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Original Studies |
Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia
Address all correspondence and requests for reprints to: Patricia A. Mote, Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia. e-mail: patm@westgate.wh.usyd.edu.au.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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It is well established that PR concentrations vary with menstrual cycle phase (4, 5, 6, 7, 8, 9, 10, 11, 12). PR content increases in both epithelial and stromal compartments during the proliferative phase and remains high during the early secretory phase. In the mid- to late secretory phase there is a decline in PR expression, which is marked in glandular cells and less evident in the stroma (5, 7, 8, 10, 12). These changes are probably related to the known effects of estrogen and progesterone on PR expression, with high levels of estrogen in the proliferative phase inducing PR synthesis, and progesterone down-regulating expression of its own receptor postovulation (1). The maintenance of stromal PR throughout the secretory phase of the menstrual cycle is suggestive of constitutive PR expression and implies the continued need for progesterone to support further growth and development in this tissue (5, 7).
The human PR is expressed as two isoforms, PRA and PRB (13, 14), that differ only in that the smaller isoform, PRA, lacks 164 amino acids from the N-terminus (15). PRA and PRB are products of a single gene and are translated from individual messenger ribonucleic acid species under the control of distinct promoters (15). Both PRA and PRB function as ligand-activated transcription factors, but it has been suggested on the basis on in vitro studies that the two proteins are not functionally equivalent. Transient cotransfection of PRB or PRA and progestin-sensitive reporter genes has shown that, in general, PRB is transcriptionally the more active of the two isoforms (16, 17). Furthermore, PRA can act as a dominant repressor of PRB activation of progestin-sensitive reporter genes (17, 18, 19) and similarly inhibits the transcriptional activity of receptors for androgens, glucocorticoids, and mineralocorticoids (19, 20). In addition, PRA has been implicated in the inhibition of ER activity; cotransfection of PRA, ER, and estrogen-sensitive reporters has shown a striking diminution of ER trans-activation (16, 21, 22).
The implications of these findings in vitro, if borne out in vivo, are that the relative levels of PRA and PRB within target cells may determine the nature and magnitude of functional responses to progesterone and estrogen (19), yet it is not known whether PRA and PRB normally reside within the same cell in vivo, nor is it known whether both PR isoforms are expressed in the various cell types that respond to progesterone within target tissues, such as the endometrium. To understand the functional significance of PRA expression in normal physiology it is essential to determine whether PRA is coexpressed with PRB, and this study now reports the development of a dual immunofluorescent staining technique and the demonstration of PRA and PRB proteins by single cell analysis in the same tissue section of human endometrium during the menstrual cycle.
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Materials and Methods |
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Archival, formalin-fixed, paraffin-embedded endometrial tissue from 26 women who had undergone hysterectomy or endometrial biopsy at Westmead Hospital (Westmead, Australia) for complaints such as menorrhagia (n = 11), dysfunctional uterine bleeding (n = 4), benign leiomyoma (n = 3), pelvic pain (n = 2), infertility (n = 2), dysmenorrhea (n = 1), endometriosis (n = 1), uterine prolapse (n = 1), or urinary stress incontinence (n = 1) were studied. The women were between the ages of 18–49 yr. All of the endometrial samples were reported as being morphologically normal. Cases that could not be accurately dated with respect to the menstrual cycle were excluded.
Histological dating of endometrium
Menstrual cycle dating was performed according to the criteria of Noyes et al. (23). The menstrual cycle was divided into seven categories: menses (days 1–4), early proliferative (days 5–7), midproliferative (days 8–10), late proliferative (days 11–14), early secretory (days 16–19), midsecretory (days 20–24), and late secretory (days 25–28).
Immunohistochemical staining
Formalin-fixed, paraffin-embedded sections were cut at 2 µm using a standard rotary microtome, mounted onto SuperFrost Plus slides (Lomb Scientific, Sydney, Australia) to which Mayer Albumen adhesive (24) had been applied, and dried at 37 C for 72 h. This was followed by storage at 4 C for no longer than 3 weeks.
