The Plasminogen Activator System, Glucocorticoid, and Mineralocorticoid Receptors in the Primate Endometrium During Artificial Menstrual Cycles

As a key mechanism in fibrinolysis and tissue remodeling, the plasminogen activator system has been suggested in the process of endometrial shedding and tissue remodeling. Previous studies have explored the role of estrogen, progesterone and androgen receptors as well as elements of the renin-angiotensin-aldosterone system in shaping the morphology of the endometrium. This study investigates the distribution and concentrations of the mineralocorticoid receptor, glucocorticoid receptor, tissue plasminogen activator, urokinase plasminogen activator and plasminogen activator inhibitor-1 within the endometrial stroma, glandular and endothelial cells of the primate endometrium during artificial menstrual cycles. Our immunohistochemistry quantification shows mineralocorticoid and glucocorticoid receptors are ubiquitously distributed within the macaque endometrium with their patterns of expression following similar fluctuations to urokinase and tissue plasminogen activators particularly within the endometrial vasculature. These proteins are present in endometrial vasculature in high levels during the proliferative phase, decreasing levels during the secretory phase followed by rising levels in the menstrual phase. These similarities could suggest overlapping pathways and interactions between the plasminogen activator system and the steroid receptors within the endometrium. Given the anti-inflammatory properties of glucocorticoids and the role of plasminogen activators in endometrial breakdown, the glucocorticoid receptor may be contributing to stabilizing the endometrium by regulating plasminogen activators during the proliferative phase and menstruation. Further, given the anti-mineralocorticoid properties of certain anti-androgenic progestins and their reduced unscheduled uterine bleeding patterns, the mineralocorticoid receptor may be involved in unscheduled endometrial bleeding.

Mineralocorticoid receptor, glucocorticoid receptor, tissue plasminogen activator, urokinase plasminogen activator, plasminogen activator inhibitor, plasminogen activator system

The uterine endometrium is composed of epithelial cells lining the uterine lumen, glandular cells, stromal cells, endothelial cells, smooth muscle cells surrounding blood vessels and supportive tissue composed of fibrin and collagen. Throughout the menstrual cycle, the endometrium undergoes morphological and biochemical changes reflecting the intricate interplay of many hormones and their receptors in response to ovarian estrogen and progesterone biosynthesis and secretion1. Alongside receptors for estrogen and progesterone hormones, previous studies have provided evidence for the role of other key hormones including androgens and corticosteroids in shaping the dynamic morphology of the endometrium2–4. Estrogen, progesterone and androgen receptors have all previously been demonstrated in the various tissues of the endometrium5. The human endometrium also contains all of the elements of the renin-angiotensin-aldosterone system (RAAS). Renin, angiotensin-converting enzyme (ACE), and Angiotensin (AT) type 1 and type 2 receptors have previously been demonstrated in glandular epithelial cells and stromal cells by immunohistochemistry6. The glucocorticoid receptor (GR) expression has been demonstrated in the stromal and endothelial cells7–9. Glucocorticoids are well known inhibitors of angiogenesis10,11. Additionally, the mineralocorticoid receptor (MR) has been shown in endometrial glandular and stromal cells, however it has not been identified in endometrial endothelial cells to the best of our knowledge although it is present in endothelial cells in other vascular beds12. Progestins with antimineralocorticoid activity such as drospirenone have been proposed as therapies to reduce unscheduled endometrial bleeding. Therefore, endometrial MR, if present in endothelial cells may have a significant role in the menstrual cycle and potentially in the process of abnormal uterine bleeding13. Dilated small blood vessels located just beneath the surface epithelium are a common finding in women with abnormal uterine bleeding14,15. Overt bleeding occurs when both the micro vessel wall and the endometrial epithelial lining are disrupted and any clot is dissolved. This suggests that there are increased local collagenase and proteases at the site of micro vessel rupture within the endometrium. The plasminogen activator system (PAS) is well established as involved in both normal and pathologic tissue remodeling16. PAS consists of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and their specific inhibitors, plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2), ubiquitously distributed throughout the body17. The interaction between tPA and uPA with their high-affinity receptors – tissue plasminogen activator receptor (tPAR) and urokinase plasminogen activator receptor (uPAR) respectively results in activation of the enzyme plasminogen by proteolytic cleavage. This in turn initiates a cascade of events resulting in plasmin, a serine protease involved in fibrinolysis of blood clots and proteolysis via activation of collagenases16–18. The endometrium contains fluctuating levels of tPA and uPA during the ovarian cycle19–22. uPA has been reported to be most active within the endometrial stroma as progesterone is withdrawn23. Previous studies have demonstrated that levels of PA activity in the endometrium as measured in uterine fluid increases during the proliferative phase and decreases during the secretory phase to increase again in the pre-menstrual phase19,22. When correlated with the ovarian cycle, this evidence indicates the plasminogen activator system is highly involved in the proliferation, differentiation and degradation of the endometrium throughout the menstrual cycle and there is an intricate interplay between cycling ovarian hormones and the plasminogen activator system24,25. Additionally, androgenic progestin-only contraceptives such as levonorgestrel have previously been shown to be associated with significant unscheduled uterine bleeding26,27. The lesser associated dysfunctional bleeding with anti-androgenic progestins suggests that there may be some intrinsic regulatory mechanisms in anti-androgenic progestins. Given the anti-mineralocorticoid properties of anti-androgenic progestins such as drospirenone, MR may potentially be involved in the interplay with uPA and tPA systems at the level of the endometrium28. This study was designed to investigate the changes in GR and MR in endometrial stroma, glandular and endothelial cells of the primate endometrium during artificial menstrual cycles and explore their potential role and effects in the menstrual cycle. We have also identified the relative levels and distribution of uPA, tPA, and plasminogen activator inhibitor-1 (PAI-1) in the same tissues during the different phases of the artificial menstrual cycle to explore the relation and role of the plasminogen activator system in the hormonal cycle.

