Circadian clock gene Per2 is not necessary for the photoperiodic response in mice

Abstract

In mammals, light information received by the eyes is transmitted to the pineal gland via the circadian pacemaker, i.e., the suprachiasmatic nucleus (SCN). Melatonin secreted by the pineal gland at night decodes night length and regulates seasonal physiology and behavior. Melatonin regulates the expression of the β-subunit of thyroid-stimulating hormone (TSH; Tshb) in the pars tuberalis (PT) of the pituitary gland. Long day-induced PT TSH acts on ependymal cells in the mediobasal hypothalamus to induce the expression of type 2 deiodinase (Dio2) and reduce type 3 deiodinase (Dio3) that are thyroid hormone-activating and hormone-inactivating enzymes, respectively. The long day-activated thyroid hormone T3 regulates seasonal gonadotropin-releasing hormone secretion. It is well established that the circadian clock is involved in the regulation of photoperiodism. However, the involvement of the circadian clock gene in photoperiodism regulation remains unclear. Although mice are generally considered non-seasonal animals, it was recently demonstrated that mice are a good model for the study of photoperiodism. In the present study, therefore, we examined the effect of changing day length in Per2 deletion mutant mice that show shorter wheel-running rhythms under constant darkness followed by arhythmicity. Although the amplitude of clock gene (Per1, Cry1) expression was greatly attenuated in the SCN, the expression profile of arylalkylamine N-acetyltransferase, a rate-limiting melatonin synthesis enzyme, was unaffected in the pineal gland, and robust photoperiodic responses of the Tshb, Dio2, and Dio3 genes were observed. These results suggested that the Per2 clock gene is not necessary for the photoperiodic response in mice.

Figure 1. Wheel-running activity rhythms of melatonin-proficient wild-type and Per2 deletion mutant mice.

(A, C) Locomotor activity of melatonin-proficient wild-type (Per2+/+) and Per2 deletion mutant (Per2 m/m) mice in a running wheel and (B, D) their periodogram analyses under constant darkness. (A, C) The bar over the records indicates the light-dark cycles. The mice were transferred from 12L12D to constant darkness (DD). The Per2 mutant mice showed shorter free-running rhythms followed by arhythmicity. Periodogram analyses were performed during the last 7 days. The peak above the diagonal line (<0.1%) indicates the significant circadian period (B).

Figure 2. Expression analysis of clock genes in the suprachiasmatic nucleus (SCN) under short/long day conditions.

Temporal changes in Per1 (A, B), Cry1 (C, D), and Cry2 (E, F) expressions in the SCN of the wild-type and Per2 mutant mice under short-day and long-day conditions. The dark and light lines in each graph represent the data of the wild-type and the Per2 mutant mice, respectively. The bars at the bottom of each graph represent the light conditions. Representative autoradiograms of the SCN are also shown. *P<0.05, **P<0.01 Per2+/+ vs. Per2 m/m at the same time point (Student’s t-test), mean ± SEM (n = 4–5). The arrowhead indicates the peak phase determined using cosinor curve fitting.

Figure 3. Temporal expression profiles of the circadian clock genes and arylalkylamine N-acetyltransferase (Aanat) gene expressions in the pineal gland.

Temporal gene expressions of Per1 (A, B), Cry1 (C, D), Cry2 (E, F), and Aanat (G, H) in the pineal gland under short-day and long-day conditions. The bars at the bottom of each graph represent the light conditions. Representative autoradiograms of the pineal gland (A, arrowhead) are also shown. *P<0.05, **P<0.01 Per2+/+ vs. Per2 m/m at the same time point (Student’s t-test), mean ± SEM (n = 4–5). The arrowhead indicates the peak phase determined using cosinor curve fitting.

Figure 4. Temporal expression profiles of the Aanat gene in wild-type (A) and Per2 mutant mice (B) under short-day and long-day conditions.

Data were replotted from Figure 3. The bars at the bottom of each graph represent the short-day (SD; blue) and long-day (LD; red) conditions. Representative autoradiograms of the pineal gland are also shown. *P<0.05, **P<0.01 LD vs. SD at the same time point (Student’s t-test), mean ± SEM (n = 4–5).

Figure 5. No clear internal coincidence timer in the mouse pars tuberalis (PT).

Temporal expression profiles of the circadian clock genes (Per1, Cry1, and Cry2) in the PT of wild-type and Per2 mutant mice. The bars at the bottom of each graph represent the light conditions. The dark and light lines in each graph represent the data of the wild-type and the Per2 mutant mice, respectively. *P<0.05, **P<0.01 Per2+/+ vs. Per2 m/m at the same time point (Student’s t-test), mean ± SEM (n = 4–5). The arrowhead indicates the peak phase determined using cosinor curve fitting.

Figure 6. The photoperiodic response of key genes regulating seasonal reproduction in wild-type and Per2 mutant mice.

The bars at the bottom of each graph represent the light conditions. The dark and light lines in each graph represent the data for the wild-type and the Per2 mutant mice, respectively. *P<0.05, **P<0.01 Per2+/+ vs. Per2 m/m at the same time point (Student’s t-test), mean ± SEM (n = 4–5). The arrowhead indicates the peak phase determined using cosinor curve fitting.