Antigen retrieval
A combination of heat and pressure was used for antigen retrieval as previously described (25). Briefly, immediately before staining, sections were deparaffinized, rehydrated to distilled water, and placed in 0.01 mol/L sodium citrate solution (pH 6.0). Pairs of slides were positioned back to back into Corning, Inc. 50-mL polypropylene centrifuge tubes (Crown Scientific, Sydney, Australia) with sufficient 0.01 mol/L sodium citrate solution to cover the tissue. The tubes were fitted with loose fitting screw caps, placed vertically into a foil-covered 500-mL beaker, and heated in a Tuttenaur 2540 EKA autoclave at 121 C and 15 psi for 30 min. After autoclaving, the sections were allowed to remain in the sodium citrate solution for a minimum of 30 min, followed by washing in three 5-min changes of phosphate-buffered saline (PBS).
Dual immunofluorescent staining
First sequence. To minimize nonspecific background staining, sections were blocked for 30 min with normal goat serum (Hunter Antisera, Jesmond, Australia) diluted 1:1 in PBS. All incubations were performed at room temperature in a humidified chamber. After removal of excess serum, the sections were incubated overnight with a mouse antihuman PR monoclonal antibody that detects PRB alone (hPRa6-IgG2b) (26) diluted 1:5 in PBS-0.5% Triton X-100 (Amresco, Solon, OH). Primary antibody incubation was followed by washing the sections in two 5-min changes each of PBS-0.5% Triton X-100 and PBS. The PR protein was detected by incubation for 30 min with a biotinylated goat antimouse antibody (DAKO Corp., Copenhagen, Denmark) diluted 1:100 in PBS, washing as described above, and a 60-min incubation with Texas Red (TXR)-avidin (Vector Laboratories, Inc., Burlingame, CA) diluted 1:250 with TXR buffer [0.1 mol/L sodium bicarbonate (pH 8.2), 0.15 mol/L sodium chloride, 0.5% (wt/vol) BSA]. During this latter step and for the remainder of the staining, sections were kept in the dark to minimize loss of fluorescence. The sections were washed, using a magnetic stirrer, for a total of 30 min in two changes of 600 mL PBS.
Blocking step. To block sites of potential cross-reactivity between the two staining sequences, sections were incubated overnight with goat antimouse Ig Fab (Cappel Antibodies, ICN Biomedical, Australia) diluted 1:200 in 1% (wt/vol) BSA-PBS and then washed in four changes of PBS for 5 min each.
Second sequence. Sections were incubated in normal goat serum diluted 1:1 in PBS for 30 min. This was followed by removal of excess serum and incubation for 4 h with the second mouse monoclonal antibody to detect human PR (hPRa7-IgG1) (26) diluted 1:10 in PBS-0.5% Triton X-100. Primary antibody incubation was followed by washing the sections in two 5-min changes each of PBS-0.5% Triton X-100 and PBS. The PR protein was detected by incubation for 30 min with a biotinylated goat antimouse antibody (DAKO Corp.), diluted 1:100 in PBS, washing as described previously, and a 60-min incubation with fluorescein isothiocyanate (FITC)-avidin (Calbiochem, Australia) diluted 1:200 with FITC buffer [0.6 mol/L sodium chloride, 0.6 mol/L sodium citrate (pH 7.0), 1% (wt/vol) BSA, and 0.2% (vol/vol) Tween-20]. The sections were washed, using a magnetic stirrer, for a total of 30 min in two changes of 600 mL PBS, mounted with Vectashield fluorescent mountant (Vector Laboratories, Inc.), and stored in the dark at 4 C.
Preparation of control cells
Breast cancer cell lines transfected with either PRA only or PRB only served as controls for specificity of the dual immunofluorescent staining technique. An MCF-7 clone expressing undetectable levels of PR (MCF-7 M11, obtained from Anna de Fazio, Garvan Institute of Medical Research, Sydney, Australia) was transfected with an expression vector encoding PRA (McGowan, E. M., et al., manuscript in press; MCF-7 M11/PRA), and the hormone receptor-negative cell line, MDA-MB-231 (American Type Culture Collection, Manassas, VA) was transfected with an expression vector encoding PRB (McGowan, E. M., et al., manuscript in press; MDA-MB-231/PRB). Approximately 1 x 107 cells were harvested with trypsin-ethylenediamine tetraacetate (Life Technologies, Inc., Victoria, Australia) washed twice in PBS and centrifuged for 5 min at 1000 rpm. The supernatant was discarded, and the cells were clotted by the addition of three drops of human plasma, three drops of thrombin (5 IU/mL; Fibrindex, Orthodiagnostic Systems, Raritan, NJ) and warming at 37 C for a few minutes until a visible clot appeared. The cells were fixed by adding 10 mL 10% buffered formalin (0.4% sodium acid phosphate and 0.56% anhydrous disodium phosphate in 10% formalin) for 45–60 min, dehydrated, cleared, and embedded in an automatic tissue processor (Tissue-Tek VIP3000, Miles Scientific, Elkhart, IN). To compare the relative signals of PRA and PRB fluorescence obtained using dual immunofluorescent staining with PR protein levels, paraffin-embedded blocks of the PR-positive breast cancer cell line, T-47D (E. G. Mason Research Institute, Worcester, MA) (27) were prepared as described above, and relative PRA and PRB fluorescence was compared to relative PRA and PRB protein levels by immunoblot analysis.
