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Endocrinology, doi:10.1210/en.2007-0856
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Endocrinology Vol. 148, No. 11 5433-5441
Copyright © 2007 by The Endocrine Society

Progesterone, But Not Estrogen, Stimulates Vessel Maturation in the Mouse Endometrium

Jane E. Girling, Fiona L. Lederman, Lisa M. Walter and Peter A. W. Rogers

Centre for Women’s Health Research, Monash University Department of Obstetrics and Gynecology and Monash Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Jane Girling, Department of Obstetrics and Gynecology, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: jane.girling{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human endometrium undergoes regular periods of growth and regression, including concomitant changes in the vasculature, and is one of the few adult tissues where significant angiogenesis and vascular maturation occurs on a routine, physiological basis. The aim of this study was to investigate the effects of estrogen and progesterone on endometrial vascular maturation in mice. Endometrial tissues were collected from early pregnant mice (d 1–4) and ovariectomized mice given a single 17β-estradiol (100 ng) injection 24 h before dissection (short-term estrogen regime) or three consecutive daily injections of progesterone (1 mg) with/without estrogen priming (progesterone regime). Experiments were then repeated with the inclusion of mice treated concurrently with progesterone and either RU486 or a vascular endothelial growth factor-A antiserum. Proliferating vascular mural cells (PVMC) were observed on d 3–4 of pregnancy, corresponding with an increase in circulating progesterone. A significant increase in PVMC and {alpha}-smooth muscle actin (labels mural cells) coverage of vessel profiles were observed in mice treated with progesterone in comparison to controls; no significant change was noted in mice treated with estrogen or with vascular endothelial growth factor antiserum. RU486 treatment did not inhibit the progesterone-induced increases in PVMC and mural cell coverage, although progesterone-induced changes in endothelial and epithelial cell proliferation were inhibited. These results show that progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium. The work illustrates the relevancy of the mouse model for understanding endometrial vascular remodeling during the menstrual cycle and in response to the clinically important progesterone receptor antagonist RU486.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONCURRENT GROWTH OF the vasculature is an essential component of endometrial regeneration after menstruation. This requires both angiogenesis (growth of new blood vessels from the existing vessels of the basalis) and vessel maturation (mural cell recruitment). In recent years, attention has been paid to the timing and mechanisms of angiogenesis in human endometrium and in animal and in vitro models (1, 2, 3). Less attention has focused on maturation of the endometrial vasculature, including the recruitment and proliferation of mural cells [pericytes and vascular smooth muscle cells (VSMC)] (4). This is despite the regular cyclic regrowth of human endometrial vasculature providing an ideal model for the study of vessel maturation and despite hypotheses suggesting that abnormal maturation may be involved in several endometrial pathologies including menorrhagia and breakthrough bleeding (4, 5, 6, 7, 8).

The recruitment of mural cells provides mechanical support to the vessel and gives it the ability to regulate blood flow through rapid alterations in its internal diameter. Mural cells are also involved in regulating angiogenesis and vessel function and interact closely with endothelial cells (9, 10, 11). Various differentiation markers have been used to examine the mural cells associated with vessels in the human endometrium. One such marker is {alpha}-smooth muscle actin ({alpha}-SMA), a contractile cytoskeletal protein found in pericytes, which is also one of earliest differentiation markers of VSMC. {alpha}-SMA is expressed throughout the vascular tree of the human endometrium (5, 6, 12) and can be found on both the conventional or "straight" arterioles and the specialized "spiral" arterioles unique to menstruating primates, which develop during the secretory phase of the menstrual cycle. Studies examining {alpha}-SMA in human endometrium have reported mixed results with either no significant changes in {alpha}-SMA expression on straight or spiral arterioles across the menstrual cycle (5, 6) or an increase in {alpha}-SMA around the spiral arterioles during the secretory phase (12).

Proliferation of VSMC also has been examined in the human endometrium using immunohistochemistry (7). Total proliferation was low during the early stages of the menstrual cycle (2–2.5% of VSMC were positive with an antibody against proliferating cell nuclear antigen), but increased significantly during the mid-late secretory stages to approximately 4%. This pattern of proliferation was maintained when the VSMC of spiral arterioles were considered separately from straight arterioles, presumably reflecting the growth and coiling of spiral arterioles in response to increases in circulating progesterone during the secretory phase. In contrast, no such role for progesterone was identified for the straight arterioles as proliferation of their VSMC remained constant across the menstrual cycle.

