Reproduction of many temperate zone birds is under photoperiodic control. The Japanese quail is an excellent model for studying the mechanism of photoperiodic time measurement because of its distinct and marked response to changing photoperiods. Studies on this animal have suggested that the mediobasal hypothalamus (MBH) is an important centre controlling photoperiodic time measurement. Here we report that expression in the MBH of the gene encoding type 2 iodothyronine deiodinase (Dio2), which catalyses the intracellular deiodination of thyroxine (T4) prohormone to the active 3,5,3'-triiodothyronine (T3), is induced by light in Japanese quail. Intracerebroventricular administration of T3 mimics the photoperiodic response, whereas the Dio2 inhibitor iopanoic acid prevents gonadal growth. These findings demonstrate that light-induced Dio2 expression in the MBH may be involved in the photoperiodic response of gonads in Japanese quail.

Local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating (DIO2) and -inactivating (DIO3) enzymes regulates seasonal reproduction in birds and mammals. Recent functional genomics analysis in birds has shown that long days induce thyroid-stimulating hormone production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells (EC) of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density, but the interface between the melatonin signal perceived in the PT and the thyroid hormone levels in the MBH remains unclear. Here we provide evidence in mice that TSH participates in this photoperiodic signal transduction. Although most mouse strains are considered to be nonseasonal, a robust photoperiodic response comprising induced expression of TSHB (TSH ␤ subunit), CGA (TSH ␣ subunit), and DIO2, and reduced expression of DIO3, was observed in melatonin-proficient CBA/N mice. These responses could not be elicited in melatonin-deficient C57BL/6J, but treatment of C57BL/6J mice with exogenous melatonin elicited similar effects on the expression of the abovementioned genes as observed in CBA/N after transfer to short-day conditions. The EC was found to express TSH receptor (TSHR), and ICV injection of TSH induced DIO2 expression. Finally, we show that melatonin administration did not affect the expression of TSHB, DIO2, and DIO3 in TSHR-null mice. Taken together, our findings suggest that melatonin-dependent regulation of thyroid hormone levels in the MBH appears to involve TSH in mammals.circadian rhythm ͉ melatonin ͉ pars tuberalis ͉ photoperiodism ͉ type 2 and 3 iodothyronine deiodinases O rganisms living outside the tropics detect and predict seasonal changes in day length (photoperiod) to adapt various metabolic and behavioral functions to the environment. This mechanism, called photoperiodism, allows animals to control the timing of reproduction so that they can raise their offspring in spring and summer when food is most abundant. Among vertebrates, birds possess a highly sophisticated photoperiodic mechanism and show robust responses to photoperiodic changes. Taking advantage of the elaborate avian photoperiodic system, we have recently revealed the gene cascade regulating the photoperiodic response of reproduction in Japanese quail (Coturnix japonica) by using a functional genomics approach (1, 2). Exposure to long days induced thyroid-stimulating hormone (TSH), a heterodimer of the TSH ␤ subunit (TSHB), and the common glycoprotein ␣ subunit (CGA, also called TSH ␣ subunit), in the pars tuberalis (PT) of the pituitary gland. TSH triggers the expression of type 2 iodothyronine deiodinase (DIO2) in the ependymal cells (EC) lining the ventrolateral walls of the third ventricle (i.e., the infundibular recess) within the mediobasal hypothalamus (MBH). DIO2 is a thyroid hormoneactivating enzyme that converts the prohormone thyroxine...

The molecular mechanisms responsible for seasonal time measurement have yet to be fully described. Recently, we used differential analysis to identify that the type 2 iodothyronine deiodinase (Dio2) gene is responsible for the photoperiodic response of gonads in Japanese quail. It was found that expression of Dio2 in the mediobasal hypothalamus is induced by light and that T(3) content in the mediobasal hypothalamus increased under long day conditions. In addition, we showed that intracerebroventricular infusion of T(3) mimics photoperiodically induced testicular growth. Because it is well known that thyroid hormone is also essential for the maintenance of the seasonal reproductive changes in a number of mammals, we examined expression of Dio2 in Djungarian hamsters and found expression in the ependymal cell layer lining the infralateral walls of the third ventricle and the cell-clear zone overlying the tuberoinfundibular sulcus. Signal intensity was high under long days and weak under short days. Although light pulse did not affect Dio2 expression, melatonin injections decreased Dio2 expression under long days. These results indicate that Dio2 may be involved in the regulation of seasonal reproduction in mammals in the same way as observed in birds.