Control sections
Control sections were treated and stained in the same way as the test sections. Controls included sections adjacent to each endometrial sample stained using PBS-0.5% Triton X-100 1) in place of both primary antibodies to control for nonspecific staining and 2) to replace the second sequence primary antibody to ensure no cross-reactivity between the two staining sequences; and dual staining of the transfected cells expressing only PRA or PRB.
Immunoblot analysis of PR
T-47D cells were harvested and stored as described previously (28). Cell pellets were thawed on ice in PEMTG buffer (29) containing 0.4 mol/L KCl and protease inhibitors (0.5 mmol/L phenylmethylsulfonylfluoride, 1.4 µmol/L pepstatin A, 100 µg/mL bacitracin, 25 mmol/L benzamidine, 86 µmol/L leupeptin, and 77 µg/mL aprotinin). Cytosol extracts were prepared, and 25 µg protein were separated by electrophoresis and transferred to nitrocellulose as previously described (29). Protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Inc., Sydney, Australia). Blots were incubated with hPRa6 and -7 (26) at saturating concentrations and goat antimouse Igs linked to horseradish peroxidase at 1:5000 (Bio-Rad Laboratories, Inc.). Previous work (not shown) has demonstrated that the use of two PR antibodies (hPRa6 and hPRa7) does not result in overestimation of PRB, as the amount of PRB detected on immunoblots with a combination of hPRa6 and hPRa7 is comparable with that detected when using hPRa6 alone. This suggests that the epitopes recognized by each antibody are in close proximity and that their binding to PRB is mutually exclusive. PR immunoreactivity was visualized using a chemiluminescent method (ECL, Amersham Pharmacia Biotech, Castle Hill, Australia). The relative intensity of the immunoreactive bands, in the linear range of the film, was visualized by densitometric scanning of x-ray films (Molecular Dynamics, Inc., Melbourne, Australia).
Fluorescent analysis
PR staining was examined using an Olympus Corp. BX 40 fluorescent microscope fitted with filters to detect both TXR (BP 545–580) and FITC (BP 450–480) fluorescence simultaneously and each of the two fluorochromes separately. All of the sections were examined in detail under individual fluorochrome excitation and using the dual filter by three observers, and the intensity per field was recorded. Staining of the epithelial and stromal elements of the endometrium across the whole section was specifically examined and compared. The intensities of the TXR and FITC signals in each field were scored according to a five-point scale: 4–5, very high; 3, high; 2, moderate; 1, low ; and 0, negative. The mean intensity of each fluorochrome for each section was calculated. The same arbitrary scales were used to show the relative intensity of signals for PRA and PRB in both stromal and glandular tissues.
Immunoperoxidase staining
After antigen retrieval, sections were placed in 3.0% (vol/vol) hydrogen peroxide for 5 min to reduce endogenous peroxidase activity and washed in three changes of distilled water. To minimize nonspecific background staining, sections were blocked for 30 min with normal goat serum (Hunter Antisera) diluted 1:1 in PBS. All incubations were performed at room temperature in a moist chamber. After removal of excess serum, the sections were incubated overnight either with the primary mouse antihuman ER monoclonal antibody (DAKO Corp.), diluted 1:50 in PBS-0.5% Triton X-100 (Amresco) or with a mixture of hPRa7 and hPRa6 at final dilutions of 1:10 and 1:5, respectively, in PBS-0.5% Triton X-100. Primary antibody incubation was followed by washing the sections in two 5-min changes each of PBS-0.5% Triton X-100 and PBS and incubation for 30 min with a biotinylated goat anti-mouse antibody (DAKO Corp.) diluted 1:100 in PBS. Sections were then washed in four 5-min changes of PBS and incubated with a streptavidin-biotin-horseradish peroxidase complex prepared in accordance with the manufacturer’s instructions (Zymed Laboratories, Inc., San Francisco, CA). Sections were washed in PBS as before, and the PR or ER proteins were visualized using diaminobenzidine (DAKO Corp.; 1 mg/mL diaminobenzidine and 0.02% hydrogen peroxide in PBS). Finally, sections were lightly counterstained with hematoxylin, dehydrated, and mounted in Normount (Fronine, Sydney, Australia). The control sections were treated in the same way, except for replacement of the primary antibody with PBS-0.5% Triton X-100.