Although a mature vasculature develops as the functionalis grows during the menstrual cycle, as yet there is no evidence that estrogen and/or progesterone are directly responsible for the vessel maturation observed. In this study, we used two established mouse models (13, 14, 15, 16) to investigate the effects of estrogen and progesterone on endometrial vessel maturation. Our aim was to examine the regulation of mural cell proliferation and recruitment in pregnant and hormone-treated mice. We hypothesized that mural cell proliferation and vessel coverage would increase during early pregnancy and after progesterone treatment (in conjunction with endothelial cell proliferation which is known to occur in this model) (16), but not after estrogen treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female mice (7–13 wk, 18–28 g, C57BL/6J x CBA) were housed under controlled environmental conditions (20 C, 12 h light per day) and provided with food and water ad libitum. The study was approved by the Monash Medical Centre Animal Ethics Committee A and was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Study one: early pregnancy
Female mice were housed overnight with stud males and the presence of a vaginal plug the following morning indicated successful mating. The day of a successful mating was considered d 1 of pregnancy. Pregnant mice were dissected on d 1–4 of pregnancy (n = 4–5/d) when circulating progesterone concentrations were increasing, but before implantation on d 5 (17).

Four hours before dissection, mice received an ip injection of bromodeoxyuridine (BrdU; 40 mg/kg body weight, 500 µl; Sigma-Aldrich Co., St. Louis, MO), enabling visualization of proliferating cells by immunohistochemistry. The uterine tissues were removed and further immersion fixed in 10% buffered formalin for 2 h before processing for paraffin sections.

Study two: short-term estrogen regime
In the second study, the hormone regime was designed to examine the short-term effects of estrogen on the endometrial vasculature (13, 14, 15). Mice were bilaterally ovariectomized after anesthesia with Avertin (25 mg/100 g body weight, 2,2,2-tribromoethanol; Aldrich Chemical Company, Milwaukee, WI; in butan-2-ol, BDH Laboratory Supplies, Poole, UK), before being separated into two treatment groups (n = 5 per group). All animals were left for 7 d after ovariectomy to allow regression of the endometrium. Mice received a single sc injection of 17β-estradiol (100 ng/100 µl peanut oil) or vehicle (100 µl peanut oil) on d 8 after ovariectomy, before dissection 24 h later, 4 h after a BrdU injection (Fig. 1Go).


Figure 1
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  		FIG. 1. Experimental protocols for exogenous hormone treatment of ovariectomized CBA/C57 mice. Study two: short-term estrogen regime: One group of mice (n = 5) was treated with a single injection of 17β-estradiol (100 ng) on d 8 after ovariectomy before dissection 24 h later (4 h after a BrdU injection). Study three: progesterone regime: One group of mice (n = 5) was treated with a single injection of 17β-estradiol (100 ng) on d 8 after ovariectomy, followed by a day without treatment and three consecutive daily injections of progesterone (1 mg). Other groups were treated with either the vehicle (n = 5), 17β-estradiol (n = 4–5), or progesterone injections only (n = 5). All animals were dissected on d 13, 4 h after a BrdU injection.

 
Study three: progesterone regime
The dose and schedule of hormones used with the third study were designed to mimic the endometrial endothelial cell proliferation observed during early pregnancy (16). Ovariectomized mice were separated into four treatment groups (n = 4–5 per group). One group of mice was treated with a single sc injection of 17β-estradiol (100 ng/100 µl peanut oil, estrogen-priming) on d 8 after ovariectomy, followed by three consecutive daily sc injections of progesterone (1 mg/100 µl peanut oil) on d 10–12, before dissection 24 h after the final hormone injection (Fig. 1Go). The other groups received vehicle (100 µl peanut oil), 17β-estradiol, or progesterone injections only. All mice were dissected on d 13 after ovariectomy. Four hours before dissection, the mice received an ip injection of BrdU.

Study four: RU486 and progesterone
To confirm that the effects seen in study three were due to progesterone, experiments were conducted using the progesterone receptor antagonist RU486 (mifepristone). Ovariectomized mice were divided into two treatment groups (n = 6–8). Mice were treated with three consecutive daily progesterone injections concurrently with daily injections of RU486 or vehicle. The experiment was conducted twice using two doses of RU486: 500 µg/100 µl/mouse/d (stock: 50 mg/ml in ethanol; working solution: 5 mg/ml in peanut oil) and 2 mg/100 µl/mouse/d (stock: 200 mg/ml in 20% ethyl acetate/80% ethanol; working solution: 25 mg/ml in peanut oil). Mice were dissected and uterine tissues were collected 24 h after the last injection, 4 h after a BrdU injection.

Study five: vascular endothelial growth factor (VEGF) antiserum
To investigate whether VEGF or VEGF-induced angiogenic changes were required for endometrial vascular maturation, studies two and three were modified and repeated using a VEGF antiserum. Short-term estrogen regime: ovariectomized mice received a single sc injection of 17β-estradiol (100 ng/100 ml peanut oil) with (n = 5) or without (n = 4) a concurrent ip injection of rabbit polyclonal VEGF antiserum (raised in rabbit against mouse VEGF164, 200 ml) (18). Mice were dissected 24 h later, 4 h after a BrdU injection. Blood collection and perfusion fixation were as described for study one. Progesterone regime: mice were treated with the single 17β-estradiol injection and three consecutive daily progesterone injections (n = 6), or the progesterone injections only (n = 6). Further groups were treated in the same way with the addition of three consecutive daily ip injections of VEGF antiserum given concurrently with the progesterone (n = 5–6 per group). Mice were dissected 24 h after the last injection, 4 h after a BrdU injection.