& MATERIALS Macaque Samples Archived paraffin-embedded sections of rhesus macaque (Macaca mulatta) uterus (n=12) were obtained from the Oregon National Primate Research Center (ONPRC). Animal husbandry was provided by the ONPRC Department of Comparative Medicine and animal use was reviewed and approved by the ONPRC Institutional Animal Care and Use Committee. The macaques were ovariectomized and treated with defined regimens of estradiol (E2) and progesterone to stimulate two artificial menstrual cycles3,5 (Fig 1). The artificial cycles were initiated by the subcutaneous insertion of a 5-cm E2 releasing Silastic capsule. After 14 days of E2 priming, a 6-cm progesterone-releasing capsule was implanted. Removal of the progesterone capsule on cycle day 28 and leaving the E2 capsule in place induced menstruation and completed cycle 1 (Fig 1). During the second cycle, the uterus was collected from animals on either cycle day 3–4 (menstruation; n=4), day 14 (E2 alone; proliferative phase; n=4) or cycle day 19–21 corresponding to the mid secretory phase (5–7 days E2 + P4; n=4). Throughout the artificial cycle E2 levels were 92+11 pg/ml and mid-secretory phase P levels were 6.97+0.96 ng/ml, which is consistent with natural menstrual cycles in rhesus macaques. In each case, the reproductive tracts were prepared by dissecting the uterus from the oviducts and cervix. The uterine body was cut into equal quarters along the longitudinal axis. Full-thickness endometrial cross-sectional slices spanning from the lumen to the myometrium were fixed overnight (~18h) in 4% paraformaldehyde, dehydrated, cleared with xylene and embedded in paraffin. 25 slides were obtained from each of the full thickness uterus samples, cut at 5um thickness. Chromogenic staining Immunohistochemical staining on tissue sections were done chromogenically as described previously by Chandra et al29. Staining steps were carried out at room temperature, unless otherwise indicated. Tissue sections (5 μm) were de-paraffinized and rehydrated followed by antigen retrieval in citrate buffer (pH 6.2, DAKO) at high temperature. The tissue sections were incubated with appropriate protein block for 30 minutes to block non-specific protein binding. Primary antibodies were applied on tissue sections and left overnight at 4°C, followed by treatment with appropriate biotinylated secondary antibodies for 30 minutes at room temperature (See Table 1). The sections were treated with ABC reagent (Vector labs, Burlingame, CA) and staining reaction was developed by using AEC chromogen-substrate kit (SkyTek labs, Mississauga, Ontario Canada). The slides were cover-slipped using accergyl mounting media (Accurate chemicals, NY). Positive controls used monkey kidney, tonsil, pancreas and tumor for each of the receptors to establish positive thresholds for analysis. Negative controls were tissues incubated without a primary antibody and no staining was observed in the analyzed tissues. Table 1: | Antibody | Source | Cat. # | Dilution | |---|---|---|---| | Primary | ||| | Mineralocorticoid receptor (MR) | abcam | ab2774 | `1:200 | | Von Willebrand Factor (VWF) | abcam | ab6994 | `1:1000 | | Plasminogen activator (tPA) | abcam | ab157469 | `1:200 | | Plasminogen activator inhibitor-1 (PAI-1) | abcam | ab66705 | `1:200 | | anti-glucocorticoid receptor antibody (GR) | abcam | ab3578 | `1:200 | | urokinase plasminogen activator (uPA) | R&D Systems | AF1310 | `1:40 | | Secondary | ||| | Anti rbt in goat | Vector | BA-1000 | `1:200 | | Anti ms in horse | Vector | BA-2000 | `1:200 | | Alexa-Fluor555 goat anti rabbit | Life Technologies | A 21428 | `1:100 | | Alexa-Fluor488 goat anti mouse | Life Technologies | A11001 | `1:100 | | Protein Block | ||| | Normal horse serum | Vector | S-2000 | 1.5% | | Normal goat serum | Vector | S-1000 | 1.5% | Immunofluorescence staining of endometrial endothelial cells Paraffin embedded tissue sections were processed as described above for mineralocorticoid receptor and von-Willebrand factor. Normal goat serum was used as a protein block. Two antibodies (mouse and rabbit origin) were diluted separately, mixed and applied on tissues with final dilutions