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Results |
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Selectivity and specificity of dual staining
Specificity of the dual immunofluorescent technique was
demonstrated by staining cell lines that express either PRA or PRB
alone. The hPRa6 antibody, which is PRB specific by immunoblot analysis
(26), strongly stained a cell line expressing only PRB (MDA-MB-231/PRB;
Fig. 1A
). The hPRa7 antibody recognizes
both PRA and PRB on immunoblot analysis (26). However, this antibody
fails to detect PRB on immunohistochemistry, as evidenced by the
absence of staining by this antibody of the PRB-expressing
MDA-MB-231/PRB cell line (Fig. 1B). This demonstrates that the epitope
on PRB recognized by hPRa7, although accessible after denaturing gel
electrophoresis and immunoblot analysis, is not accessible in fixed
tissue even after antigen retrieval. This suggests that the presence of
the additional N-terminal sequence in PRB may affect the conformation
of the region of the molecule in which the hPRa7 epitope is located in
such a way to reduce its accessibility in immunohistochemistry.
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Similarly, MCF-7 M11/PRA cells expressing only PRA proteins exhibited
exclusively green staining under dual excitation, confirming that
hPRa6, the PRB-specific primary antibody used in the first sequence,
did not detect the PRA isoform (Fig. 1D). When the two PR-expressing
transfected cell lines were mixed in an MCF-7 M11/PRA:MDA-MB-231/PRB
ratio of 1:5, only orange or green nuclei were observed under dual
fluorescent excitation, with no evidence of any yellow nuclei (Fig. 1E). Assessment of the relative levels of PRA and PRB
To determine whether both fluochromes were equally efficient in
detecting antigen, adjacent sections of the same specimen were stained
with the same antibody (hPRa6 or hPRa7). One section was visualized
using TXR, and the other using FITC, and the fluorescence intensity was
evaluated. Equivalent fluorescent intensity was observed (data not
shown), indicating that the efficiency of antigen detection by each
fluochrome was the same. To ensure that the red and green signals
obtained by dual immunofluorescent staining accurately reflected the
amounts of PRA and PRB protein present, relative PR isoform expression
in T-47D cells, which contain both PRB and PRA, was compared by
immunohistochemical and immunoblot analyses. Paraffin-embedded T-47D
cells stained by dual immunofluorescence revealed the majority of cells
to be either deep orange (PRB only) or yellow (PRA and PRB; Fig. 1F).
The mean intensity for each fluorochrome was scored over the entire
section (10 fields at x200 magnification), using the scale described
in Materials and Methods, as 4.65 for TXR (PRB) and 1.5 for
FITC (PRA), corresponding to a PRB:PRA ratio of 3.1. Immunoblot
analysis confirmed the predominant expression of PRB in this cell line
(Fig. 1G), and quantitation by densitometry showed the ratio of PRB:PRA
to be 3:1, consistent with the results observed by dual staining.
Expression of PRA and PRB in the endometrium
PRA and PRB isoforms were coexpressed in the nuclei of most
PR-positive cells. PRB was visualized as a red color under TXR
excitation (Fig. 2A), whereas FITC
fluorescent excitation of the same section revealed PRA proteins to be
green (Fig. 2B). When the same section was viewed with simultaneous
excitation of both fluorochromes, nuclei that expressed predominantly
PRA proteins were green, whereas nuclei that expressed primarily PRB
proteins were orange (Fig. 2C). Nuclei coexpressing both PRA and PRB
proteins in similar concentrations were yellow (Fig. 2C). In general,
there was homogeneous expression of the two isoforms in nuclei within
the same tissue compartment during most stages of the menstrual cycle,
although there were exceptions, as discussed below.
