| Disappearance of the progesterone receptor (PGR) from the uterine luminal epithelium (LE) is essential for cyclicity and pregnancy in pigs. In humans, three PGR mRNA isoforms (PGR-A, PGR-B and PGR-C) arise from alternative transcription start sites, conferring distinct biological functions. The objective was to identify and characterize PGR mRNA and protein isoforms in the pig during the estrous cycle and pregnancy. Primer pairs for porcine PGR were developed from porcine genomic and human mRNA sequences and used to amplify porcine PGR fragments from cDNA.;Primer pairs for quantitative real-time PCR (qRT-PCR) were originally developed from porcine genomic and mRNA sequence and used to amplify porcine PGR fragments from cDNA. There was a tendency for an effect of d on uterine PGR-B expression (P < .10), because PGR-B mRNA fold change was greater on day 0 (d 0; 0.52 +/- 0.07) and d 5 (0.51 +/- 0.07) compared with d 7.5 (0.31 +/- 0.07) and d 15 (0.30 +/- 0.05) (remaining d were intermediate). The PGR-AB mRNA remained low through d 13 (0.13 +/- 0.01; d 0 to 13; cyclic and pregnant) and increased on d 15 in both pregnant (0.90 +/- 0.07) and cyclic (0.41 +/- 0.07) pigs (P < 0.001). The PGR-AB mRNA remained elevated in pregnant pigs on d 17 (0.33 +/- 0.06). The existence of PGR-B and possibly PGR-A isoforms were detected by Northern Blot analysis. PGR-C, however, was not detected. Transcription initiation sites were detected in PGR by RPA: PGR-B.1, 291-314; PGRB.2 , 379; PGR-A, 1046; PGR-C.1, 2559; PGR-C.2, 2631. We conclude that PGR isoform mRNA abundance may change during the estrous cycle and pregnancy potentially leading to functional differences in PGR action. We also observed the presence of PGR-B, PGR-A and PGR-C in qRT-PCR and ribonuclease protection assay analyses, although some transcripts may be too unstable to be consistently detected by Northern blot analysis.;Three PGR antibodies were identified that were expected to cross-react with porcine PGR: a PGR-B-specific antibody and two PGR-AB antibodies which targeted the region of PGR protein common to PGR-B, PGR-A and PGR-C isoforms. The presence of PGR-B and PGR-A proteins was confirmed by immunoblotting, but none of the antibodies were able to detect PGR-C. Additionally, the molecular weights of the proteins identified, though consistent with previous findings in humans and pigs, were observed to be greater than predicted from amino acid sequence. Finally, abundance of PGR-B and PGR-A isoforms was assessed on d 0, d 8 and d 12 (n = 3 samples per day), with no differences detected between the days. The three antibodies were then used for immunohistochemistry localization of PGR isoforms to specific cell types. The PGR-B specific antibody detected strong nuclear staining in the LE and uterine glandular epithelium (GE), decreasing in intensity from d 8 to d 12. Both PGR-AB antibodies, however, detected no nuclear staining in the LE and only sporadic staining of the stroma nuclei. Antibodies to PGR-AB also showed cytoplasmic staining in the uterine LE, GE and stroma that was not observed using the PGR-B antibody, with strongest staining on the apical surface of the LE on d 12. The PGR-A isoform, therefore, may have a more diffuse cytoplasmic staining than the strictly nuclear staining of PGR-B. Taken together, these results may indicate varying levels of biological activity rather than protein abundance of the two isoforms on days 8 and 12 of the estrous cycle in porcine endometrium.;The project concludes that porcine PGR has high nucleotide and amino acid conservation with human, mouse and cow sequences. The project concludes that abundance of mRNA and abundance of protein are, at times, uncoupled in this system, with PGR-C present as an mRNA transcript but not a translated protein. Abundance of mRNA and abundance of protein, at other times, appear to act in tandem, with PGR-B mRNA and PGR-B protein nuclear staining in the LE and GE both decreasing from d 8 -- d 12. New questions to be investigated were identified for further study to increase understanding of PGR isoform expression in the porcine endometrium. (Abstract shortened by UMI.). |