as listed in Table 1. The slides were incubated overnight at 4°C. The tissues were treated with a mixture of Alexa fluor conjugated secondary antibodies (Alexa-fluor 555 goat anti rabbit and 488 goat anti-mouse) and left in dark for an hour on the following day. This was followed by application of ABC reagent (Vector labs, Burlingame, CA). The slides were mounted using Vectoshield mounting media with DAPI (Vector labs, Burlingame, CA) in the dark. Slides were visualized under Nikon Eclipse DR-Ri2 fluorescence microscope. MR was stained green and VWF red. Nucleus showed blue staining with DAPI. The images were overlapped to see double staining of the antibodies. Antibodies used in the study are detailed in Table 1. Image J Analysis All immunohistochemical stained slides were reviewed under Olympus BX50 microscope at bright field. All images to be analyzed were captured at 20x magnification under identical, standardized settings using Olympus DP70 camera and saved in TIFF format. Four images were collected of endometrial glandular tissue alone, endometrial vasculature alone, and endometrial stroma from random sections of each tissue section. Images of glandular tissue were analyzed by tracing around glands of various shapes and sizes in each image using free selection tool. Images of stroma were analyzed by outlining a homogenous section of stromal tissue using the oval tool and analyzing four different areas within each section. Images of vasculature were analyzed for the intensity of staining by outlining the endothelial layer using the free selection tool within four random sections of endometrial stroma. Selection areas could not be standardized for size due to differences in the size of the glands and vessels; therefore, percent area was used for comparison, which is unaffected by the size of the selection area. ImageJ analysis software (NIH, Bethesda, MD) was used to analyze the images for intensity of staining in endometrial glands, stroma and vasculature. Two independent observers analyzed four images from each macaque endometrial tissue (glandular, stromal and vasculature) for each of the analytes GR, MR, tPA, uPA and PAI-1 during the proliferative, secretory and menstrual phases. Each image was converted into a binary (black and white) image on Image J and analyzed for integrated density and area fraction by standardized thresholds determined by positive and negative controls. Data were reported as percentage of pixels in the selection that have been highlighted by the set threshold. Statistical Analysis The data points collected by both observers for each tissue and analyte were averaged and the mean, standard deviation (SD), and variance (s2) were determined for GR, MR, tPA, uPA, and PAI-1 during the proliferative (n=4), secretory (n=4) and menstrual phases (n=4). Data were subjected to ANOVA with Tukey’s multiple comparison test using GraphPad Prism version 6.00 for windows, GraphPad software (La Jolla, California). Variance (s2) was calculated as the sum of squared differences from the mean Σ(xi – x)2 divided by the size of the data set (n-1). Significance was tested at p<0.05 as compared to *proliferative phase and to ^secretory phase where proliferative phase was compared to both secretory and menstrual phases; and the secretory phase was compared to both the proliferative and menstrual phases with significant difference denoted by * as compared to proliferative phase and by ^ as compared to secretory phase (Table 2). Table 2 – | Positive Controls | Proliferative | Secretory | Menses | ANOVA (p) | | |---|---|---|---|---|---| | tPA | ||||| | Stroma | 14.9 ± 3.6 (s2=13.3) | 6.0 ± 1.9* (s2=3.9) | 20.1 ± 4.2^ (s2=18.0) | <0.0008 | | | Glands | 50.6 ± 6.5 (s2=42.3) | 28.7 ± 6.3* (s2=39.6) | 38.0 ± 15.6 (s2=242.0) | 0.04 | | | Vasculature | 38.6 ± 7.9 (s2=62.8) | 12.9 ± 10.1*(s2=102.7) | 30.9 ± 10.5 (s2=111.1) | 0.01 | | | Monkey Kidney | 52.2 | |||| | uPA | ||||| | Stroma | 17.0 ± 1.2 (s2=1.3) | 10.4 ± 2.3* (s2=5.4) | 21.1 ± 3.0^ (s2=8.7) | 0.0003 | | | Glands | 40.2 ±13.3 (s2=175.7) | 14.7 ± 5.8* (s2=33.5) | 22.1 ± 7.7 (s2=59.6) | 0.01 | | | Vasculature | 21.2 ±3.7 (s2=13.5) | 17.6 ±9.5 (s2=91.0) | 20.3 ±6.1 (s2=37.5) | 0.74 | | | Monkey Tumor | 39.7 | |||| | PAI-1 | ||||| | Stroma | 15.8 ±2.4 (s2=5.9) | 6.9 ± 2.1 (s2=4.5) | 31.8 ± 7.3*^ (s2=54.0) | 0.0001 | | | Glands | 80.5 ±3.9 (s2=15.9) | 49.8 ± 23.0* (s2=528.6) | 72.3 ± 6.5 (s2=42.4) | 0.03 | | | Vasculature | 26.6 ±8.6 (s2=74.7) | 13.3 ± 7.0 (s2=49.3) | 36.8 ±6.9^ (s2=46.9) | 0.005 | | | Monkey Kidney | 54.7 | |||| | GR | ||||| | Stroma | 12.5 ±1.7 (s2=3.1) | 13.7 ± 3.2 (s2=10.1) | 9.8± 1.4 (s2=2.1) | 0.09 | | | Glands | 27.0 ±10.9 (s2=118.5) | 12.8 ± 10.9 (s2=119.0) | 12.1 ± 4.0 (s2=16.4) | 0.08 | | | Vasculature | 21.5 ±2.1 (s2=4.4) | 17.1 ±4.4 (s2=19.0) | 16.0 ±4.7 (s2=22.5) | 0.17 | | | Monkey Tonsil | 36.7 | |||| | MR | ||||| | Stroma | 14.1 ±2.2 (s2=5.0) | 14.1 ±3.4 (s2=11.4) | 10.9 ±6.0 (s2=36.0) | 0.5 | | | Glands | 17.1 ±6.1 (s2=37.4) | 12.1 ±9.4 (s2=89.0) | 11.4 ±5.3 (s2=28.5) | 0.5 | | | Vasculature | 25.5 ±5.6 (s2=31.1) | 17.3 ±3.8 (s2=14.7) | 18.9 ±5.8 (s2=33.3) | 0.1 | | | Monkey Pancreas | 56.6 | Values are expressed as Mean ± SD (variance s2) of n=4/group. Data were subjected to ANOVA with Tukey’s multiple comparison test at p<0.05. Significance was compared to *proliferative phase and to ^secretory phase uPA: urokinase Plasminogen activator tPA: tissue Plasminogen activator PAI-1: Plasminogen Activator Inhibitor-1 GR: Glucocorticoid receptor MR: Mineralocorticoid receptor

Mineralocorticoid Receptor Mineralocorticoid receptor (MR) staining was observed in endometrial endothelial cells during all three phases of the artificial menstrual cycle in rhesus macaques through colocalization with vWF by both immunofluorescence and immunohistochemistry (Fig 2–4). The endometrial endothelial MR expression fluctuates during the different phases of the menstrual cycle with increased staining by percent area in the proliferative phase and decreased staining in the secretory phase and intermediate levels during the menstrual phase (Fig 5A). The percent area averaged by the two observers was 25.5% in the proliferative phase, 18.9% in the menstrual phase, and 17.3% in the secretory phase (Fig 5A, Table 2). Endometrial glandular MR showed highest staining by percent area in the proliferative phase with similar levels during menses and secretory phases (Fig 5B). The percent area averaged by the two observers was 17.1% in the proliferative phase, 11.4 % in the menstrual phase, and 12.1% in the secretory phase (Fig 5B, Table 2). Stromal cell MR showed similar levels of staining during proliferative and secretory phases and decreased staining during menses (Fig 5C). The percent area averaged by the two observers was 14.1% in the proliferative phase, 14.1% in the secretory phase and 10.9 % in the menstrual phase (Fig 5C, Table 2). The immunohistochemical results for each tissue and the time in the artificial cycle are shown in Figure 4. While a qualitative analysis among the groups showed the above discussed trends, the differences in MR expression within the different compartments of the endometrium throughout the different phases of the menstrual cycle failed to reach statistical significance. MR expression was also analyzed by immunofluorescence, which showed the lack of MR staining in the endothelial cells during the secretory phase with the majority of MR expression noted within the stroma. In contrast, MR was noted to be expressed within endothelial cells of endometrial vasculature during the proliferative and menstrual phases as shown by vWF co-localization (Fig 3). Glucocorticoid Receptor The glucocorticoid receptor (GR) immunohistochemistry results demonstrated fluctuating levels during the different phases of the menstrual cycle with highest level of expression during the proliferative phase. When analyzed within endometrial vasculature, the averaged percent area was 21.5% in the proliferative phase, 17.1% in the secretory phase, and 16.0% in the menses phase (Fig 7A, Table 2). The differential expression of GR within each artificial menstrual phase showed a similar pattern when analyzed within glands. The percent area averaged by the two observers was 27.0%, 12.8% and 12.1% during the proliferative, secretory and menstrual phases