functional placenta is established. In both the menstrual and the estrous cycle the absence of implantation induces the degeneration of the corpus luteum in the ovary and subsequently the progesterone level drops.