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) despite the presence of PRA in
these cells (Fig. 2F), demonstrating no cross-reaction between
components of the sequential stain. Inclusion of the second sequence
primary antibody (hPRa7) in a dual stained adjacent section of the same
tissue shown in Fig. 2, D and E, demonstrated the presence of PRA
proteins (Fig. 2F). Expression of PRA and PRB during the proliferative phase of the menstrual cycle
Glandular epithelium. During the proliferative phase
of the menstrual cycle, high levels of both PRA and PRB were noted in
the glands, as evidenced by intense yellow staining of glandular cell
nuclei (Fig. 3A). The cell to cell
expression of PR was homogeneous; the majority of cells within the
glands were positive for both PRA and PRB (Fig. 3A). The intensity of
PR staining increased during the proliferative phase, reaching maximal
levels in the mid- to late proliferative phase (Fig. 4A), and a similar increase in levels of
PRA and PRB was evident.
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), and PRA was
consistently observed to be the predominant isoform expressed in the
stroma (Figs. 3A and 4B). The intensity of PR staining in the stroma
increased during the proliferative phase; however, in contrast with the
glands, there was a greater relative increase in the PRA isoform (Fig. 4B). Expression of PRA and PRB during the secretory phase of the menstrual cycle
Glandular epithelium. During the early secretory phase, PR
protein levels in the glands were lower than those during the
proliferative phase, and glandular cells displayed greater
heterogeneity, with adjacent cells frequently appearing yellow or
yellow/green, consistent with a reduction in PRB expression in some
cells (Fig. 3B). However, by the midsecretory phase of the menstrual
cycle, although overall PR protein concentrations were still further
reduced, the majority of glands were stained orange, implying a
predominant expression of the PRB isoform (Figs. 2C and 3C). By the
late secretory phase the majority of glands were negative (Fig. 3D),
except for a low level expression of PRB persisting in some cells.
To determine whether the predominance of PRB in the glands in the
midsecretory phase of the cycle could be due to new PRB synthesis under
the influence of estrogen, the question of whether ER was present at
the same time in these sections was examined. Immunohistochemical
staining revealed ER staining in both stroma and epithelial glands in
the midsecretory phase (Fig. 3E), suggesting that estrogen binding to
ER in the glands may cause a second increase, primarily in PRB, in this
phase of the cycle.
Stroma. PR was expressed in the stroma throughout the
secretory phase of the menstrual cycle, and PRA was the predominant
isoform present in these cells (Fig. 3, C and D, and Fig. 4B). Levels
of PRA were similar in the stroma during the early to midsecretory
phases and declined in the late secretory phase (Fig. 4B). PRB was
reduced markedly during the early secretory phase (Fig. 4B), recovered
slightly in the midsecretory phase, and was virtually undetectable by
late secretory phase (Figs. 3D and 4B); this was similar to the pattern
of fluctuation in PRB noted in the glands in the secretory phase,
except that the magnitude of the changes seen in the stroma were less
pronounced.
Expression of PRA in predecidual cells of endometrial stroma
PR was expressed in a cell-specific manner in the endometrial
stroma during the secretory phase. Cells that displayed PR positivity
were large and displayed characteristic morphological features of
predecidualization, such as nuclear vesiculation and swelling of the
cytoplasm associated with increased glycogen storage (30) (Fig. 3F).
The small stromal granulocyte cells were largely PR negative.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro studies show the A and B isoforms of PR to demonstrate unique properties and to activate different target genes (15, 31). Transfection experiments have shown that PRA can act as a dominant negative inhibitor of PRB, dependent upon cell and promoter context (18, 19), and that it is generally considered to be the weaker of the two PR isoforms in activation of target gene transcription (19). Additionally, PRA can inhibit the transcriptional efficiency of ER (16, 21), a repression dependent on the absolute levels of PRA (16). As it was not known whether PRA is coexpressed in vivo with PRB, the significance of the inhibitory activity of PRA documented in vitro was difficult to evaluate. This study, using dual immunofluorescent localization of the PR isoforms, has now revealed that PRA and PRB are coexpressed in nuclei of PR-positive cells, and this suggests that the relative levels of PRA and PRB within endometrial cells may determine the nature and magnitude of functional responses to progesterone and estrogen during the menstrual cycle.
