Research articleOnapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium
Introduction
Progesterone (P4), which is produced in the CL, is the main steroid hormone regulating the estrous cycle and maintaining gestation in many species, including cows [1]. This hormone influences target cells via membrane nongenomic receptors (PGRMC and mPR) [2], [3] and nuclear P4 receptor (PGR) through genomic mechanisms. Nuclear PGR occurs as the following two main isoforms: A (PGRA) and B (PGRB) [4]. The same gene encodes both isoforms, but transcription starts from two different promoters. PGRB is longer than PGRA because of the presence of additional amino acids at the N-terminus. The length of this N-terminal region varies from 128 amino acids in chickens [5] to approximately 164 amino acids in humans [6]. Moreover, both isoforms also differ in their activities. PGRB is a gene activator that is dependent on P4, whereas PGRA is a weak gene activator that acts as a potent inhibitor of PGRB, thereby reducing the effects of P4 [7].
Bovine endometrial tissue shows the highest levels of protein expression of both the PGRA and PGRB isoforms on Days 1 to 5 and 17 to 20 of the estrous cycle. The lowest levels of expression these proteins are observed on Days 6 to 16 [8]. The endometrial concentration of P4 is the highest on Days 1 to 5 and then declines on Days 11 to 16 and 17 to 20 [8]. Endometrial PGR messenger RNA (mRNA) is expressed mainly in the subepithelial stroma and in the superficial glands throughout the estrous cycle, and it is also localized to the luminal epithelium but at much lower concentrations [9], [10]. Progesterone antagonists repress the biological activity of P4 by inhibiting PGR activation. These compounds have greater affinity for the receptor than for P4 alone and compete with the hormone for receptor binding [10]. They are valuable tools that are often used in reproductive research aimed at the further elucidation of the role of P4 in physiological and pathologic processes [11]. The most popular PGR antagonists are onapristone (ZK299) and mifepristone (RU486) [12]. These compounds represent two groups of antagonists that differ in terms of their interactions with receptors. The binding of ZK299 to PGR prevents its association with its target DNA [13]. In contrast, RU486 is capable of inducing a high-affinity association between the receptor and target DNA after receptor binding. Thus, it is believed that RU486 interferes with a receptor activation step downstream of DNA binding and that ZK299 impairs receptor binding to DNA [14]. In addition to its inhibitory effect, RU486 also displays partial agonist activity. The binding of RU486 to PGRA causes this receptor to become inactive but does not affect the target gene. However, during interaction with the PGRB isoform, it uses energy from the breakdown of cyclic adenosine monophosphate (cAMP) and is converted into a highly active receptor agonist [15], [16], [17].
The aim of this study was to examine whether P4 and its antagonists, ZK299 and RU486, regulate PGRA and PGRB mRNA and protein levels in the bovine uterus. Our findings may be essential to determine whether the final physiological effect evoked by an antagonist depends on the PGR isoform that is bound to it.
Section snippets
Endometrial collection
Uteri from healthy cyclic cows and heifers were harvested at a commercial slaughterhouse within 20 minutes after their deaths and transported to the laboratory in ice-cold PBS within 1 hour after their deaths. The stage of the estrous cycle was estimated by morphologic observations of the ovaries and uteri, as described previously [18], [19]. Endometrial tissues from Days 6 to 10 of the estrous cycle, which have the highest levels of PGR protein, and from 17 to 20 days of the estrous cycle,
PGFM medium concentrations
The addition of AA increased PGFM levels after 6 hours of stimulation in the endometrial slices harvested on Days 6 to 10 (P < 0.05) and on Days 17 to 20 (P < 0.01) and after 24 hours of stimulation in the endometrial slices harvested on Days 6 to 10 (P < 0.01) and on Days 17 to 20 (P < 0.01; Fig. 1) compared with the control. The increase in PGF2α secretion from the slices incubated with AA indicated the viability of the sections and their potential responses to the factors evaluated.
mRNA levels of PGR isoforms
Discussion
These data indicate that P4 and PGR antagonists have different effects on the mRNA and protein expression levels of both the PGRAB or PGRA and PGRB isoforms in the bovine endometrium, depending on the phase of the estrous cycle. Progesterone at the 10−4 M dose increased PGRAB mRNA levels in the samples on Days 6 to 10 of the cycle. This observation suggests that the mRNA levels of the PGR isoforms depend on P4 concentrations in cows, similar to an observation made by Misao [28] in humans. These
Acknowledgments
The authors would like to thank Professor W.J. Silvia (University of Kentucky, KY, USA) for the PGFM antiserum. This study was supported by a grant (N N311 113638) from the Ministry of Science and Higher Education and by the Polish Academy of Sciences.
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