Fig. 5.
Generation of 7α-hydroxypregnenolone in the pineal gland. (A) Biosynthetic pathway of 7α-hydroxypregnenolone from cholesterol. (B) Expressions of StAR, P450scc, and P4507α in the pineal gland. Total RNA extracted from the tissues at ZT6 and ZT18 were mixed and subjected to reverse transcription with (+) or without (–) reverse transcriptase (RTase), followed by PCR amplification. (C and D) Production of 7α-hydroxypregnenolone from pregnenolone in the pineal and diencephalon lysates. (C) The homogenate from the pineal gland or the diencephalon were incubated with [3H]pregnenolone for 40 min, and each extract was subjected to HPLC with a reversed-phase column. (D) Radioactivity of 7α-hydroxypregnenolone fraction was plotted against incubation time. Data are the mean ± SEM (n = 6). (E) Effect of light on 7α-hydroxypregnenolone release from the pineal gland. The chicks were exposed to 20-min light pulse (≈300 lx; white fluorescent lamps) from CT6, CT14, or CT22, and then pineal glands were isolated and pooled (eight pineal glands) to culture for 3 h under the light. For dark control group, all procedures were performed in the dark. The amounts of 7α-hydroxypregnenolone released into the medium were measured by gas chromatography/mass spectrometric (GC-MS) analysis. Data are the mean ± SEM (n = 4). (F) Effect of intracerebroventricular injection of 7α-hydroxypregnenolone on chick locomotor activity. 7α-hydroxypregnenolone (0 ng, n = 14; 10 ng, n = 5; 200 ng, n = 9) was injected at ZT14-16, and the locomotor activity of each individual was recorded for 20 min. Data are the mean ± SEM P < 0.005 by one-way ANOVA. (G) Effect of light on locomotor activity. Locomotor activity of individual chick was recorded for 10 min before (under infrared light) and after (under white LED light; ≈300 lx) the 10-min light exposure at CT6–8, CT14–16, or CT22–24 (Left). Data are the mean ± SEM (n = 6–8). Light/dark ratio of the activity of each chick was plotted in Right (P < 0.05 by one-way ANOVA). (H) Effect of pinealectomy on light-dependent stimulation of locomotor activity. Locomotor activity of pinealectomized (n = 6) or sham-operated (n = 7) chick was recorded for 10 min before and after the 10-min light pulse at CT14–16, and light/dark ratio of the activity was plotted as in G. *P < 0.05 by Student's t test.