Expression and Activation of Protein Kinase C Isozymes by Prostaglandin F2(alpha) in the Early- and MidLuteal Phase Bovine Corpus Luteum

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). PCK(alpha), (beta)I, (beta)II, (epsilon), and (mu) isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKC(gamma),(eta), (lambda), and (theta)isozymes failed to detect protein bands in the luteal samples. PKC(beta)II and (epsilon)isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKC(epsilon) was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKC(epsilon) was 1.16 (plus-minus) 0.13. This ratio was higher than the detected ratio for PKC(beta)I and (mu) at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCK(alpha) and (beta)II. The amount of PKC(beta)II was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 (plus-minus) 0.2) than in the Day-4 CL (0.35 (plus-minus) 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, (alpha) (Day 4 = 0.93 (plus-minus) 0.16, Day 10 = 0.97 (plus-minus) 0.09), (beta)I (Day 4 = 0.54 (plus-minus) 0.073, Day 10 = 0.48 (plus-minus) 0.74), and (mu) (Day 4 = 0.21 (plus-minus) 0.042, Day 10 = 0.21 (plus-minus) 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 PGF2(alpha). 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 PGF2(alpha)(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 PGF2 (alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF2(alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF2(alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the (epsilon)and (beta)II isozymes of PKC at this stage and not due to an inability of the PGF2(alpha) receptor to activate the isozymes expressed in the early CL.