Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor alpha knockout mouse uterus

Abstract

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor alpha knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor alpha modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.

Figure 1

Schematic diagram of hormonal and surgical regimen used to induce decidual response. The top portion of the figure is a schematic representing the trends in progesterone (P) and estradiol (E) levels in the mouse during the estrous cycle and early pregnancy. Estradiol levels peak just before ovulation during the estrous phase and then drop, but remain above the basal level through the periimplantation period. The progesterone level begins to rise after ovulation and is maintained at a plateau if implantation occurs. To mimic this cycle, animals are given a priming dose of estradiol on days 1, 2, and 3 (E). After 2 days with no injection, mice are treated with progesterone and a low dose of estradiol (PE) on days 6–13. Six hr following the third P injection, the uterine lumen is traumatized as described in the Materials and Methods section to mimic implantation. The tissue is collected and analyzed on the day following the final injection. The hormonal regimen was altered to determine the role of estrogen by excluding estrogen from all injections (no E), or cotreating with antiestrogen ICI 182,780 (anti-E) with all injections.

Figure 2

PR-A and PR-B isoforms are present in all three ERα genotypes. Cytosol from ERKO, heterozygous (H), and WT animals was analyzed by SDS/PAGE/Western blot as described in Materials and Methods. Two PR isoforms, PR-A and PR-B, are detected with anti-PR antibody h928, but not when primary antibody was not used (control). Size (in kDa) of ¹⁴C-labeled molecular weight markers (mw) is indicated.

Figure 3

Calcitonin and amphiregulin mRNA induction by progesterone in the uterus is ERα independent. (A) Uterine poly(A)⁺ RNA (3 μg/lane) was analyzed by Northern blot as described in Materials and Methods. WT or ERKO mice were ovexed and then treated as described with vehicle (C) or estrogen primed for 1 day and then treated with progesterone for 3 days (P). Duplicate samples were prepared from identically treated animal sets and analyzed in adjacent lanes. The Northern blot was probed with calcitonin cDNA (CALCI) or cyclophilin riboprobe (CYP). The RNA size markers (in kb) are indicated on the upper left. (B) Total uterine RNA (10 μg/lane) was analyzed by Northern blot as described in Materials and Methods. WT or ERKO mice were ovexed and then treated as described with vehicle, progesterone alone (PROG), or estradiol and progesterone (EST + PROG) and sacrificed 4 hr later. Duplicate samples were prepared from identically treated animal sets and analyzed in adjacent lanes. The Northern blot was probed with amphiregulin riboprobe (Top) or cyclophilin riboprobe (Bottom). The position of RNA size markers is indicated in kb.

Figure 4

Decidualization occurs in ERKO uterus. WT and ERKO mice were treated as described in Fig. 1 and in Materials and Methods. Uteri (Lower) were traumatized on the third day of PE treatment as described. The WT uterus pictured (Lower) shows a decidual reaction in one horn, indicating that oil may have entered only one horn.

Figure 5

Decidualization is estrogen dependent in the WT but not in the ERKO. (A) WT and ERKO mice were given the hormonal regimen described above with the addition of the antiestrogen ICI 182–780 each day. Uteri (Lower) were traumatized as described. (B) WT and ERKO mice were treated as described in Fig. 4 with the omission of estrogen at each injection. Both uteri shown were traumatized as described.

Figure 6

Fold increase in uterine weight in responding uteri. Uteri were weighed and the average fold increase in weight of responding uteri was calculated in comparison with nontraumatized control uteri of the same genotype treated with the same hormonal regimen. Numbers of determinations: WT E, PE, n = 5; WT P only, n = 3; WT ICI, n = 10; ERKO E, PE, n = 5; ERKO P only, n = 2; ERKO ICI, n = 5.