BrdU/{alpha}-SMA double immunohistochemistry for visualization of proliferating mural cells
After dewaxing and rehydration, sections (3 µm) were incubated with 3% H2O2 in methanol (10 min) to quench endogenous peroxidase and protein blocking agent (PBA; Thermo Electron Corp., Immunon Shandon, Pittsburgh, PA) to prevent nonspecific binding. Sections were incubated with mouse monoclonal {alpha}-SMA (0.18 µg/ml, 1 h; DakoCytomation, Carpenteria, CA) and DakoCytomation mouse Envision HRP (30 min). Staining was visualized by aminoethyl carbazole (Zymed, Invitrogen, San Francisco, CA). Slides were then microwaved in trisodium citrate buffer (20 min) and incubated in 0.1 M HCl (45 min). PBA was applied (10 min) before incubation in monoclonal sheep anti-BrdU (8 g/ml, 1 h; Biodesign International, Saco, ME) and donkey antisheep IgG (4 g/ml, 1 h; Jackson ImmunoResearch Laboratories, distributed by ALS, Melbourne, Australia). Immunostaining was visualized after incubation in LSAB2 AP-streptavidin (15 min; DakoCytomation) by vector blue alkaline phosphatase (10 min; Vector Laboratories, Burlingame, CA). A negative isotype-matched control was prepared by replacing the {alpha}-SMA primary antibody with mouse IgG2a (Chemicon, Temecula, CA) and the BrdU primary antibody with sheep IgG (Sigma-Aldrich Co.) at the same concentrations as that of the primary antibodies.

Proliferating vascular mural cells (PVMC) were counted (on a single section) using a x 20 objective lens and expressed as a count of positively stained cells per mm2 of endometrial tissue; pericytes and VSMC were not differentiated. A proliferating cell was only classified as being a mural cell if its nucleus was completely surrounded by {alpha}-SMA. This provided a conservative measure of the level of mural cell proliferation.

CD31/{alpha}-SMA double immunohistochemistry for visualization of blood vessels and mural cells
Immunostaining for {alpha}-SMA was as in the previous protocol. Sections were then incubated in 0.1% (1 mg/ml) pepsin in 3% acetic acid (10 min, 37 C), PBA (10 min), rat monoclonal antimouse CD31 (5 µg/ml, 1 h, 37 C, BD PharMingen, San Diego, CA) and biotinylated goat antirat IgG (1:200, 1 h; Chemicon). CD31 immunostaining was visualized after incubation in alkaline phosphatase conjugated streptavidin (15 min; DakoCytomation) and vector blue alkaline phosphatase (10 min). A negative isotype-matched control was prepared by replacing the {alpha}-SMA primary antibody with mouse IgG2a (Chemicon) and the CD31 primary antibody with rat IgG2a (BD PharMingen) at the same concentrations as that of the primary antibodies.

From a single section from each uterus, the vessel profiles in the endometrium were classified as either having no {alpha}-SMA, minimal {alpha}-SMA, extensive {alpha}-SMA, or being completely surrounded by {alpha}-SMA (Fig. 2Go, A–D). The number of vessel profiles in each classification was then expressed as a percentage of total vessel profiles.


Figure 2
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  		FIG. 2. Representative photomicrographs illustrating CBA/C57 mouse endometrium immunostained with double immunohistochemistry protocols to visualize endothelial cells (antibody against CD31, blue staining) and mural cells (antibody against {alpha}-SMA, brown staining) (A–D), mural cells (antibody against {alpha}-SMA, brown staining) and proliferating cells (antibody against BrdU, blue staining) (E–I), and mural cells (antibody against {alpha}-SMA, blue staining) and progesterone receptor (brown staining) (J). Note: {alpha}-SMA immunostaining also stains the smooth muscle cells of the myometrium. Vessel profiles in mouse endometrium were classified as either having no {alpha}-SMA (A), minimal {alpha}-SMA (B), extensive {alpha}-SMA (C), or being completely surrounded by {alpha}-SMA (D). I, Photomicrograph illustrating transition of vessel from an arteriole to capillary. J, Photomicrograph illustrating progesterone receptor immunostaining in mural cells. PEC, black arrow; proliferating pericyte, white arrow; proliferating VSMC, red arrow; progesterone receptor-positive mural cell, dashed black arrow; endothelial cell, gray arrow. e, Luminal epithelium; g, gland; m, myometrium; s, stroma. Scale bars: A–I, 20 µm; J, 10 µm.