respectively within glands (Fig 7B, Table 2). The stromal differences were relatively small and the percent area was 12.5%, 13.7, and 9.8% during proliferative, secretory, and menstrual phases, respectively (Fig 7C, Table 2). Similar to MR, the observed qualitative trends of GR failed to show a statistically significant difference between hormonal phases. The immunohistochemical localization of the GR in endometrial vasculature, glands, and stroma during each phase of the artificial menstrual cycles are shown in Figure 6. Tissue Plasminogen Activator and Urokinase Plasminogen Activator The plasminogen activator system proteins uPA, tPA and plasminogen-activator inhibitor (PAI-1) were examined separately within the different compartments (glandular, stromal and vasculature) of the macaque endometrium during the different phases of the menstrual cycle. Both uPA and tPA showed notably higher levels during the proliferative phase, followed by the menstrual phase, and secretory phase within endometrial endothelial cells and glandular epithelial cells. The average percent area of tPA within endometrial endothelial cells was 38.6%, 30.9%, and 12.9% during the proliferative, menstrual, and secretory phases respectively with significant difference between proliferative and secretory phases (Fig 9A, Table 2). The average percent area of tPA within endometrial glandular epithelium was 50.6%, 38.0%, and 28.7% during the proliferative, menstrual and secretory phases respectively and reached statistical significance between proliferative and secretory phases (Fig 9B, Table 2). A similar pattern was demonstrated in the differential expression of uPA within the vasculature and glandular compartments of the endometrium during the artificial hormonal phases. The average percent area of uPA within endometrial endothelial cells was 21.2%, 20.3%, and 17.6% during the proliferative, menstrual, and secretory phases respectively, however these findings failed to reach statistical significance (Fig 11A, Table 2). The average percent area of uPA within endometrial glandular epithelium was 40.2%, 22.1%, and 14.7% during the proliferative, menses and secretory phases respectively and the differences were statistically significant between proliferative and secretory phases (Fig 11B, Table 2). The pattern of tPA and uPA expression within the stroma showed statistically significant higher levels during the menstrual phase, followed by the proliferative phase, and secretory phase. The percent area of tPA within the stroma was 20.1%, 14.9%, and 6.0% in the menstrual, proliferative, and secretory phases respectively with significant difference between all groups (Fig 9C, Table 2). The percent area of uPA within the stroma was 21.1%, 17.0%, and 10.4% in the menstrual, proliferative, and secretory phases respectively with again significant different between all groups (Fig 11C, Table 2). The immunohistochemical localization of tPA and uPA during each phase of the artificial hormonal cycles are shown in Figures 8 and 10. Plasminogen Activator Inhibitor-1 PAI-1 in the vasculature showed the highest levels of staining during the menstrual phase (36.8%), followed by the proliferative phase (26.6%) and secretory phase (13.3) (Fig 13A, Table 2). This pattern was also present within the stroma with the highest level of staining observed during the menstrual phase (31.8%), followed by the proliferative (15.8%) and secretory phases (6.9%) (Fig 13C, Table 2). PAI-1 in glandular epithelium conversely had the highest level of staining during the proliferative phase (80.5%), followed by the menstrual (72.3%) and secretory phases (49.8%) (Fig 13B, Table 2). The observed differences in PAI-I levels between the different hormonal phases were statistically significant between secretory and menstrual phases within the endometrial endothelial cells, between secretory and proliferative phases within endometrial glands and between both proliferative and secretory phases as compared to the menstrual phase within the endometrial stroma (Table 2). The immunohistochemical staining within each tissue during each artificial hormonal cycle is demonstrated in Figure 12.