The molecular mechanisms underlying photoperiodic time measurement are not well understood in any organism. Relatively recently, however, it has become clear that thyroid hormones play an important role in photoperiodism, and in a previous study we reported that long daylengths in Japanese quail increase hypothalamic levels of T(3) and of the thyroid hormone-activating enzyme, type 2 iodothyronine deiodinase. The present study extends these observations to measure gene levels of the thyroid hormone-inactivating enzyme, type 3 deiodinase. Levels decreased after exposure to long days, but increased under short days. Changes in the two genes were then analyzed during the precisely timed photoinduction that occurs in quail exposed to a single long day. The two gene switches are the earliest events yet recorded in the photoinduction process, and overall, these reciprocal changes offer the potential to regulate active brain thyroid hormone concentrations rather precisely at the site in the brain where photoinduction is triggered.

Photorefractoriness is the insensitivity of gonadal development to the stimulatory effects of long photoperiods in birds and to the inhibitory effects of short photoperiods in small mammals. Its molecular mechanism remains unknown. Recently, it has been shown that reciprocal expression of thyroid hormone-activating enzyme [type 2 deiodinase (Dio2)] and -inactivating enzyme [type 3 deiodinase (Dio3)] genes in the mediobasal hypothalamus is critical for photoperiodically induced gonadal growth. Since thyroid hormones are required not only for photoinduction, but also for the induction of photorefractoriness, we examined the expression of these genes in relation to photorefractoriness in birds and mammals. Transfer of birds to long photoperiods induced strong expression of Dio2. This was maintained in tree sparrow when they later became photorefractory, but decreased somewhat in quail. In hamsters, transfer to long photoperiods also induced strong expression of Dio2. High values were not maintained under long photoperiods, and, indeed, expression decreased at the same rate as in animals transferred to short photoperiods. There was no renewed expression of Dio2 associated with testicular growth as animals became refractory to short photoperiods. Expression of Dio3 was high under short photoperiods and low under long photoperiods in all the animals examined, except for the short photoperiod-refractory hamsters. Our present study revealed complex regulation of deiodinase genes in the photoinduction and photorefractory processes in birds and mammals. These gene changes may be involved in the regulation of photorefractoriness, as well as photoinduction.

In birds, the mediobasal hypothalamus (MBH) including the infundibular nucleus, inferior hypothalamic nucleus, and median eminence is considered to be an important center that controls the photoperiodic time measurement. Here we show expression patterns of circadian clock genes in the MBH, putative suprachiasmatic nucleus (SCN), and pineal gland, which constitute the circadian pacemaker under various light schedules. Although expression patterns of clock genes were different between long and short photoperiod in the SCN and pineal gland, the results were not consistent with those under night interruption schedule, which causes testicular growth. These results indicate that different expression patterns of the circadian clock genes in the SCN and pineal gland are not an absolute requirement for encoding and decoding of seasonal information. In contrast, expression patterns of clock genes in the MBH were stable under various light conditions, which enables animals to keep a steady-state photoinducible phase.

In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.

To compare the crystalline-state conformations of MαNP acid esters with those existing in the solution state, X-ray crystallographic analyses of 22 MαNP esters and two MαNP acids were carried out. It was found that all 27 conformers observed in the solid state have structures in which the 2-CH 3 group and the 2Ј-H of the naphthyl group are synperiplanar and are almost always located in the same plane. In addition, the CH 3 -O7-C2-C1 moiety in all cases has an antiperiplanar structure within the MαNP plane. From further analyses, 22 conformers adopt the so-called syn structure, in which the O7-C2-C1-O6 moiety is synperiplanar. On the other hand, the remaining five conformers have the so-called anti structure. With regard to the rotational conformation around C1ЈЈ-O5, all conformers have structures similar to the synperiplanar conformation. In the so-called syn conformers, the interatomic distance d(H8Ј-O6) is distributed between 2.48 Å and 2.96 Å, while the distance d(H8Ј-O7) varies between 2.26 Å and 2.56 Å, indicating the weak bifurcated hydrogen