 
CD31/BrdU double immunohistochemistry for visualization of blood vessels and proliferating endothelial cells (PEC) (study 4)
Immunostaining for CD31 and BrdU was as in the previous protocols except that BrdU-positive cells were visualized using 3,3-diaminobenzidine (Sigma-Aldrich Co.). PEC (on a single section from each animal) were counted and expressed as a count of positively stained cells per square millimeter of endometrial tissue. Proliferating luminal and glandular epithelial cells were counted (on one section per animal) and expressed as a percentage of the total number of luminal and glandular epithelial cells.

Progesterone receptor/{alpha}-SMA double immunohistochemistry for visualization of receptor in mural cells
Immunostaining for {alpha}-SMA antibody was as above except immunostaining was visualized with LSAB2 AP-streptavidin (15 min) and vector blue alkaline phosphatase (10 min). Slides were then microwaved in trisodium citrate buffer (30 min), PBA (10 min), rabbit monoclonal antiprogesterone receptor (1:100 dilution, ~1.5 µg/ml, 1 h, 37 C, SP2 clone; LabVision Corp., Fremont, CA) and biotinylated swine antirabbit IgG (1:200, 30 min; DakoCytomation). Immunostaining was visualized using 3,3-diaminobenzidine (5 min). A negative isotype-matched control was prepared by replacing the {alpha}-SMA primary antibody with mouse IgG2a (Chemicon) and the progesterone receptor primary antibody with rabbit IgG (Sigma-Aldrich Co.) at the same concentrations as that of the primary antibodies.

Statistics
Statistical analysis was performed using SPSS for Windows, version 11.0 (SPSS Inc., Chicago, IL). A P value of less than 0.05 was considered significant. Unless otherwise indicated, data are expressed as means ± SEM. For the pregnancy data and progesterone regime (with/without VEGF antiserum), PVMC data were analyzed using Kruskal-Wallis (KW) nonparametric tests. If a significant difference was detected, pairs of means were analyzed using Mann-Whitney (MW) tests. For the short-term estrogen regime and the RU486 experiments, PVMC and PEC data were analyzed using Mann-Whitney tests. Proliferating luminal and glandular epithelial cell data were analyzed using t tests. Data illustrating the percentage of vessel profiles with different amounts of {alpha}-SMA coverage were analyzed initially using a two-way ANOVA with treatment group and level of actin coverage as fixed factors. If a significant interaction term was obtained, indicating that the proportion of vessel profiles with differing {alpha}-SMA coverage varied with treatment group, individual levels of actin coverage were further analyzed using individual t tests or a one-way ANOVA with treatment group as the fixed factor, followed by least significant difference post hoc tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferating mural cells
Low numbers of PVMC were observed in pregnant mice, and ovariectomized mice treated with estrogen or progesterone (details below). Based on their morphology and association with the blood vessel in question, some PVMC could be easily classified as either a pericyte of VSMC (Fig. 2Go, E–H). However, this was not true in the majority of cases and the two cell types have not been distinguished further during quantification of PVMC.

Study one: pregnancy.
The number of PVMC in the mouse endometrium changed significantly over the first 4 d of pregnancy (KW(3) = 12.2, P = 0.007). PVMC were observed in only one animal on d 1 [median = 0.0 (range = 0.0–15.9) PVMC/mm2] and d 2 [0.0 (0.0–2.6) PVMC/mm2] of pregnancy. Mice on d 3 and 4 of pregnancy all had PVMC [d 3: 12.0 (4.4–36.2) and d 4: 24.5 (7.8–49.2) PVMC/mm2]. Significant differences between groups are illustrated in Fig. 3Go.


Figure 3
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  		FIG. 3. Endometrial mural cell proliferation in CBA/C57 mice on d 1–4 of pregnancy. Horizontal bars indicate median values (n = 4–5). Each dot represents an individual mouse. Days that do not share a letter in common are significantly different (P ≤ 0.05).

 
Study two: short-term estrogen regime.
A small number of PVMC were observed in three of five mice treated with a single injection of 17β-estradiol [vehicle: median = 0.0 (range: 0.0–0.0), 24 h E: 3.0 (0.0–6.3) PVMC/mm2; Fig. 4Go]; however, there was no significant increase in comparison to the vehicle-treated animals (MW = 5.0, P = 0.151).


Figure 4
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  		FIG. 4. Endometrial endothelial cell proliferation in hormone-treated ovariectomized CBA/C57 mice. Study two: short-term estrogen regime: Mice were treated with a single injection of 17β-estradiol or vehicle before dissection 24 h later. Study three: progesterone regime: Mice were treated with a single injection of 17β-estradiol, a no-treatment day, and three consecutive daily injections of progesterone before dissection 24 h after the final hormone injection. Other groups were injected with vehicle, 17β-estradiol, or progesterone injections only. Horizontal bars indicate median values (n = 4–5). Each dot represents an individual mouse. Treatment groups that do not share a letter in common are significantly different (P ≤ 0.05).

