Changes in Levels of Messenger Ribonucleic Acid for Cytochrome P450 Side-Chain Cleavage and 3β-Hydroxysteroid Dehydrogenase during Prostaglandin F2α-Induced Luteolysis in Cattle1

Regression of the CL causes a dramatic decrease in plasma progesterone levels. To test the hypothesis that the decrease in progesterone involves the down-regulation of mRNA encoding the steroidogenic enzymes, cytochrome P450 side-chain cleavage (P450scc) and/or 3β-hydroxysteroid dehydrogenase (3β-HSD), levels of plasma progesterone and luteal mRNA for P450scc and 3β-HSD were measured and correlated during induced luteolysis. Holstein heifers (n = 25) were injected with 25 mg prostaglandin F2α (PGF2α) on Day 6 or 7 of the estrous cycle (Day 0 = estrus) to induce luteal regression. To determine acute changes in plasma progesterone during luteolysis, jugular blood samples were obtained from 5 heifers hourly for 12 h, beginning immediately before injection of PGF2α, and assayed for progesterone by RIA. A significant decrease in plasma progesterone levels was observed as early as 1 h after the PGF2α injection (3.62 vs. 2.72 ng/ml, p < 0.05). Progesterone levels continued to decline with time through 12 h after administration of PGF2α. The other 20 animals were ovariectomized at 0 (n = 6), 2 (n = 4), 12 (n = 4), or 24 (n = 6) h after PGF2α. Levels of P450scc and 3β-HSD mRNA were determined in extracts of total luteal RNA by ribonuclease (RNase) protection assay. By 2 h after PGF2α, 3β-HSD mRNA levels had decreased by about 40% as compared with levels at time 0 (p < 0.05), and a further significant decrease occurred between 2 and 12 h. During the first 12 h after PGF2α injection, levels of 3β-HSD mRNA and plasma progesterone were highly correlated (r = 0.76, p < 0.005). No further significant decrease in 3β-HSD mRNA was observed at 24 h post-PGF2α. In contrast, levels of P450scc mRNA were not significantly different from levels at time 0 at either 2 or 12 h post-PGF2α and the correlation between P450scc and plasma progesterone was not significant (r = 0.38, p < 0.25). A significant decrease in P450scc mRNA was not observed until 24 h post-PGF2α, about 12 h after plasma progesterone had reached basal levels. In situ hybridization of riboprobes for P450scc and 3β-HSD to luteal tissue was conducted to compare morphological changes in the CL with P450scc and 3β-HSD mRNA levels during luteolysis. Results from in situ hybridization confirmed the results of RNase protection assays by showing a much more rapid loss of mRNA for 3β-HSD than of mRNA for P450sccc; the findings also indicated that the decline in message levels occurred in both large and small luteal cells and revealed that signs of cellular degeneration (pycnotic bodies, vacuoles in the cytoplasm) were not apparent until 24 h after the induction of luteolysis, when both plasma progesterone and 3β-HSD mRNA levels were low. The fact that cellular degeneration of luteal tissue occurred much later than the decline in plasma progesterone and mRNA for 3β-HSD suggests that loss of steroidogenic function precedes cellular degeneration. These results show that acute decreases in plasma progesterone during the initial stage of luteolysis are correlated with reduced levels of mRNA for 3β-HSD, but not for P450scc and suggest that the decrease in mRNA for 3β-HSD may be involved in the decline in plasma progesterone during luteolysis.