The results of this study provide the first insights into the relative expression of PRA and PRB in the glandular epithelial and stromal cells of the human endometrium. There are previous studies of PRA and PRB expression in the uterus using immunoblot analysis of whole cell extracts (32, 33), but no clear information on the cell types expressing the protein species revealed on immunoblots can be obtained using this technique. Immunohistochemical studies have also attempted to examine PRA and PRB expression by staining of adjacent sections with antibodies that recognized PRB only or both PR isoforms (34), but this approach is limited by the inability to compare PRA and PRB staining within individual cells and the difficulty of making quantitative comparisons between adjacent sections stained using different antibodies. Consequently, to date, it has not been possible to simultaneously reveal PRA and PRB within the same tissue section, and the dual immunohistochemical technique reported in this study has been helpful in this regard. It must be noted, however, that the samples used in this study, although morphologically normal, may have been obtained from patients with some concurrent uterine dysfunction. Although an effect of such concurrent dysfunction on expression and relative levels of PRA and PRB cannot be ruled out, there was remarkable consistency in the results obtained within the cohort regardless of the underlying clinical presentation.
Regulation of PRA and PRB during the menstrual cycle
Regulation in the proliferative phase. During glandular proliferation, levels of both PRA and PRB proteins increased markedly, reaching similar high concentrations by late proliferative phase, suggesting that the known induction of PR by estrogen (2, 4, 14, 35) in the glandular epithelium during the proliferative phase is reflected by increases in both PRA and PRB. Similarly, in the stroma, levels of both PRA and PRB proteins were increased during the proliferative phase, but in this tissue, the increase in PRA expression was greater than that in PRB, and throughout the proliferative phase PRA was the dominant isoform.
Although both PRA and PRB are increased by estrogen, there is evidence
in the literature for preferential up-regulation of PRB by estrogen in
T47D human breast cancer cells (29), in human endometrial tissue (33),
in chicken spleen and lung (36), and in the freshwater turtle oviduct
(37), implying that PRB, in some circumstances, may be the more
sensitive isoform to this hormone. However, there is also evidence that
estrogen can have a greater stimulatory effect on PRA protein levels
than PRB in chicken oviduct (38), suggesting that estrogen stimulation
of PRA and PRB is likely to be cell, tissue, and species specific. It
is also likely that mechanisms other than those under ER
control can
influence PR expression, as it is known that PR can be expressed in
ER-negative breast tumors (39), PR expression can be regulated by
growth factors (40), and ER knockout mice continue to express a low
level of PR messenger ribonucleic acid (41). In this study both PRB and
PRA were increased in the glandular epithelium during the proliferative
phase, although given that the immunofluorescent detection of these
proteins was only semiquantitative, it cannot be excluded that there
was a difference in the magnitude of the increase in the two isoforms.
It was notable in the stroma, however, that there was a greater
increase in PRA protein expression than PRB during the proliferative
phase.
Regulation in the secretory phase. In the glandular epithelium, expression of both PRA and PRB was reduced during the secretory phase of the cycle under the influence of rising serum progesterone levels, consistent with the known down-regulating effect of progestins on PR expression (1). Interestingly, dual immunofluorescent localization of PR revealed that there was discordance in the down-regulation of each isoform. In the early secretory phase, loss of PRB was evident, and glands expressing a predominance of PRA were noted, as shown by marked numbers of glandular nuclei staining green, whereas in the midsecretory phase, PRB was the predominant isoform expressed in most glands. These results are consistent with the possibility that there was an initial down-regulation of PRB protein by progesterone in the early secretory phase, but this loss was compensated for during the midsecretory phase by the second estrogen peak that is known to be present at this time (42). Conversely, loss of PRA protein in the glands was less marked during the early secretory phase, but levels of PRA decreased continuously during the secretory phase, and there was no apparent rise coincident with the second estrogen peak, as was observed for PRB. The discordance of progesterone down-regulation of PRA and PRB in the glandular epithelium suggests differential sensitivities of the two isoforms to the effects of progesterone during the secretory phase of the menstrual cycle. The maintenance of PRB expression during the midsecretory phase was an unexpected observation, but is suggestive of the continued need for progesterone action, mediated by PRB, in glandular tissue at this time.