 
Study three: progesterone regime.
In study three, PVMC were observed only in progesterone-treated mice [vehicle: median = 0.0 (range 0.0–0.0) PVMC/mm2, E only: 0.0 (0.0–0.0) PVMC/mm2, P only: 7.2 (4.4–27.0) PVMC/mm2, E + P: 20.5 (6.4–27.4) PVMC/mm2, KW(3) = 15.7, P = 0.001, Fig. 4Go]. There was no significant effect of estrogen priming on progesterone-induced mural cell proliferation (MW = 6.0, P = 0.22).

Study four: RU486 and progesterone.
In the first experiment, RU486 treatment did not significantly reduce PVMC or affect the proportion of vessel profiles covered with differing amounts of {alpha}-SMA. A second experiment was conducted administering a higher dose of RU486. Results were similar for both experiments; only data from the second experiment are presented.

There was no significant difference between the number of PVMC in mice treated with progesterone with/without RU486 [P + vehicle: median = 3.7 (range 0.0–8.3) PVMC/mm2, P + RU486: 7.0 (3.3–12.5) PVMC/mm2; MW = 11.5, P = 0.10] (Fig. 5AGo). Proliferating epithelial cells and PEC were also counted (Fig. 5Go, B–D). PEC were significantly reduced by concurrent RU486 treatment [P + vehicle: median = 69.3 (range 11.2–156.6) PEC/mm2, P + RU486: 10.3 (0–19.3) PEC/mm2; MW = 3.0, P = 0.01]. RU486 significantly reduced the progesterone-induced inhibition of epithelial cell proliferation in glandular (P + vehicle: 0.9 ± 0.6 PEC/mm2; P + RU486: 8.6 ± 1.8 PEC/mm2; t = 3.71, P = 0.003) but not luminal (P + vehicle: 2.6 ± 1.3 PEC/mm2; P + RU486: 5.7 ± 1.8 PEC/mm2; t = 1.35, P = 0.21) epithelium.


Figure 5
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  		FIG. 5. Endometrial cell proliferation in mice treated with three consecutive daily injections of progesterone concurrently with daily injections of RU486 or vehicle before dissection 24 h after the final hormone injection. A, Mural cell; B, endothelial cell; C, luminal epithelial cell; and D, glandular epithelial cell proliferation. Horizontal bars indicate median values (n = 6–7). Each dot represents an individual mouse. Treatment groups that do not share a letter in common are significantly different (P ≤ 0.05).

 
Study five: VEGF antiserum.
Concurrent use of VEGF antiserum had no effect on PVMC in mice treated with either the short-term estrogen or progesterone regimes. Short-term estrogen regime: 24 h E: median = 4.8 (range: 0.0–9.5), 24 h E + VEGF antiserum: 0.0 (0.0–12.0) PVMC/mm2; MW = 8.5, P = 0.73. Progesterone regime: P only: median = 6.9 (range 0.0–24.8) PVMC/mm2, P + VEGF antiserum: 3.0 (0.0–6.8) PVMC/mm2, E + P: 3.6 (0.0–20.3) PVMC/mm2, E + P + VEGF antiserum: 2.5 (1.1–2.7) PVMC/mm2, KW(3) = 1.91, P = 0.59).

Proportion of vessel profiles covered by {alpha}-SMA
The proportions of vessel profiles with no, minimal, extensive, or complete {alpha}-SMA coverage were quantified in pregnant and hormone-treated mice. Only a small proportion of vessel profiles were completely surrounded by {alpha}-SMA, and a large number of these were vessels surrounded by a distinct layer of VSMC. These vessel profiles tended to be seen in small clusters close to the endometrial/myometrial border (Fig. 2Go, D–H). In some uterine sections, the transition of a vessel from arteriole (completely surrounded by a layer of VSMC) to capillary (irregular pericyte coverage) within the endometrium could be seen in a single vessel profile (Fig. 2IGo).

Study one: pregnancy.
A significant interaction term was obtained after two-way ANOVA with day of pregnancy and level of {alpha}-SMA as the fixed factors (F(9,68) = 2.07, P = 0.049), suggesting that the proportion of endometrial vessel profiles with different levels of {alpha}-SMA coverage changed across the first 4 d of pregnancy. However, the P value only just reached significance and when each level of actin coverage was analyzed separately using one-way ANOVA, no significant effect of day of pregnancy was detected (no {alpha}-SMA coverage: d 1: 41 ± 8%, d 2: 31 ± 6%, d 3: 33 ± 8%, d 4: 20 ± 3%, F(3,16) = 2.19, P = 0.15; minimal {alpha}-SMA coverage: d 1: 42 ± 6%, d 2: 47 ± 6%, d 3: 47 ± 5%, d 4: 54 ± 4%, F(3,16) = 0.91, P = 0.47; extensive {alpha}-SMA coverage: d 1: 11 ± 1%, d 2: 17 ± 5%, d 3: 15 ± 5%, d 4: 19 ± 3%, F(3,16) = 1.16, P = 0.36; complete {alpha}-SMA coverage: d 1: 6 ± 1%, d 2: 5 ± 1%, d 3: 4 ± 1%, d 4: 7 ± 1%, F(3,16) = 1.76, P = 0.20; Fig. 6Go).


