Molecular Mechanism of Photoperiodic Time Measurement in the Brain of Japanese Quail

In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic‐pituitary‐gonadal axis. Recent investigations of Japanese quail show that long‐day‐induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T₄) to bioactive 3,5,3′‐triiodothyronine (T₃). The T₃ content in the MBH is approximately 10‐fold higher under long than short days and conditions, and the intracerebroventricular infusion of T₃ under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T₃ from T₄ by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T₃ and T₄ into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long‐day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.     

Molecular mechanism of the photoperiodic response of gonads in birds and mammals

Appropriate timing of various seasonal processes is crucial to the survival and reproductive success of animals living in temperate regions. When seasonally breeding animals are subjected to annual changes in day length, dramatic changes in neuroendocrine-gonadal activity take place. However, the molecular mechanism underlying the photoperiodic response of gonads remains unknown for all living organisms. It is well known that a circadian clock is somehow involved in the regulation of photoperiodism. Recently, rhythmic expression of circadian clock genes was observed in the mediobasal hypothalamus (MBH) of Japanese quail. The MBH is believed to be the center for photoperiodism. In addition, long-day-induced hormone conversion of the prohormone thyroxine (T(4)) to the bioactive triiodothyronine (T(3)) by deiodinase in the MBH has been proven to be important to the photoperiodic response of the gonads. Although the regulating mechanism for the photoperiodic response of gonads in birds and mammals has long been considered to be quite different, the long-day-induced expression of the deiodinase gene in the hamster hypothalamus suggests the existence of a conserved regulatory mechanism in avian and mammalian photoperiodism.     

Comparative analysis of the molecular basis of photoperiodic signal transduction in vertebrates

In temperate zones, the reproductive physiology of most vertebrates is controlled by changes in photoperiod. Mechanisms for the regulation of photoperiodic gonadal responses are known to differ between mammals and birds: in mammals, melatonin is the photoperiodic signal messenger, whereas in birds, photoperiodic information is received by deep brain photoreceptors. Recently, the molecular mechanism of photoperiodism has been revealed by studies on Japanese quail, which exhibit a most remarkable responsiveness to photoperiod among vertebrates, and molecular cascades involved in photoperiodism have been elucidated. Long-day stimulus induces expression of the β-subunit of thyroid stimulating hormone (TSH-β) in the pars tuberalis (PT) of the pituitary gland, and TSH derived from the PT regulates reciprocal switching of genes encoding types 2 and 3 deiodinases (Dio2 and Dio3, respectively) in the mediobasal hypothalamus (MBH) by retrograde action. Dio2 locally converts prohormone thyroxine (T(4)) to bioactive triiodothyronine (T(3)) in the MBH, which subsequently stimulates the gonadal axis. These events have been confirmed to occur in mammals with seasonal breeding, such as hamsters and sheep, suggesting that similar mechanisms are involved among various vertebrates. In addition, nonphotoperiodic mice also appeared to possess the same molecular mechanisms at the hypothalamo-hypophysial level. It has been noted that melatonin regulates the above-mentioned key genes (Dio2, Dio3, and TSH-β) in mammals, while photoperiod directly regulates these genes in birds. Thus, the input pathway of photoperiod is different between mammals and birds (i.e., melatonin versus light); however, the essential mechanisms are conserved among these vertebrates.     

Differential response of type 2 deiodinase gene expression to photoperiod between photoperiodic Fischer 344 and nonphotoperiodic Wistar rats

The molecular basis of seasonal or nonseasonal breeding remains unknown. Although laboratory rats are generally regarded as photoperiod-insensitive species, the testicular weight of the Fischer 344 (F344) strain responds to photoperiod. Recently, it was clarified that photoperiodic regulation of type 2 iodothyronine deiodinase (Dio2) in the mediobasal hypothalamus (MBH) is critical in photoperiodic gonadal regulation. Strain-dependent differences in photoperiod sensitivity may now provide the opportunity to address the regulatory mechanism of seasonality by studying Dio2 expression. Therefore, in the present study, we examined the effect of photoperiod on Dio2 expression in photoperiod-sensitive F344 and photoperiod-insensitive Wistar rats. A statistically significant difference was observed between short and long days in terms of testicular weight and Dio2 expression in the F344 strain, while no difference was observed in the Wistar strain. These results suggest that differential responses of the Dio2 gene to photoperiod may determine the strain-dependent differences in photoperiod sensitivity in laboratory rats.     

Hypothalamic expression of thyroid hormone-activating and -inactivating enzyme genes in relation to photorefractoriness in birds and mammals

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.     