Role of Mineralocorticoid and Glucocorticoid Receptors in the Morphologic Changes of the Endometrium The endometrial morphology undergoes well described changes during artificial hormonal cycles comprised of estradiol, estradiol plus progesterone and during progesterone withdrawal in the macaque3. Both estrogen receptor (ER) and progesterone receptor (PR) in the endometrial tissues reflect the changes in circulating hormone levels during normal and artificial cycles in rhesus macaques, tightly correlating with morphological changes within the endometrium3–4. Endometrial morphologic changes following the use of exogenous estrogen and progestins for contraceptive purposes are well described and reflect dynamic changes in the ER and PR in the endometrial tissue26,30. Progesterone receptor modulators significantly alter endometrial morphology in humans and macaques5,26,31. The morphologic changes occurring with synthetic androgenic progestins and progesterone receptor antagonists also appear to involve the endometrial androgen receptor (AR)31–33. Adrenal corticoids have been studied in previous reports and their receptors have been identified in luminal and glandular epithelial cells, both in undifferentiated and decidualized stromal cells, smooth muscle cells surrounding blood vessels and in the endothelial cells7,9,34. The GR has been demonstrated in human stromal cells, vascular endothelial cells, uterine natural killer cells and mast cells similar to estrogen, progesterone and androgen receptors7–9,35. We have characterized the localization of MR and GR in this study, which generally show similar expression and distribution of each receptor in the macaque stroma across the hormonal cycles. Previous studies have demonstrated sub-chronic administration of cortisol over one menstrual cycle in the cynomologous monkey retarded the secretory endometrium and reduced endometrial ER by 50 %36. Hypercortisolism such as in Cushing’s disease has not been associated with endometrial abnormalities37. There is limited evidence for effects of cortisol and aldosterone on endometrial morphology despite both the GR and MR being present in the endometrial stroma. Aldosterone, a mineralocorticoid has been shown to be involved in decidualization but most likely via the renin-angiotensin-aldosterone system, not the MR35. Progesterone receptor modulators such as ulipristal acetate have limited binding to the GR and do not interact with the MR38. One of the well-established roles of the activated GR is the upregulation of anti-inflammatory proteins in the nucleus and repression of pro-inflammatory proteins. Inflammation is thought to play a major role in the initiation of multiple downstream biologic events that culminate in the loss of integrity in the endometrium with resultant menstruation39. The influx of leukocytes just prior to the onset of menstruation contributes cytokines that can potentiate multiple proteases in the endometrium. Metalloproteinases are associated with the dissolution of endometrial extracellular matrix resulting in loss of tissue cohesion and menstruation. Plasmin, activated by tPA and uPA is involved in breaking down a wide range of extracellular matrix components by activating various matrix metalloproteinases (MMP)40. MMPs are upregulated during progesterone withdrawal and involved in endometrial breakdown resulting in menstruation in women and female monkeys41–43. Our study shows similar expression patterns between GR and the plasminogen activator system proteins uPA and tPA, particularly within the endometrial vasculature (Fig 6–11), with high levels during the proliferative phase and decreasing levels during the secretory phase followed by a rise again during the menstrual phase. The similarities in the distribution and fluctuations of these proteins may signify important interactions between these pathways. Given the known anti-inflammatory properties of GR and the role of tPA and uPA in endometrial breakdown, GR may be contributing to stabilizing the endometrium against tPA and uPA during the proliferative phase prior to menstruation. Activation of the GR by glucocorticoids has been shown to increase GR associated genes in human umbilical vein endothelial cells44. Glucocorticoids have been found to increase PAI-1 gene and mRNA in human endothelial cells from the femoral head and a human ovarian cancer cell line respectively, in vitro45,46. The major function of GR activation is angiogenesis 1. Our finding of decreased GR in endothelial cells in the menstrual phase of the macaque cycle suggests reduction in angiogenesis in the endometrium during menstruation. The effect of GR activation on endothelial cell function and the plasminogen activator system is suggested by the finding of increased inactivation of cortisol by 11-β steroid dehydrogenase 2 in the endometrium in women with heavy menstrual bleeding that would increase fibrinolysis1. MR has been demonstrated in endometrial glandular and stromal cells. We have identified MR in human immortalized endometrial endothelial cells and MR has been demonstrated in endothelial cells in other vascular beds9,12 47. We now have evidence for the first time that MR is present in the monkey endometrial glandular stromal and endothelial cells. Demonstration of MR within the endometrial vasculature holds potential implications for unscheduled uterine bleeding patterns, particularly in setting of hormonal contraceptive use. Given the anti-mineralocorticoid properties of certain progestins used for contraception and their association with decreased abnormal uterine bleeding patterns, MR antagonism particularly may in part explain these effects12,13,47. MR and GR qualitatively appear to be upregulated during the proliferative phase in comparison to the menstrual and secretory phases particularly within the vasculature and glands (Fig 4–7, table 2). The inconsistent results within the stroma compared to vasculature and glands may be the result of the artifactual increased unfilled areas within secretory phase slides from edematous changes in the secretory