Figure 6
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  		FIG. 6. Proportion of vessel profiles in endometrium from CBA/C57 mice on d 1–4 of pregnancy (n = 4–5/d) with differing levels of {alpha}-SMA coverage. Black bars, No {alpha}-SMA; white bars, minimal {alpha}-SMA; gray bars, extensive {alpha}-SMA; and hatched bars, profiles completely surrounded by {alpha}-SMA.

 
Study two: short-term estrogen regime.
Despite a significant interaction term after two-way ANOVA (F(3,36) = 3.79, P = 0.021), individual t tests were unable to detect an effect of treatment group on the proportion of vessel profiles with no (vehicle: 54 ± 6%, E only: 33 ± 2%, t(7) = 2.0, P = 0.081), minimal (vehicle: 32 ± 7%, E only: 45 ± 4%, t(7) = 1.3, P = 0.22), extensive (vehicle: 9 ± 1.5%, E only: 16 ± 4%, t(7) = 1.4, P = 0.22), or complete (vehicle: 5 ± 0.4%, E only: 6 ± 1%, t(7) = 0.55, P = 0.598) {alpha}-SMA coverage (Fig. 7Go).


Figure 7
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  		FIG. 7. Proportion of vessel profiles with differing levels of {alpha}-SMA coverage in endometrium of hormone-treated ovariectomized CBA/C57 mice. Study two: short-term estrogen regime: Mice were treated with a single injection of 17β-estradiol (n = 5) or vehicle (n = 4) before dissection 24 h later. Study three: progesterone regime: Mice were treated with a single injection of 17β-estradiol, a no-treatment day, and three consecutive daily injections of progesterone before dissection 24 h after the final hormone injection. Other groups were injected with vehicle, 17β-estradiol, or progesterone injections only (n = 4–5). Black bars, No {alpha}-SMA; white bars, minimal {alpha}-SMA; gray bars, extensive {alpha}-SMA; and hatched bars, profiles completely surrounded by {alpha}-SMA. For each level of actin coverage: treatment groups that do not share a letter in common are significantly different (P ≤ 0.05).

 
Study three: progesterone regime.
A significant interaction term was obtained after two-way ANOVA (F(9,76) = 35, P < 0.001) reflecting the different proportions of vessels with different levels of {alpha}-SMA coverage within a particular hormone-treated group. Using 1-way ANOVA, there was a significant decrease in the percentage of vessel profiles with no {alpha}-SMA (vehicle: 58 ± 2%, E only: 49 ± 5%, P only: 21 ± 2%, E + P: 12 ± 1%, F(3,18) = 35.6, P < 0.001) and a significant increase in the percentage of vessels with minimal (vehicle: 27 ± 2%, E only: 33 ± 5%, P only: 44 ± 2%, E + P: 52 ± 4%, F(3,18) = 10.9, P < 0.001) and extensive {alpha}-SMA coverage (vehicle: 6 ± 0.5%, E only: 10 ± 2%, P only: 27 ± 3%, E + P: 27 ± 5%, F(3,18) = 9.6, P < 0.001) in progesterone-treated groups in comparison to vehicle and E-only groups (Fig. 7Go). There was no significant effect of treatment group on the percentage of vessel profiles that were complete surrounded by {alpha}-SMA (vehicle: 10 ± 1%, E only: 7 ± 1%, P only: 8 ± 1%, E + P: 9 ± 1%, F(3,18) = 0.37, P = 0.77). There was no significant effect of estrogen priming on progesterone-induced changes in mural cell coverage.

Study four: RU486 and progesterone.
No significant interaction term was obtained after two-way ANOVA with treatment group and level of {alpha}-SMA as the fixed factors (F(3.56) = 0.3, P = 0.82) indicating that there was no significant difference in the proportion of vessel profiles with different levels of mural cell coverage between mice treated with/without RU486 (no {alpha}-SMA coverage: P + vehicle: 25 ± 4%, P + RU486: 28 ± 5%; minimal {alpha}-SMA coverage: P + vehicle: 44 ± 1%, P + RU486: 41 ± 3%; extensive {alpha}-SMA coverage: P + vehicle: 25 ± 4%, P + RU486: 25 ± 5%; complete {alpha}-SMA coverage: P + vehicle: 6 ± 1%, P + RU486: 5 ± 1%).

Study five: VEGF antiserum.
There was no significant interaction term after two-way ANOVA in the mice treated with the short-term estrogen regime with/without the inclusion of VEGF antiserum (F(3,32) = 0.31, P = 0.82).

