Our study shows that EPs and FP are localized in different cell types of the rat uterus suggesting their involvement in normal uterine physiology. Although PG receptors are G protein coupled receptors, they are also localized to cytoplasm and nucleus [5, 7, 10, 17, 18]. We observed EPs and FP in cytoplasm and membranes, although some nuclear staining was seen in EP1 and EP4. Nuclear PG receptors have been reported to perform intracrine signaling by activating transcription [18, 19].
The mRNA data shows that EPs and FPs are regulated by E2. It has been reported that P4 does not regulate mRNA of EPs, but upregulated EP2, EP3 and EP4 proteins in rat cervix . In the uterus of ovariectomized rodents EP2 mRNA increased when given P4 alone or co-treated with E2 [3, 13]. We did not have mRNA data to compare, but upregulation of EP2 protein levels by P4 was not found in our study. EP2 mRNA expression was downregulated by E2 in mice  which is in agreement with our observation in rats. Previous studies have shown the regulation of EP2, EP3 and EP4 mRNAs by E2 in pseudopregnant endometrium  and EP2, EP3 and EP4 protein by P4 in rat cervix  showing steroidal regulation of EPs. However, most conclusions in previous studies on mice are based only on RNA levels [2, 3, 11, 13, 21] and few studies shows the regulation of protein levels [5, 12]. We showed that the ERα selective agonist PPT downregulates all EPs and FP mRNAs, whereas the ERβ agonist DPN downregulates EP2 and EP4 mRNAs. We did not observe any differences in protein expression after PPT or DPN treatment. This could be due to the short duration of the treatments. These observations indicate that ERα may play a major role in the regulation of EPs and FP mRNA expression. It is known that ERα is the dominant ER subtype in rat uterus . The expression of EP2 and EP4 are decreased also by DPN in addition to E2 and PPT. Thus, ERβ could be involved in relaxation of the uterine smooth muscle cells. The mRNA level of FP was decreased by E2 and PPT, but not DPN treatment, indicating an ERα mediated FP regulation. None of the PG receptors inducing contraction are affected by DPN.
Our study shows the expression of EPs and FP proteins are differentially regulated by ovarian steroids. Interestingly, EP2 and EP4 proteins are exclusively regulated by E2 but EP1 and EP3 proteins are regulated only by a combination of E2 and P4. FP protein levels appear to be insensitive to ovarian steroids. Thus, the contraction mediating receptors are regulated exclusively by a combination of E2 and P4, while the relaxatory receptors are regulated only by E2. EP2 regulates epithelial differentiation and implantation [3, 12, 23]. E2 is known to induce proliferation and changes in epithelium in preparation for implantation . Thus, regulation of EP2 by E2 in epithelium may be important for implantation. Further, EP2, EP3 and EP4 are known to play vital roles in decidualization . Our findings show that administration of P4 after E2 priming upregulates EP1 and EP3, indicating the role of both steroids in the regulation of EPs in stroma during decidualization. These receptors may be regulated via a P4 mediated pathway . EP4, upregulated in stroma by E2 in our study, is known to trigger decidualization under the direction of ovarian steroids . PG receptors can constrict (EP1 and EP3) or dilate (EP2) blood vessels [23, 25, 26]. Treatment with E2 increased EP2 protein in blood vessels indicating that uterine blood flow could be regulated by E2 via EP2. It was recently reported that the expression of EP2, EP3 and EP4 increased in human endometrium during the mid-secretory phase, coinciding with increased stromal edema, blood vessel permeability and blood flow . We could also show that E2 upregulates the expression of EP2 and EP3 proteins in myometrium. Interestingly, when treated with E2 in combination with P4, there is an upregulation of EP1 and EP3 also in myometrium. It appears that E2 and P4 act synergistically to increase the expression of EP1 and EP3 and estrogen priming seems to be especially important for EP3. Thus, EPs may modulate the contractile and relaxatory functions under the regulation of steroid hormones.
E2 downregulates the mRNA levels of EPs and FP, but their protein levels were either upregulated or unchanged suggesting a multilayered regulation of RNA and protein. This apparent discrepancy has to be interpreted with caution. It is not possible to compare mRNA results with protein, as RNA data shows the total expression while immunohistochemistry detects the protein in a cross section. However, it is interesting to note that such discrepancies have been reported before [1, 12, 20, 27]. Although there are justifications based on the techniques used, we speculate that there may be a post-transcriptional regulation. Estrogen is known to regulate mRNA stability of many genes , and could be the possible regulator. A weakness of the present study is that only RNA data from studies 1 and 4 are analyzed. It would have added strength to the study if RNA levels from studies 2 and 3 had been available, enabling studies of long duration of E2 treatment and E2 and P4 combined effects.
The exact mechanisms of the steroidal regulation of PG receptors at transcription and translation are not clear. There are reports showing the presence of response elements in PG receptor genes that could be utilized by E2 and P4 to regulate their expression. EP1 has an estrogen response element and AP1 element [29, 30], EP2 has a progesterone receptor binding element , EP3 and EP4 have SP1 binding sites and the FP promoter contains AP1 and SP1 sites [32, 33]. Thus, it is possible that E2 and P4 directly or indirectly regulate the expression of PG receptors through these binding sites. E2 can also bind to G-protein coupled estrogen receptor-1 (GPER) to activate kinase mediated pathways  which may also regulate the expression of PG receptors.