phase in comparison to the relatively compacted stromal tissue during menses and proliferative phases. Immunofluorescent staining for MR within the macaque endometrium shows a shift from being primarily localized within the endothelial cells in the proliferative phase to stromal cells in the secretory phase (Fig 3). These results are consistent with the data extrapolated from immunohistochemistry analysis where MR shows higher levels of MR expression within endothelial cells during the proliferative phase and decreasing levels during the secretory phase. This could be due to a shift in MR expression from endothelial cells to stromal cells during the secretory phase. A similar pattern of expression has previously been demonstrated with uPA in the endometrium by real time PCR quantification of uPA mRNA showing higher level of expression within stromal cells in the secretory phase and no distinguishable expression during the proliferative phase21. In these studies, although uPA mRNA was exclusively isolated from stromal cells, immunohistochemical analysis of uPA showed localization in both stromal and epithelial cells with particularly more intense staining in the secretory phase than the proliferative phase. These findings suggest uPA is likely produced in the stromal cells during the secretory phase and then presumably binds to other cells of the endometrium in a paracrine way21. These studies did not analyze MR, however our data suggest a similar pattern of expression for MR, with a shift from being primarily localized within stromal cells, particularly during the secretory phase to being localized within epithelial and endothelial cells during the proliferative and menses phases (Fig. 3). Given the observed similarities in the fluctuation patterns of MR, GR and the plasminogen activator system proteins uPA and tPA in our study, MR may also be primarily expressed by stromal cells during the secretory phase then bind to glandular, epithelial and endothelial cells of the endometrium as deduced for uPA in previous studies. MR and GR appear to display nuclear expression within the cells of endometrial stroma and glands when observed on a subcellular level in higher magnification (Fig. 14). We have also colocalized vWF and MR within the endometrial vasculature along DAPI overlay of the nuclei, which show nuclear localization of MR throughout all phases of the menstrual cycle (Fig 3D, 3H, 3L). In the current study, we have chosen to analyze slides at 20x magnification to be able to analyze a larger surface area to add to the power of the study, however there may be differential subcellular localization of MR and GR between the different phases of the menstrual cycle better characterized at higher magnifications. Differential subcellular localization of these receptors may further elucidate their level of activity and function within the menstrual cycle. In the current study, we cannot draw conclusions on the subcellular compartmentalization of these receptors during the different phases of the menstrual cycle, but this may be a future direction for the study. Previous studies have also explored the role of the androgen receptor in the endometrium. Synthetic androgenic progestins used in combination oral contraceptives are known to alter the endometrial morphology to an atrophic or secretory morphology26. This has been reported to be due to their interaction with the progesterone receptor but the androgen receptor is involved in the observed endometrial morphologic changes. Previous research has demonstrated the interaction of progesterone receptor antagonists with the androgen receptor evidenced by the blockage of their activity by flutamide48,49. Administration of the progesterone receptor modulator, asoprisnel has also been shown to increase endometrial androgen receptor content associated with unique endometrial morphology in monkeys50. Progesterone receptor modulators can result in a unique endometrial morphology that is a benign finding. Levonorgestrel, an androgenic progestin has been shown to decrease endometrial vascular endothelial cell plasminogen activator inhibitor-1 activity in vitro via the androgen receptor51. Role of Plasminogen Activator System in the Morphological Changes of the Endometrium Our data support a role for the plasminogen activator system in the onset of menstruation. Levels of tPA and uPA were noted to be higher during the menstrual phase compared to the proliferative and secretory phases of the cycle (Fig 8–11, Table 2). The onset of menstruation, the shedding of the endometrium in response to withdrawal of ovarian estrogen and progesterone has previously been associated with local anoxia brought about by vasoconstriction of the spiral arteries1. Matrix metalloproteinases are upregulated just prior to and during menstruation due to progesterone withdrawal in both monkey and human endometrium 1,25,42,43,52–54. The resulting fragmentation of the extracellular matrix leads to loss of cellular adhesions, cellular apoptosis and shedding of the superficial layer of the endometrium. As a key mechanism in fibrinolysis and tissue remodeling, the plasminogen activator system is highly involved in the process of endometrial shedding and tissue remodeling20,55. The plasminogen activator system receptors and regulators have been studied within the endometrium and demonstrated to show fluctuating levels during different phases of the menstrual cycle influenced by the cyclic ovarian hormones23. However, information on their localized fluctuations within different compartments of the endometrium is not well established. Previous studies have demonstrated uPA and uPAR mRNA expression to be variable with probable shift from epithelial to stromal expression between proliferative and secretory phases23. Additionally in these studies, there is a discordance in the localization of mRNA and the corresponding protein within endometrial components. Specifically, uPA has been shown to be exclusively expressed by stromal cells, but demonstrated within both epithelial and stromal cells by immunohistochemistry, with