Exogenous T3 mimics long day lengths in Siberian hamsters

Siberian hamsters (Phodopus sungorus) exhibit seasonal cycles of reproduction driven by changes in day length. Day length is encoded endogenously by the duration of nocturnal melatonin (Mel) secretion from the pineal gland. Short-duration Mel signals stimulate reproduction and long-duration signals inhibit reproduction. The mechanism by which Mel signals are decoded at the level of neural target tissues remains uncharacterized. In Siberian hamsters, exposure to short day lengths or injections of Mel in long days results in a decrease in hypothalamic expression of type 2 iodothyronine deiodinase (Dio2) mRNA. Dio2 catalyzes the conversion of the thyroid hormone thyroxine to triiodothyronine (T3). Thus exposure to short and long day lengths should decrease and increase hypothalamic T3 concentrations, respectively. We tested the hypothesis that exogenous T3 administered to short-day hamsters would mimic exposure to long day lengths with respect to gonadal stimulation. Hamsters gestated and raised in short day lengths that exhibited photoinhibition of the testes were given daily subutaneous injections of T3 or saline vehicle for 4 wk beginning at week 12 of life. The results indicate that exogenous T3 induced gonadal growth in short-day hamsters and delayed spontaneous gonadal development by an interval equal to the number of weeks during which T3 was administered. T3 injections delayed gonadal regression if given coincident with the transfer of hamsters from long to short day lengths. These results suggest that T3 mimics long day exposure in Siberian hamsters and may serve as an intermediate step between the Mel rhythm and the reproductive response.     

T3 implantation mimics photoperiodically reduced encasement of nerve terminals by glial processes in the median eminence of Japanese quail

Photoperiodically generated triiodothyronin (T₃) in the mediobasal hypothalamus (MBH) has critical roles in the photoperiodic response of the gonads in Japanese quail. In a previous study, we demonstrated seasonal morphological changes in the neuro-glial interaction between gonadotrophin-releasing hormone (GnRH) nerve terminals and glial endfeet in the median eminence (ME). However, a direct relationship between photoperiodically generated T₃ and seasonal neuro-glial plasticity in the ME remained unclear. In the present study, we examined the effect of T₃ implantation into the MBH on the neuro-glial interaction in the ME. T₃ implantation caused testicular growth and reduced encasement of nerve terminals in the external zone of the ME. In contrast, no morphological changes were observed in birds given an excessive dose of T₃, which did not cause testicular growth. These results support the hypothesis that thyroid hormone regulates photoperiodic GnRH secretion via neuro-glial plasticity in the ME. T. Yoshimura was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture, Japan.     

Circadian Clock Gene Per2 Is Not Necessary for the Photoperiodic Response in Mice

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.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.     

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.)

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5′-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3′,5′-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5′-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1 mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5′-deiodination in liver is potently stimulated by 1 mM DTT. The marked biochemical differences between 5′-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.     

Metabolism of the thyroid hormones

This review covers the current knowledge about the various metabolic pathways involved in the conversion of thyroid hormones to the thyromimetically active and inactive iodothyronines. The concerted mechanism of systemic and local production of iodothyronines by tissue-specific iodothyronine deiodinase isozymes will ultimately determine the expression of thyroid hormone action. This is exemplified for the regulation of synthesis and release of TSH by iodothyronines at the pituitary level. Iodothyronine metabolites, e.g. Triac, rT3 and T3 amine may modulate TSH secretion, and alterations of local pituitary deiodination (e.g. iopanoate inhibition) influence diurnal TSH secretion without changing TRH-dependent episodic TSH secretion pattern. A summary of structure-activity relationships of greater than 200 naturally occurring and synthetic ligands of rat liver type I iodothyronine deiodinase isozyme propylthiouracil-sensitive) in vitro allows the design of iodothyronine analogues which either serve as specific substrates or antagonists of iodothyronine binding and metabolizing proteins. Furthermore, a complete picture of the ligand-complementary active site of the type I isozyme can be derived. A synthetic 'structurally optimized' iodothyronine-analogue flavonoid inhibitor of the type I deiodinase is able to displace T4 from binding to thyroxine-binding prealbumin and leads to unexpected organ-specific alterations of thyroid hormone metabolism and expression of thyroid hormone actions in an animal model. Therefore, for a complete understanding of thyroid hormone metabolism and action, thyroid hormone transport, cellular compartmentalization, and alternate pathways also have to be considered.     

Changes in pituitary responsiveness during the ovulatory cycle of the Japanese quail, in vitro

Anterior pituitary glands from ovulating Japanese quail (Coturnix coturnix) were used to investigate variation in sensitivity to chicken luteinizing hormone-releasing hormone (cLHRH I; Gln8-LHRH). Grouping the pituitaries by ovulatory stage provided preliminary evidence of changes in sensitivity to LHRH during the ovulatory cycle. Pituitaries taken from quail before the preovulatory LH surge were responsive to cLHRH I, while pituitaries from the other times of the cycle showed minimal response to cLHRH I. Female pituitary glands release less LH than those of males. These data indicate a change in sensitivity to LHRH in the female quail that may be due to changes in gonadal steroids or the pool of releaseable LH from the pituitary.     

