11. A Possible Mechanism of Inactivation of C17-C20 Lyase in Rat Ovary by a Single Injection of hCG
Keiko Suzuki
Keywords: P450, down-regulation, cholesterol, StAR, ROI
The activity of C17-C20 lyase in immature rat ovary which had been pretreated with PMSG was down-regulated completely in 6 h by a single injection of hCG. Since cycloheximide and actinomycin D prevented the decrease to some extent, the transcription of genes and newly protein syntheses were supposed to be involved in the process. At first testosterone in the ovary was measured by radioimmunoassay. In the experiment, testosterone increased 30 min after hCG treatment, but decreased to the basal level at 1 h. Lyase is a P450 enzyme and has a heme at the active center. Therefore heme oxygenase was investigated, because the enzyme destroys heme. At first the transcriptional level of heme oxygenase-1 was examined by northern blot analyasis. However, it did not change until 6 h after the injection of hCG . And although zinc protoporphyrin, an inhibitor of heme oxygenase, was injected to the rat 1 and 3 h after hCG treatment, the down-regulation of lyase was not prevented. Therefore heme oxygenase was not involved in the down-regulation. Next the transcription of steroidogenic acute regulatory protein (StAR) was examined similarly. The level of mRNA of StAR increased 20 min after the injection of hCG, and increased further until 2 h. Finally, to examine the possible involvement of reactive oxygen intermediates (ROI) in this process, rats were consecutively pretreated with dimethyl sulfoxide at 12 and 3 h before the injection of hCG. As a result, the down-regulation of lyase at 6 h after hCG injection was protected to some extent in these animals. Therefore ROI was possibly involved in the down-regulation.
In this system, a huge amount of cholesterol is supplied to the cholesterol side chain cleavage enzyme (SCC) transiently by hCG injection as shown by the transient increase of testosterone. And the transcriptional increase of StAR indicates cholesterol might have been supplied further continuously. It is known that P450 enzymes such as lyase generate ROI in this process and thus microsomal enzymes are destroyed by the ROI. This mechanism is possibly working in the down-regulation of lyase.