the conclusion that uPA is likely produced by stromal cells then bound to epithelial cells23. Our current study examines these proteases in the different compartments of the endometrium throughout the menstrual cycle to show the differential concentration and localization of these receptors. Our study demonstrates tPA, uPA and PAI-1 are present in the lowest concentrations within the macaque endometrial stroma and glands during the secretory phase of the ovarian cycle (Fig 9, 11, 13, Table 2). These findings are consistent with the observed suppression of uPA protein and mRNA in endometrial stromal cells by progesterone as previously described20,56–59. In these studies, both bound and unbound forms of tPA and PAI-1 were demonstrated to be decreased during the secretory phase of the menstrual cycle in endometrial tissue extracts analyzed by ELISA. Our study demonstrates a similar progesterone effect by immunohistochemistry analysis of the macaque endometrium, where stromal tPA, uPA and PAI-1 concentrations decrease in the secretory phase under the influence of progesterone (Figures 9,11,13, Table 2). PAI-1 has previously been identified in the human endometrium as the primary regulator of the plasminogen activator system58 We have found that PAI-1 peaks during the proliferative phase within glands, likely as a mechanism to inactivate tPA and uPA in order to provide glandular tissue stability during proliferation (Fig 13B, Table 2). Within the vasculature, PAI-1 levels peak during the menstrual phase when blood vessels breakdown and endometrial shedding is greatest. The increased PAI-1 during menstruation may be a regulatory mechanism to prevent uncontrolled fibrinolysis and excessive bleeding (Fig 13A, Table 2)20,60. Vascular Breakdown and the Role of tPA, uPA and PAI-1 as Related to MR Our quantification based on immunohistochemistry analysis shows the MR and GR are present at higher levels within endometrial vasculature during the proliferative phase, which correlates with the peak of plasminogen activator proteins uPA and tPA within endometrial vasculature. The endometrial increase in MR during the proliferative phase at the peak of uPA and tPA within the endometrial vasculature may be a synergistic process in fibrinolysis. Our results show that PAI-1 also peaks during the proliferative phase within glands, likely as the principal inactivator of tPA and uPA to provide glandular and stromal cell stability during this phase. Endothelial cell PAI-1 levels peak during the menstrual phase when the breakdown of blood vessels and extracellular matrix is greatest, again likely as a mechanism to prevent uncontrolled fibrinolysis and modulate tissue remodeling20. MR and GR generally show the same pattern of expression and distribution within the macaque endometrium and their pattern of expression follows similar fluctuations to uPA and tPA particularly within the endometrial vasculature (Fig 5, 7, 9, 11, Table 2), with high levels noted during the proliferative phase, decreasing levels during the secretory phase followed by rising levels again during the menstrual phase. The similarities in the fluctuation patterns of these receptors and ligands may indicate overlapping pathways and interactions between these systems. MR and GR may be involved in the fibrinolysis and proteolysis pathways along with uPA and tPA. Progestins with antimineralocorticoid activity may be hindering this interaction with resulting reduction in unscheduled uterine bleeding patterns as seen with drospirenone13. However, mechanisms involved in these interactions are speculative and currently unknown with only limited indirect evidence. Our conclusions at this stage are mainly based on the qualitative analysis of these receptors.

MR has been identified within the endometrial endothelial cells of rhesus macaques for the first time to our knowledge. MR qualitatively appears to be upregulated during the proliferative phase in comparison to the menstrual and secretory phases, although these results have failed to show statistical significance, likely limited by our small sample size (n=12). Presence of MR within the vasculature of the endometrium holds important implications. Given the anti-mineralocorticoid properties of certain progestins used for contraception and their associated reduction in unscheduled uterine bleeding patterns, MR antagonism particularly may in part explain these effects by its potential interaction with the plasminogen activator system in endothelial cells as suggested by our study. GR is present along with MR in all endometrial tissues and may be involved in modulating inflammatory cell influx particularly during menses. Given uPA and tPA are fibrinolytic precursors, the upregulation of MR during the proliferative phase at the peak of uPA and tPA within the endometrial vasculature may indicate a synergistic process in clot breakdown. The anti-mineralocorticoid activity exhibited by certain progestin contraceptives may be controlling unscheduled uterine bleeding by abolishing this interaction. The fluctuations and patterns observed in the plasminogen activator system components as demonstrated in immunohistochemistry confirm in vitro data that progesterone decreases plasminogen activity in the secretory phase with significant increase in tPA, uPA and PAI-1 during the menstrual phase associated with active tissue remodeling and fibrinolysis to maintain liquidity of the menstrual effluent. Progesterone appears to be the significant factor in these changes since estradiol levels are maintained throughout the artificial cycles and progesterone is removed initiating the menstrual phase of the cycle. FUNDING This work was supported by Primate Center Core (grant# P51 OD011092). Footnotes CONFLICTS OF INTEREST The authors have no conflicts of interest for disclosure. ETHICS APPROVAL Animal husbandry was provided by the ONPRC Department of Comparative Medicine and animal use was reviewed and approved by the ONPRC Institutional Animal Care and Use Committee.

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