Dynamic expressions of hypothalamic genes regulate seasonal breeding in a natural rodent population

Seasonal breeding is a universal reproductive strategy in many animals. Hypothalamic genes, especially type 2 and 3 iodothyronine deiodinases (Dio2/3), RFamide‐related peptide 3 (Rfrp‐3), kisspeptin (Kiss‐1) and gonadotropin‐releasing hormone (GnRH), are involved in a photoperiodic pathway that encodes seasonal signals from day length in many vertebrate species. However, the seasonal expression patterns of these genes in wild mammals are less studied. Here, we present a four‐year field investigation to reveal seasonal rhythm and age‐dependent reproductive activity in male Brandt's voles (Lasiopodomys brandtii) and to detect relationships among seasonal expression profiles of hypothalamic genes, testicular activity, age and annual day length. From breeding season (April) to nonbreeding season (October), adult male voles displayed a synchronous peak in gonadal activity with annual day length around summer solstice, which was jointly caused by age structure shifts and age‐dependent gonadal development patterns. Overwintered males maintained reproductive activity until late in the breeding season, whereas most newborn males terminated gonadal development completely, except for a minority of males born early in spring. Consistently, the synchronous and opposite expression profiles of Dio2/3 suggest their central function to decode photoperiodic signals and to predict the onset of the nonbreeding season. Moreover, changes in Dio2/3 signals may guide the actions of Kiss‐1 and Rfrp‐3 to regulate the age‐dependent divergence of reproductive strategy in wild Brandt's vole. Our results provide evidence on how hypothalamic photoperiod genes regulate seasonal breeding in a natural rodent population.     

Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of 1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; 2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and 3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final 125I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. 125I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.     

Photoperiodic induction in quail as a function of the period of the light-dark cycle: implications for models of time measurement.

The earliest detectable event in the photoperiodic response of quail is a rise in luteinizing hormone (LH) secretion beginning at about hour 20 on the first long day. The timing of this rise was measured in castrated quail after entrainment to short daylengths which cause significant phase angle differences in the circadian system: (1) LD 2:22 and LD 10:14, and (2) LD 3:21 (T = 24 hr) and LD 3:24 (T = 27 hr). The quail were then exposed to 24 hr of light (by delaying lights-off), and the time of the first LH rise was measured; it was similar in all schedules. Quail were also entrained to LD 3:21 or LD 3:24 and then given a single 6-hr nightbreak 6-12, 7-13, or 13-19 hr after dawn. The earlier pulse was marginally more inductive in the 27-hr cycle. Thus the entrainment characteristics of the photoinducible rhythm (phi i) in quail appear very different from those of the locomotor circadian rhythm, and raise doubts as to whether phi i is a primary circadian oscillator.     

Influence of different photoperiods on development of gonad, cloacal gland and circulating thyroid hormones in male Japanese quail Coturnix coturnix japonica.

One day old chicks of Japanese quail were exposed to different photoperiods (LD, 8:16, 13.5:10.5, 16:8 and LL) and observations (testes weight, cloacal gland size, body weight and circulating thyroxine and triiodothyronine) were taken at the age of 3, 5, 7, 9 and 16 weeks. Results indicate that immediate reproductive development occurred in birds exposed to long photoperiods (greater than 12 hr). Growth under LD 8:16, was not apparent till 7th week and by 16 weeks, degree of gonadal development was similar in all the birds, irrespective of photoperiodic treatment. Whereas body weight of the intermediate and long day (LD 13.5:10.5, 16:8 and LL) treated birds increased upto 5th week and remained constant thereafter. But the chicks maintained under short day length (LD 8:16), showed spontaneous increase till the end of the study and birds were much heavier compared to all other groups. Plasma T4 concentration increased with increasing age till 9th week and remained unaltered thereafter. On the other hand T3 level did not change till 7th week followed by a decline. It is suggested that the initiation and degree of gonadal growth in quail depends on the availability of daily photoperiod, until the achievement of full breeding condition. Peak level of T4 observed in 9 week old birds may be involved in the development of photorefractoriness at that age.     

Bioenergetic impact of tissue-specific regulation of iodothyronine deiodinases during nutritional imbalance

The regulation of energy homeostasis by thyroid hormones is unquestionable, and iodothyronine deiodinases are enzymes involved in the metabolic activation or inactivation of these hormones at the cellular level. T3 is produced through the outer ring deiodination of the prohormone T4, which is catalyzed by types 1 and 2 iodothyronine deiodinases, D1 and D2. Conversely, type 3 iodothyronine deiodinase (D3) catalyzes the inner ring deiodination, leading to the inactivation of T4 into reverse triiodothyronine (rT3). Leptin acts as an important modulator of central and peripheral iodothyronine deiodinases, thus regulating cellular availability of T3. Decreased serum leptin during negative energy balance is involved in the down regulation of liver and kidney D1 and BAT D2 activities. Moreover, in high fat diet induced obesity, instead of increased serum T₃ and T₄ secondary to higher circulating leptin and thyrotropin levels, elevated serum rT3 is found, a mechanism that might impair the further increase in oxygen consumption.