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Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼492nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

An analysis of the circadian and circannual patterns of luteinizing hormone (LH) secretion in male European starlings (Sturnus vulgaris) held in outdoor aviaries is presented. On the basis of six daily time points, eight calendar dates, and seven individuals per sampling unit, we present a mathematical model developed to compare the two temporal patterns of secretion, the circadian and the circannual one. The superposition of four orthonormal trigonometric base functions, plus a constant, is used to examine the daily fluctuations. The contribution of the second harmonic was not the same at each calendar date. From the subsequent regression functions, hourly estimates of LH concentration were calculated and used to construct circannual cubic splines and an LH(t,T) function over the whole year. The model features indicate strong circannual and circadian components in the data. In addition, there appear to be two daily time regions of LH secretion: morning and afternoon. In particular, the afternoon maxima occur during photorefractoriness and early gonadal development; the morning maxima at the onset of photoinduction and late gonadal development. At the peak of gonadal activity (end of April), the highest LH levels occur during the photic input phase. It appears, then, that the circadian acrophase of LH secretion in male starlings fluctuates over the annual cycle of gonadal activity in a highly predictive and biologically interpretable manner.

This perspective considers first the general issue of seasonality and how it is shaped ecologically. It asks what is the relative importance of "strategic" (photoperiod-dependent) versus "tactical" (supplemental) cues in seasonality and what neural circuits are involved? It then considers recent developments as reflected in the Special Issue. What don't we understand about the photoperiodic clock and also the long-term timing mechanisms underlying refractoriness? Are these latter related to the endogenous annual rhythms? Can we finally identify the opsins involved in photodetection? What is the present position with regard to melatonin as "the" annual calendar? An exciting development has been the recognition of the involvement of thyroid hormones in seasonality but how does the Dio/TSH/thyroid hormone pathway integrate with downstream components of the photoperiodic response system? Finally, there are the seasonal changes within the central nervous system itself--perhaps the most exciting aspect of all.

It has been demonstrated using Fos immunocytochemistry that copulation activates specific cell populations in the mammalian brain. Prior to this study, no similar work has been carried out in birds. In mammals, Fos has identified brain circuits activated by genital (penile)/somatosensory and by olfactory/vomeronasal stimuli. Such inputs, of course, should play little or no role in birds (no penis, little or no role for olfaction) and a differential responsiveness could therefore be expected. Male Japanese quail (Coturnix japonica) were allowed to interact freely with adult females and the presence of active sexual behavior, including cloacal contact movements, was confirmed in each case. Control subjects were exposed to a domestic chick (same size as an adult quail) and no sexual behavior was observed. Copulation induced the appearance of Fos-like immunoreactive (FLI) cells in the preoptic area, the hyperstriatum ventrale, parts of the archistriatum, and the nucleus intercollicularis. Induction of FLI cells was observed throughout the rostral to caudal extent of the preoptic region of males from the level of the tractus septomesencephalicus to the level of the anterior commissure, and in the rostral part of the hypothalamus to the level of the supraoptic decussation. The FLI cells did not lie directly adjacent to the third ventricle, but were located 500-1000 microns from the ventricle wall at the level of the lateral edge of the medial preoptic nucleus or, in more caudal sections, in a position ventrolateral to the bed nucleus striae terminalis. It is unlikely that the Fos induction in males resulted from copulation-induced endocrine changes because copulation did not affect plasma levels of luteinizing hormone or testosterone. It is concluded that the responses were due to copulation-associated somatosensory inputs and/or to stimuli originating from the female.

The hypothesis that thyroid hormones are required for the expression of normal cycles of reproductive activity was tested in Soay rams, a widely used subject for studies on seasonality. Six rams were thyroidectomized and six sham-operated in October, just before the expected peak of reproductive activity. All were then placed in long days (18 h light and 6 h dark, 18L:6D) for 12 weeks and then transferred to short days (8L:16D). All animals exhibited an initial short-term increase in testicular size and serum concentrations of follicle-stimulating hormone and luteinizing hormone then, as expected, these parameters decreased in sham-operated animals for the remainder of the time they were maintained on long days. Four weeks after transfer back to short days, gonadotrophin concentrations in the control animals were significantly raised above the lowest level, and scrotal size increased after eight weeks. By contrast, in thyroidectomized rams all parameters remained high throughout the experiment. Thyroidectomy therefore abolished seasonal changes in reproductive activity, indicating a requirement for thyroid hormones for the normal expression of such patterns. It is unlikely that this was related to any generalized metabolic disturbances caused by thyroidectomy, as body masses were similar in both groups of animals throughout the experiment.

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment ‘sensor’ involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra ( λ max = ∼490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response ( λ max = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.

Plasma levels of luteinizing hormone (LH), testosterone (T), and 5 alpha-dihydrotestosterone (DHT) were measured in relation to age and social/breeding status among free-living male Australian magpies. Magpies live in territorial groups of up to 20 individuals and due to a largely age-related dominance hierarchy among males, many individuals are prevented from breeding. Adult plumage is not attained until the fourth year, but males can produce motile sperm in their first year. Plasma levels of LH and T peaked just prior to egg-laying among breeding males, but DHT levels remained steady or declined slightly during the breeding period. Adults (greater than 3.5 years) had significantly higher levels of both LH and androgens than did subadults (less than 3.5 years) during the breeding season. Nonbreeding adults had similar levels to those found among the breeding adults, but breeding subadults had higher levels of both LH and androgens than the nonbreeding subadults. These results are interpreted in terms of the opportunities for each of these classes to obtain copulations and are discussed in relation to age-related hormonal secretion in other species.

