To investigate the possible role of PR in regulating rapid effects on tubal CBF, we studied the effects on wildtype mice of low concentrations of progesterone, a specific agonist for the PR as well as a PR receptor antagonist. We also investigated the effect of progesterone on mice lacking both isoforms of the PR. All our results support the hypothesis that the PR mediates the rapid response of progesterone on CBF in the fallopian tube.
An involvement of the PR in the long-term regulation of CBF has previously been suggested [6, 8, 36]. For example Nakahari et al. 2011 injected female guinea pigs subcutaneously with progesterone three times over 1.5 days and the guinea pigs were killed 13 hours after the last injection. Progesterone treatment in vivo decreased the CBF and this reduction was blocked if the guinea pigs were injected with RU486 . As the animals were injected in vivo, and the response studied 1.5 days after the initial treatment, it is not possible to deduce if the regulation was direct (i.e. via PR within the fallopian tube) or indirect via stimulation of PR in other tissues. In another study Mahmood et al. investigated the effects of progesterone on human fallopian tubes in vitro and found that progesterone reduced the CBF with 40-50% 24 h after administration . This suggests a direct effect of progesterone on cells within the fallopian tube. Neither of these studies, however, studied rapid responses, in the time frame required if the cilia are to respond and adjust their beating according to locally excreted progesterone from the travelling cumulus complex. The present study shows that within 10 minutes after administration of low levels of progesterone, there is a decrease in CBF, and this decrease is blocked if the ciliated cells are pretreated with RU486, an antagonist for the PR. Furthermore, an important piece of evidence, not presented earlier, is the lack of response in PR knockout mice. This strongly support the specificity of the action found in the wildtype mice, i.e. that the main effect of the agonists and the antagonist is indeed through the PR and not via some other receptor, such as the mPRs. Supportive of a role of the PR and not the mPRs was furthermore our finding that R5020, which bind the PR but not mPRs, gave effects similar to progesterone. Taken together, this is the first study that provides comprehensive evidence for a role of the PR in the rapid regulation of CBF in the fallopian tube of any mammal.
In a study by Wessel et al. 2004, progesterone reduced CBF 15 minutes after administration in vitro to cow fallopian tubes, but in contrast to our study, the effect of progesterone was not reversed by RU486 , leading the authors to suggest the involvement of another receptor. Importantly, in their study, 20 μM of both progesterone and RU486 was used, and it is quite possible that equimolar concentrations of antagonist did not provide sufficient competition. Furthermore, the concentration of progesterone used was very high compared to the maximum serum levels of progesterone (100 nM) in the lutheal phase found in normally cycling women  and in relation to the binding affinity of progesterone to PR, which is in the nM range (the kd 1-5 nM ), thus raising questions about specificity. The same argument may be also raised regarding the study by Mahmood et al. . Indeed, high non-physiological concentrations of steroids can lead to stimulation of several receptors or have non-specific effects on perturbation of cell membranes [37, 38]. For example progesterone is a fairly potent agonist for the androgen receptor, and at higher concentrations, progesterone is also reported to act receptor-independently with membrane vesicles, leading to decrease membrane fluidity, induced aggregation of these vesicles and renders cells permeable to hydrophilic molecules . On the other hand, the local concentration of progesterone in the follicular fluid may is considerably higher than the serum levels. In the peritoneal fluid of women the concentration of progesterone one week after ovulation reached over 900 nM [40, 41]. Thus, the ciliated cells in the fallopian tube are exposed to rather high levels of progesterone during the post-ovulatory phase, perhaps particularly so in the mouse where the ovary and the fallopian tube both are located within a common fluid-filled bursa. Hence, it is still an open question what concentration range of progesterone that is physiologically relevant here. Nevertheless, we show here that 10 nM is sufficient to affect the CBF, and this is by no means an unlikely physiological concentration.
The exact localization of the PR in fallopian tube has been debated. There is immunohistochemistry data suggesting that both in the airways and in the oviduct, the PR is located to the lower half of the cilia rather than in the nucleus of the ciliated cell [13, 42]. The specificity of the unusual staining in the airways is supported by the movement of the PR-staining to the nucleus after progesterone administration. On the other hand, there are also reports on immunoreactivity to PR-antibodies in the nuclear compartments of luminal epithelial, stromal, and smooth muscle cells in the mouse fallopian tube . More research is apparently needed to clarify the localization of PR in the fallopian tube.
In ciliated cells of the airways, progesterone reduces the CBF with maximum reduction found 24 hours after treatment a response that is evidently genomic in nature . This conclusion is based on a much slower response, a clear translocation of the PR to the nucleus after progesterone administration, and that both the translocation and the effect on CBF could be blocked by the antagonist for PR RU486. Furthermore, the CBF was also blocked by the transcription inhibitor, actinomycin D. A puzzling observation with the study on airways , also acknowledged by the authors, is that a much higher concentration of progesterone was required (20 μM) than in the fallopian tube (10 nM) studied here.