Despite a significant interaction term after two-way ANOVA (F(9,80) = 2.1, P = 0.047) of the progesterone regime, individual one-way ANOVA were unable to detect an effect of treatment group on the proportion of vessel profiles with different {alpha}-SMA coverage. The only coverages nearing significance were vessel profiles completely surrounded by {alpha}-SMA (P only: 10 ± 2%, P + VEGF antiserum: 6 ± 1%, E + P: 6 ± 1%, E + P+ VEGF antiserum: 6 ± 2%, F(3,16) = 2.8, P = 0.072).

Progesterone receptor expression in endometrial mural cells
Progesterone receptor immunoreactivity was examined in mural cells in representative sections from pregnant and progesterone (and vehicle)-treated mice. Low levels of progesterone receptor immunostaining was observed in mural cells in both pregnant (particularly d 3 and 4) and progesterone-treated animals (Fig. 2JGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has established that progesterone stimulates vessel maturation in the mouse endometrium. This occurs concurrently with angiogenesis, which has previously been described in this model of progesterone action (16). RU486 had mixed antagonistic effects on progesterone-induced endometrial vascular changes. Although endothelial cell proliferation was inhibited, no changes in progesterone-induced vascular maturation were observed. The effects of estrogen on vessel maturation were also examined. Although estrogen is known to stimulate endothelial cell proliferation in the mouse endometrium within 24 h of treatment (15), the results from this study do not support a role for estrogen in endometrial vascular maturation, at least within the short time frame of the model used. In addition, unlike endometrial endothelial cell proliferation, which was significantly reduced when ovariectomized mice were estrogen primed before progesterone treatment (16), mural cell proliferation and recruitment were not significantly affected by estrogen pretreatment. Although the mechanisms by which estrogen modifies the endothelial cell response to progesterone are unknown, they occur without modifying the mural cell response.

Previous analysis of human endometrial samples has suggested a role for progesterone in endometrial vascular maturation. This was based on the observed coiling, thickening, and maturation of the endometrial spiral arterioles, including proliferation of VSMC and increased {alpha}-SMA coverage (5, 6, 7, 12) during the progesterone-dependent secretory phase of the menstrual cycle. Further correlative evidence in an animal model is provided by the current study. Mural cell proliferation was observed on d 3 and 4 of pregnancy, which is also when endothelial cell proliferation was observed (16). However, changes in mural cell coverage of endometrial vessels did not reach significance. We hypothesize that this is due to the presence of a stable, mature vasculature in normal cycling mice before pregnancy in contrast to that in the ovariectomized animals before progesterone treatment.

Although we have shown that progesterone stimulates vascular maturation in the endometrium, we were unable to block the increases in mural cell coverage and proliferation using the progesterone receptor antagonist RU486. In contrast, treatment with RU486 blocked endothelial cell proliferation and prevented the inhibition of glandular epithelial cell proliferation, which are also known to occur in response to progesterone (16, 19). The latter result is consistent with observations in women receiving long-term low dose RU486 treatment, although these studies also saw decreased proliferation in the stromal compartment (20, 21). It is interesting to note that, although no specific changes have been noted in the endometrial stroma of RU486-treated women, an increase in thick-walled muscular vessels was seen in the endometrium of women treated with the selective progesterone receptor modulator Asoprisnil (22).

It is possible that the aforementioned results indicate the effects of progesterone on endometrial vascular remodeling are mediated by nongenomic pathways (23); however, we believe it is more likely to represent the partial agonist properties of RU486. Although RU486 is generally considered to be a pure antagonist, it has been ascribed agonist properties in a species-, tissue-, cell-, dose-, and duration-of-treatment-specific manner (24, 25). The agonist and antagonist activities of RU486 are thought to be due to the interaction and recruitment of coregulators to the progesterone receptor complex and modulation of estrogenic effects (24, 26). Recent studies using the progesterone receptor activity indicator mouse illustrate that RU486 reduces uterine progesterone receptor activity and expression acutely in ovariectomized animals, but increases both activity and expression after 3 d of treatment (27). The temporal changes in progesterone receptor expression in the mouse uterus in response to RU486 may explain the partial antagonist effects observed in the current study, which also administered RU486 over 3 d (although concurrently with progesterone). RU486 is of considerable clinical interest with potential uses for contraception, reduction of breakthrough bleeding associated with progestin-only contraceptive use, and treatment of uterine leiomyoma. Thus, although using RU486 did not confirm that progesterone induced the vascular maturation changes observed in these studies, it did provide a mouse model that will allow clinically relevant mechanistic studies on the endometrial vasculature in response to RU486.