In the stroma, there was little or no decrease in PRA expression in the early and midsecretory phases; PRA was always the predominant isoform observed, and there were minimal changes in the expression of this isoform throughout the secretory phase. Levels of PRB protein, however, fluctuated similarly to the fluctuations in PRB levels in the glandular epithelium, but at a lower magnitude. There was also a suggestion that PRA increased in the stroma in midsecretory phase; as ER was detectable at that time, and this coincided with the known timing of a second serum peak of estrogen (42), this may be due to estrogen stimulation of PRA in the stroma. The persistence and predominance of PRA in the stroma during the secretory phase suggest that previous demonstrations of PR in this tissue throughout the secretory phase (5, 7, 8, 9, 10, 12) are attributable to PRA. The mechanisms underlying the persistence of PRA in the stroma in the secretory phase of the cycle are not known, but could involve resistance of PRA in stromal cells to the down-regulating effects of progesterone, consistent with evidence in other species that PR can be resistant to down-regulation under some circumstances. For example, turtle hepatic PR is not down-regulated by progesterone (43), and the human myometrium continues to express PR throughout pregnancy despite extremely high progesterone levels during this period (44).
PRA and PRB expression and progesterone action in the uterus
There were distinct differences in the relative expression of PRA and PRB between epithelial glands and stroma of the endometrium, which may be associated with different roles for the isoforms in the different compartments of the uterus. The highest levels of PRA and PRB in the cycle were detected in the glandular epithelial cells during the late proliferative phase of the cycle. This suggests that when progesterone is released from the corpus luteum after ovulation, both isoforms are available to bind the ligand and to play a role in mediating the effects of progesterone in the glands, such as glycogenesis (45, 46) and basal vacuolation (30, 46). Progesterone also plays a role in inhibiting estrogen-mediated mitosis and stimulating secretory functions in the glandular epithelium (9): glandular secretions are initially released at the beginning of the midsecretory phase, on days 19–20, and reach a peak on day 21. By the end of the midsecretory phase (day 24), secretions in the glands diminish and become dissipated (46). The presence of high levels of both PR isoforms in the late proliferative phase in glandular epithelial cells, which exhibit well characterized responses to progesterone, not only implicates both PR isoforms in these progesterone responses, but also argues against the importance in these cells in vivo of the PRB inhibitory activity of PRA demonstrated in vitro and discussed above. It is possible, however, that PRA may be the PR isoform responsible for the progesterone inhibition of estrogen-mediated mitoses in glandular epithelial cells, which would be consistent with the ability of PRA to inhibit ER activity in vitro (16, 21, 22).
There was a persistence of PRB in the glands in the midsecretory phase,
suggesting that this PR isoform may be involved in the secretion of the
major secretory protein of the glandular epithelium, human
pregnancy-associated endometrial 2-globulin, which is
maximally produced in the late secretory phase (47). Alternatively,
continued progestational effects on the glands in the late secretory
phase of the menstrual cycle may be mediated at least in part by the
paracrine influence of PR-expressing stromal cells (9, 48).
The predominance of PRA in the stroma throughout the cycle suggests this isoform to be the principal isoform involved in mediation of the known progesterone effects in the stroma, such as stromal edema (46), stromal mitoses (45, 46), and predecidualization (46), during the secretory phase of the menstrual cycle. The detection of PRA in predecidual cells, but not stromal granulocytes, which do not undergo predecidualization, is consistent with this. The results of this study are consistent with the view that PRA may mediate specific pathways of progesterone action in uterine stroma. Although gene targets of PRA in the uterus are yet to be described, there is some evidence in other systems of preferential activation of gene expression by PRA (15, 31).
In summary, this study has demonstrated that PRA and PRB are coexpressed in target cells of the human uterus. PRA and PRB were expressed in comparable levels in glandular epithelium before subnuclear vacuolization and glycogenolysis, implicating both isoforms in this process, whereas the persistence of PRB, but not PRA, in the glands during the midsecretory phase is suggestive of the possibility that PRB may play a more significant role than PRA in glandular secretion during the late secretory phase of the cycle. In the stroma, predominance of the PRA isoform throughout the cycle implicates this isoform in progesterone-mediated events such as stromal edema, stromal mitosis, and predecidualization. Taken together, the results of this study support the view that PRA and PRB mediate distinct pathways of progesterone action in the glandular epithelium and stroma of the human uterus throughout the menstrual cycle.
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Acknowledgments |
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Footnotes |
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Received November 24, 1998.
Revised March 31, 1999.
