Onapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium

Abstract

The aim of this study was to examine whether progesterone (P₄) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P₄, ZK299, or RU486 at a dose of 10⁻⁴, 10⁻⁵, or 10⁻⁶ M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P₄ (10⁻⁴ M; P < 0.01) and RU486 (10⁻⁶; P < 0.001) and was decreased by ZK299 (10⁻⁵; P < 0.05). In contrast, PGRB mRNA was decreased by the all P₄ (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10⁻⁴ M; P < 0.01 and 10⁻⁵ M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10⁻⁵ M; P < 0.001). PGRB mRNA was decreased by P₄ (10⁻⁴ and 10⁻⁵ M; P < 0.001), ZK299 (10⁻⁴ and 10⁻⁵ M; P < 0.001), and RU486 (10⁻⁴ M; P < 0.01 and 10⁻⁶ M; P < 0.001) and was increased by ZK299 (10⁻⁶ M; P < 0.001) and RU486 (10⁻⁵ M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10⁻⁴ M; P < 0.05 and 10⁻⁵ M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10⁻⁵ M; P < 0.05 and 10⁻⁵ M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10⁻⁵ M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.

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.