Although results from the current study do not support a direct role for the specific endothelial cell mitogen VEGF-A in endometrial vessel maturation, VEGF-A is known to have indirect effects on maturation in other systems. For instance, the presence/absence of VEGF-A determines the response of vessels to the key maturation factors angiopoietin (Ang)-1 and Ang-2, which are ligands for the receptor Tie-2 (11). Several studies have examined Ang-1, Ang-2, and Tie-2 expression in the human endometrium, but the results have differed considerably, with immunostaining reported variously in the epithelium, stroma, and uterine natural killer cells (4, 28, 29, 30, 31). Although Ang-1, Ang-2, and Tie-2 are clearly present in the endometrium and can be attributed a significant role in endometrial vascular remodeling based on information from other systems, no specific functional studies relating to the endometrial vasculature have been conducted in vivo. This is also true of other significant maturation factors, such as platelet-derived growth factor (PDGF)-B/PDGF receptor-β (PDGFR-β) and transforming growth factor-β (TGF-β), which are also present in the endometrium (7, 32, 33). PDGF-B is important in the recruitment of pericytes, which express PDGFR-β (11, 34), whereas TGF-β is a pleiotrophic growth factor that is believed to promote production of extracellular matrix and induce the differentiation of mesenchymal cells to mural cells (11). The current mouse model, in which endometrial vessel maturation occurs in response to exogenous progesterone, will provide an invaluable system in which to investigate the hormonal interactions and mechanisms of action of specific maturation factors.

Progesterone-stimulated mural cell proliferation was observed at only low levels. These low levels are unlikely to account for the increased mural cell coverage observed in progesterone-treated animals and raise the question as to the source of the recruited mural cells. The source of mural cells can vary depending on their location within the body (9, 10). They may transdifferentiate from endothelial cells, differentiate from mesenchymal cells such as stromal cells, myofibroblasts, and pericytes in situ, or differentiate from bone marrow precursors or macrophages. The source of VSMC and pericytes within the endometrium is unknown, although this question could be addressed using the various transgenic mice models now available.

A limitation of the current studies was the inability of the mural cell marker ({alpha}-SMA) used to differentiate between pericytes and VSMC. Although individual cell types could only be differentiated with certainty in some instances (based on their morphology), we hypothesize that the mural cell recruitment observed in response to progesterone treatment largely involved changes in pericyte number. The proportion of vessel profiles within the endometrium that were completely surrounded by {alpha}-SMA did not change among the different hormone-treated animals examined, and the majority of vessel profiles that had distinctive VSMC (based on morphology) fell within this category. Vessel profiles with a distinctive layer of VSMC tended to be situated in small clusters close to the myometrial-endometrial border and presumably represent a stable population of arterioles within the endometrium. This implies that the changes observed in terms of the proportion of vessel profiles with no, minimal, and extensive actin may largely represent changes in pericyte number. Rapid changes in endometrial pericyte number have been observed previously in rodents (35). During pregnancy, the percentage of vessel profiles with pericytes (visualized using electron microscopy) increased from 5% on d 4 to 74% on d 5, dropping to 14% on d 6. When a hormonal regime designed to mimic pregnancy was used, the percentage of capillary profiles with pericytes increased from 6% in ovariectomized mice to 15% after 3 d of progesterone injections. The percentage of capillaries with pericytes increased further to 69% if an estradiol injection (to mimic the nidatory estrogen surge) was given at the same time as the last progesterone injection (35). As well as illustrating the potential for rapid changes in endometrial pericyte number, these results highlight the need for future studies to develop simple methods to differentiate endometrial pericytes from VSMC and consider their relative contribution to endometrial vascular development and remodeling. Unfortunately, of the various pericyte markers currently available (such as {alpha}-SMA, NG2, and PDGFR-β) none are completely specific to pericytes, and their expression varies depending on the tissue, species, or developmental stage in question (36). Electron microscopy, which reveals the pericyte completely surrounded by basement membrane, is still the most reliable form of identification.

In conclusion, results from this research have demonstrated that progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium in a manner that could not be inhibited by the progesterone receptor antagonist RU486. Further use and development of the mouse models described in these studies will provide a system which is easy to manipulate and can be used to elucidate the mechanisms of the actions of progesterone to control endometrial vascular maturation.


   Acknowledgments
 
We thank the staff of the Monash Medical Centre Animal House for technical help and assistance. We also thanks Profs. Hilary Critchley, David Baird, and Rodney Kelly (University of Edinburgh, Edinburgh, UK) for advice pertaining to the use of RU486.


   Footnotes
 
This work was supported by National Health and Medical Research Council Grant No. 384195 to P.R. and J.G. The salary of P.R. is paid by National Health and Medical Research Council Fellowship Grant No. 143805.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: Ang, Angiopoietin; BrdU, bromodeoxyuridine; KW, Kruskal-Wallis; MW, Mann-Whitney; PBA, protein blocking agent; PDGF, platelet-derived growth factor; PDGFR-β, PDGF receptor-β; PEC, proliferating endothelial cell; PVMC, proliferating vascular mural cell; {alpha}-SMA, {alpha}-smooth muscle actin; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cell.

Received June 26, 2007.

Accepted for publication July 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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