Tissue- and hormone-dependent progesterone receptor distribution in the rat uterus

Abstract

Background: Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals.

Methods: The uteri were collected from 1) ovx rats treated with E2 and/or P; 2) immature rats, intact cycling rats and animals pregnant day 8 and 18; 3) ovx rats treated with E2 or an estrogen receptor (ER)alpha agonist or an ERbeta agonist. Two antibodies were used, one detecting PRA+B and another one specific for PRB. Real-time PCR was used to determine mRNA levels for PRAB and PRB in experiment 3.

Results: In stroma and myometrium faint staining was detected in ovx controls (OvxC), whereas E2 treatment resulted in strong staining. In contrast to this, in luminal epithelium (LE) the staining was strong in the OvxC group, whereas E2 treatment during the last 24 hrs before sacrifice caused a decrease. Similar to OvxC the LE of the immature animals was strongly stained. In the pregnant rats LE was negative, well in agreement with the results seen after E2 treatment. In the pregnant animals the stroma and decidua was strongly stained for PRAB, but only faint for PRB, indicating that PRA is the most expressed isoform in this state. The increase in stromal and myometrial immunostaining after E2 treatment was also found after treatment with the ERalpha agonist PPT. The ERbeta agonist DPN caused a decrease of the PR mRNA levels, which was also found for PRAB and PRB immunostaining in the GE.

Conclusion: Stromal and myometrial PRAB levels are increased via ERalpha, as shown by treatment with E2 and the ERalpha agonist PPT, while the levels in LE are decreased. The uterine stroma of pregnant rats strongly expressed PRAB, but very little PRB, which is different to E2 treated ovx animals where both PRAB and PRB are strongly expressed. The ERbeta agonist DPN decreased the mRNA levels of PRAB and PRB, as well as the PRAB protein level in GE. These results suggest that ERbeta signals mainly down-regulate PR levels in the epithelial cells. ERalpha, on the other hand, up-regulates PR levels in the stroma and myometrium while it decreased them in LE. Thus, the effects from E2 and PPT on the mRNA levels, as determined by PCR, could be annihilated since they are increased and decreased depending on cell type. The distribution and amount of PR isoforms strongly depend on the hormonal milieu and cell type within the rat uterus.

Figure 1

Immunohistochemistry experiment 1. Representative images of immunohistochemical results from each hormone treatment group in experiment 1, PRAB A-H, PRB I-O. Treatment groups are as follows: OvxC: A and I, 24E: B and J. 24P: C and K, 48E: D and L, 48P: E and M, 24E+24P: F and N, 24P+24E: G and O. Magnification bar 30 μm in images A-H and 50 μm in images I-O.

Figure 2

Scoring results PRAB experiment 1. Results from manual scoring of PRAB immunohistochemistry results in stroma (upper panel), luminal epithelium (LE, middle panel) and myometrium (bottom panel). The "box-and-whisker plot" represents the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. An asterisk indicates a significant (p < 0.05) difference compared to the OvxC group.

Figure 3

Scoring results PRB experiment 1. Results from manual scoring of PRB immunohistochemistry results in stroma (upper panel), luminal epithelium (LE, middle panel) and myometrium (bottom panel). The "box-and-whisker plot" represents the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. An asterisk indicates a significant (p < 0.05) difference compared to the OvxC group.

Figure 4

Immunohistochemistry experiment 2. Representative images of immunohistochemical results from the immature (A), cycling (B,C) and pregnant rats (D-H). Image F shows PRB immunostaining while all the others show PRAB. The arrow in image H indicates the negative cells in the placenta. Magnification bar 30 μm in all images.

Figure 5

Immunohistochemistry experiment 3. Representative images of immunohistochemical results from the agonist treated rats, PRAB in left columnand PRB in right column. Representative images are shown from a rat in each treatment group as follows: OvxC (A,B), E2 (C,D), PPT (E,F) and DPN (G,H). The insert shows the myometrium from the same uterus. Magnification bar 30 μm.

Figure 6

Image analysis PRAB experiment 3. Results from image analysis of PRAB immunohistochemistry results in stroma (upper panel), luminal epithelium (LE, second panel), myometrium (third panel) and glandular epithelium (GE, bottom panel). The "box-and-whisker plot" represents the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. An asterisk indicates a significant (p < 0.05) difference compared to the OvxC group.

Figure 7

Image analysis PRB experiment 3. Results from image analysis of PRB immunohistochemistry results in stroma (upper panel), luminal epithelium (LE, second panel), myometrium (third panel) and glandular epithelium (GE, bottom panel). The "box-and-whisker plot" represents the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. An asterisk indicates a significant (p < 0.05) difference compared to the OvxC group.

Figure 8

Real-time PCR experiment 3. Representative real-time PCR experiments for expression of PRAB (upper panel) and PRB (bottom panel) mRNAs in uterus from rats subjected to treatment with E2, PPT and DPN. The values of relative expression of target genes were normalized against RPLP0 and displayed in arbitrary units. The "box-and-whisker plot" represents the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. An asterisk indicates a significant (p < 0.05) difference compared to the OvxC group.