The expression of progesterone receptor coregulators mRNA and protein in corpus luteum and endometrium of cows during the estrous cycle
Introduction
Progesterone (P4) is a steroid sex hormone produced mainly by the corpus luteum (CL), follicle and uterus during the estrous cycle (Niswender et al., 2000). It is a key regulator of reproductive processes in females of many species of mammals, including cows. The physiological effect of P4 is carried out through a genomic mechanism via specific nuclear progesterone receptors (PGRs) and through a non-genomic mechanism by interaction with a specific membrane progesterone receptor. The nuclear PGR occurs in two main isoforms: isoform A (PGRA) and isoform B (PGRB). Both isoforms are transcribed from the same gene but under the influence of two different promoters (Mulac-Jericevic and Conneely, 2004). Isoform PGRB is longer than PGRA by approximately 164 nucleotides in humans; in other species, this difference varies from 128 to 165 amino acids (Mulac-Jericevic and Conneely, 2004). It has been established that PGRB acts mainly as an activator of progesterone-responsive genes, but when both receptors are expressed, PGRA acts as a repressor of PGRB activity (Wen et al., 1994, Pieber et al., 2001). The inactive form of the PGR receptor is associated with a complex of chaperone proteins (Cheung and Smith, 2000). Connection of the hormone to the receptor causes dissociation of the protein complex and translocation of the receptor to the cell nucleus. Inside the nucleus, the receptors dimerize and bind to hormone response elements (HREs) located in the promoters of target genes. The last step in PGR activation is attachment to the receptor of additional transcriptional regulatory elements known as coregulators, a group of proteins that interact with the receptor complex without binding to the DNA of the target gene sequence (Glass and Rosenfeld, 2000). These proteins are characteristic regulators of any member of the nuclear receptor superfamily while there is no data on their involvement in regulation of membrane receptors (Lonard and O’Malley, 2012). The coregulators consist of two groups of proteins: coactivators, proteins that enhance the transcription of target genes, and corepressors, proteins that inhibit the transcription of such genes (Xu et al., 1999). Coactivators have an intrinsic histone acetyltransferase (HAT) activity that, by acetylation of histones, loosens chromatin and causes greater availability of transcription factors and polymerase at the appropriate gene sequence (Tyler and Kadonaga, 1999). Corepressor proteins cooperate with histone deacetylases (HDACs) and remove acetyl groups from histones, thereby increasing chromatin condensation, and transcription of the target gene is not initialized (Lazar, 2003). Both coactivators and corepressors may exhibit variable expression in the estrous cycle, as shown in the human endometrium (Shiozawa et al., 2003). Therefore, it is possible that these changes may affect P4 action or functions of PGR in cows. Thus, the aim of these studies was to evaluate expression of mRNA and protein of the coactivator P300/CBP-associated factor (PCAF) and the corepressor Nuclear Receptor Corepressor 1 (NCOR1) in luteal and endometrial tissue during the estrous cycle in cows.
Section snippets
Tissue collection
Corpora lutea (n = 5) and endometrial tissue (n = 5) from non-pregnant cows and mature heifers were harvested at a commercial slaughterhouse on days 2–5, 6–10, 11–16, and 17–20 of the estrous cycle. Days of the estrous cycle were determined by criteria reported by Ireland et al. (1980) and Fields and Fields (1996), respectively. Immediately after collection, the whole CLs and 1–2 prepared strips of each endometrium, weighing approximately 5 g were frozen in liquid nitrogen, transported to the
Progesterone concentrations
The luteal concentration of P4 was higher during days 6–16 of the estrous cycle than during days 2–5 (P < 0.01) or 17–20 (P < 0.01) (Fig. 1A). The endometrial concentration of P4 was the highest on days 2–5 of the estrous cycle, followed by a decrease on days 6–10 (P < 0.01) to the lowest level on days 11–20 (P < 0.05) (Fig. 1B).
Expression of PCAF and NCOR1 mRNA in CL and endometrium during the estrous cycle
The PCAF expression in CL was higher during days 6–16 of the estrous cycle than during days 2–5 (P < 0.01) or 17–20 (P < 0.01) (Fig. 2A). In endometrium, it was higher on days 2–10
Discussion
The data obtained in these experiments show that the highest levels of PCAF mRNA and protein in CL appeared on days 6–16 of the estrous cycle, and the lowest levels appeared at the beginning and end of the cycle. A correlation of PCAF mRNA and protein levels with the concentration of P4 in CL was also shown. The mRNA and protein level for PCAF was not correlated with previously received the levels of mRNA and proteins of PGRA and PGRB isoforms in CL (Rekawiecki et al., 2015) (data not shown).
Acknowledgements
We thank Dr. S. Okrasa (University of Warmia and Mazury, Olsztyn, Poland) for the antibodies against progesterone antiserum. The study was supported by a grant (UMO-2015/17/B/NZ4/02440) from the National Science Centre and by The Polish Academy of Sciences. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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