Expression and Activation of Protein Kinase C Isozymes by Prostaglandin F2α in the Early- and Mid-Luteal Phase Bovine Corpus Luteum

Abstract

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKα, βI, βII, ε, and μ isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCγ, η, λ, and θ isozymes failed to detect protein bands in the luteal samples. PKCβII and ε isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCε was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCε was 1.16 ± 0.13. This ratio was higher than the detected ratio for PKCβI and μ at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKα and βII. The amount of PKCβII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 ± 0.2) than in the Day-4 CL (0.35 ± 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, α (Day 4 = 0.93 ± 0.16, Day 10 = 0.97 ± 0.09), βI (Day 4 = 0.54 ± 0.073, Day 10 = 0.48 ± 0.74), and μ (Day 4 = 0.21 ± 0.042, Day 10 = 0.21 ± 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF2α. Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF2α (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF 2α. Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF2α. Therefore, if PKC, an intracellular mediator associated with the luteal PGF 2α receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the ε and βII isozymes of PKC at this stage and not due to an inability of the PGF2α receptor to activate the isozymes expressed in the early CL.