Accepted May 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-disrupted mice. Proc Natl Acad Sci USA. 94:11008–11012.2-globin’ (2-PEG),
during the menstrual cycle. J Reprod Fertil. 82:665–672.[Abstract]
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 E. M. McGowan, A. J. Russell, V. Boonyaratanakornkit, D. N. Saunders, G. M. Lehrbach, C. M. Sergio, E. A. Musgrove, D. P. Edwards, and R. L. Sutherland Progestins Reinitiate Cell Cycle Progression in Antiestrogen-Arrested Breast Cancer Cells through the B-Isoform of Progesterone Receptor Cancer Res., September 15, 2007; 67(18): 8942 - 8951. [Abstract] [Full Text] [PDF]
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R. O. Burney, S. Talbi, A. E. Hamilton, K. C. Vo, M. Nyegaard, C. R. Nezhat, B. A. Lessey, and L. C. Giudice Gene Expression Analysis of Endometrium Reveals Progesterone Resistance and Candidate Susceptibility Genes in Women with Endometriosis Endocrinology, August 1, 2007; 148(8): 3814 - 3826. [Abstract] [Full Text] [PDF]
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 K. Diedrich, B.C.J.M. Fauser, P. Devroey, G. Griesinger, and on behalf of the Evian Annual Reproduction (EVAR) The role of the endometrium and embryo in human implantation Hum. Reprod. Update, July 1, 2007; 13(4): 365 - 377. [Abstract] [Full Text] [PDF]
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 M. Schumacher, R. Guennoun, A. Ghoumari, C. Massaad, F. Robert, M. El-Etr, Y. Akwa, K. Rajkowski, and E.-E. Baulieu Novel Perspectives for Progesterone in Hormone Replacement Therapy, with Special Reference to the Nervous System Endocr. Rev., June 1, 2007; 28(4): 387 - 439. [Abstract] [Full Text] [PDF]
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 R. L. Arnett-Mansfield, J. D. Graham, A. R. Hanson, P. A. Mote, A. Gompel, L. L. Scurr, N. Gava, A. de Fazio, and C. L. Clarke Focal Subnuclear Distribution of Progesterone Receptor Is Ligand Dependent and Associated with Transcriptional Activity Mol. Endocrinol., January 1, 2007; 21(1): 14 - 29. [Abstract] [Full Text] [PDF]
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S. C. Hewitt The Five W's of Progesterone Receptors A and B: Now We Know Where and When. Endocrinology, December 1, 2006; 147(12): 5501 - 5502. [Full Text] [PDF]
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P. A. Mote, R. L. Arnett-Mansfield, N. Gava, A. deFazio, B. Mulac-Jericevic, O. M. Conneely, and C. L. Clarke Overlapping and Distinct Expression of Progesterone Receptors A and B in Mouse Uterus and Mammary Gland during the Estrous Cycle Endocrinology, December 1, 2006; 147(12): 5503 - 5512. [Abstract] [Full Text] [PDF]
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 R. Pilka, I. Oborna, V. Lichnovsky, P. Havelka, H. Fingerova, P. Eriksson, S. Hansson, and B. Casslen Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients Hum. Reprod., December 1, 2006; 21(12): 3146 - 3156. [Abstract] [Full Text] [PDF]
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M. J. Brayman, J. Julian, B. Mulac-Jericevic, O. M. Conneely, D. P. Edwards, and D. D. Carson Progesterone Receptor Isoforms A and B Differentially Regulate MUC1 Expression in Uterine Epithelial Cells Mol. Endocrinol., October 1, 2006; 20(10): 2278 - 2291. [Abstract] [Full Text] [PDF]
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 R. Shao, B. Weijdegard, K. Ljungstrom, A. Friberg, C. Zhu, X. Wang, Y. Zhu, J. Fernandez-Rodriguez, E. Egecioglu, E. Rung, et al. Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E59 - E72. [Abstract] [Full Text] [PDF]
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 J J Brosens and B Gellersen Death or survival - progesterone-dependent cell fate decisions in the human endometrial stroma. J. Mol. Endocrinol., June 1, 2006; 36(3): 389 - 398. [Abstract] [Full Text] [PDF]
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N. S. Macklon, R. L. Stouffer, L. C. Giudice, and B. C. J. M. Fauser The Science behind 25 Years of Ovarian Stimulation for in Vitro Fertilization Endocr. Rev., April 1, 2006; 27(2): 170 - 207. [Abstract] [Full Text] [PDF]
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 S. Tulac, M. T. Overgaard, A. E. Hamilton, N. L. Jumbe, E. Suchanek, and L. C. Giudice Dickkopf-1, an Inhibitor of Wnt Signaling, Is Regulated by Progesterone in Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1453 - 1461. [Abstract] [Full Text] [PDF]
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