Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) beta-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor-cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

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.

It is now known that circadian clocks are localized not only in the central pacemaker but also in peripheral organs. An example of a clock-dependent peripheral organ is the ovary of domestic poultry in which ovulation is induced by the positive feedback action of ovarian progesterone on the neuroendocrine system to generate a preovulatory release of LH during a daily 6-10 h "open period" of the ovulatory cycle. It has been assumed previously that the timing of ovulation in poultry is controlled solely by a clock-dependent mechanism within the neuroendocrine system. Here, we question this assumption by demonstrating the expression of the clock genes, Per2 (Period 2) and Per3, Clock, and Bmal1 (brain and muscle Arnt-like protein 1), in preovulatory follicles in laying quail. Diurnal changes in Per2 and Per3 expression were seen in the largest preovulatory follicle (F1) but not in smaller follicles. We next sought to identify clock-driven genes in preovulatory follicles focusing on those involved in the synthesis of progesterone. One such gene was identified, encoding steroidogenic acute regulatory protein (StAR), which showed 24-h changes in expression in the F1 follicle coinciding with those of Per2. Evidence that StAR gene expression is clock driven was obtained by showing that its 5' flanking region contains E-box enhancers that bind to CLOCK/BMAL1 heterodimers to activate gene transcription. We also showed that LH administration increased the promoter activity of chicken StAR. We therefore suggest that the timing of ovulation in poultry involves an LH-responsive F1 follicular clock that is involved in the timing of the preovulatory release of progesterone.