Changes in concentrations of hypothalamic gonadotrophin-releasing hormone (GnRH), pituitary and plasma LH, testicular mass, in-vitro release of testosterone, body mass and migratory activity were measured in male garden warblers (Sylvia borin) kept from November to June under a constant photoperiod of 12·8 h. Under such conditions garden warblers gradually change from the photorefractory to the photosensitive state and gonadal recrudescence then occurs. Hypothalamic GnRH content was low from December to March, but increased in April to reach the highest levels in June. The spontaneous increase in GnRH was paralleled by increases in pituitary LH content, testicular mass and in-vitro testosterone release. Body mass decreased 1 month and nocturnal activity 2 months before the spontaneous increase in GnRH. Ovine LH increased in-vitro testosterone release over basal release at all times. The results suggest that in garden warblers (1) changes in hypothalamic GnRH content can occur under constant photoperiodic condition, (2) the gradual change from the photorefractory to the photosensitive state is not characterized by a gradual increase in hypothalamic content of GnRH (cf. starlings), and (3) Leydig cells are capable of testosterone release even during the photorefractory state. Journal of Endocrinology (1991) 128, 339–345

Birds show a circadian rhythm in melatonin secretion and, as expected, the pattern of output changes with photoperiod. Somewhat surprisingly then, in view of the mechanisms in mammals, birds do not seem to use this seasonal message in the photoperiodic control of reproduction. Some further experiments are needed, however, because in birds the pineal gland is not the only source of melatonin. Another difference from mammals is that birds detect the photoperiodic light not with the retina but by brain photoreceptors, which probably lie in the hypothalamus. An action spectrum for these receptors has now been obtained for the quail and this shows a peak absorption at 492 nm, suggesting that the photoreceptor is rhodopsin-based. The sensitivity of the brain receptors to 500 nm light was calculated at 2 X 10(4) photons mm-2s-1. For light to induce the photoperiodic response it must be interpreted by the bird's clock as a long day. This happens if the light falls 12-20 h after dawn and coincides with a rhythm of photosensitivity. The subsequent neuroendocrine response to the light signal is both precise and relatively long-term. A single 4 h light pulse initiates a wave of gonadotropin secretion lasting for 10 days. The light stimulus can be replaced by a brief (2 min) daily electrical stimulus given to the hypothalamus 10-12 h after dawn. Over the next few years it should be possible to disentangle further the neural processes involved.

The challenge is how to provide our researchers with ready and unimpeded access to all types of research information, whether it is held on paper or electronically. This has been, of course, the role of research libraries for hundreds of years so why is it a particular challenge at the present time? May I suggest that the problems are acute because a number of factors are all occurring at the same time. In this paper I will mention some of these.

The rapid photoperiodic response in Japanese quail is so precise that it allows neural analyses of how photoperiodic information is transduced into an endocrine response. After transfer from short [SD; 6L:18D (6:18 hr light/dark cycle)] to long (LD; 20L:4D) days, luteinizing hormone (LH) first rises 20 hr after dawn. Using Fos immunocytochemistry, we examined the basal tuberal hypothalamus (BtH) to determine the relationship between brain cell activation and the first endocrine changes. Two separate cell populations within the BtH expressed Fos-like immunoreactivity (FLI) by hour 18 of the first LD. Importantly, this activation occurred before the LH rise. Median eminence activation appeared within glial cells, whereas activated infundibular nucleus cells were neuronal, providing support to the view that gonadotropin-releasing hormone (GnRH) release can be controlled at the terminals by glia. The FLI induction parallels LH changes, suggesting that gene expression may be involved in events preceding photostimulation and is the earliest photoperiodically stimulated physiological change yet reported.Additional experiments provided further support for this hypothesis. First, photoperiodically induced activation is not a result peculiar to castrates because intact birds displayed similar results. Second, the critical length of 14 hr of light had to be exceeded to cause both BtH activation and a LH rise 30 hr from dawn. Finally, valuable evidence of the response specificity was provided by using a unique property of the quail photoperiodic clock in which exposure to 10L:26D, but not 10L:14D, causes photoinduction. The 36 hr paradigm increased both plasma LH and BtH activation.

The extent to which circadian rhythms are involved in photoperiodic time measurement in quail is enigmatic, and earlier investigations have produced results consistent with an hourglass clock or one involving damped circadian oscillators. To address the problem further, the present authors carried out a direct comparison between the clocks in quail and those in starlings. Starlings possess strongly self-sustaining circadian oscillators. In Experiment 1, comparisons of testicular growth were made between the two species when birds were exposed to light:dark (LD) 6:30, LD 6:18, and LD 18:6. Starlings grew their testes rapidly under both LD 6:30 and LD 18:6, and they became photorefractory (under LD 6:18, the testes remained undeveloped). Quail grew their testes rapidly under LD 18:6 but did not do so under LD 6:30 or LD 6:18. In Experiment 2, entrainment of the activity rhythm under cycles of LD 6:30 was investigated by measuring the phase of the rhythm after release into constant darkness (DD). Birds were exposed to either 10 cycles or 11 cycles of LD 6:30 prior to DD. Starlings maintained their 24-h rhythmicity under LD 6:30 and always free ran from the phase of the subjective day By contrast, quail showed circadian activity approximately 24 h after every light pulse and free ran from the phase of the last light pulse received. In Experiment 3, phase response curves (PRCs) were generated to 6-h light pulses. The species were strikingly different; starlings produced a Type 1 PRC, whereas quail produced a Type 0 PRC. More important, in quail the 6-h light pulse had the same effect regardless of circadian time and in almost every case activity free ran from the position of the 6-h light pulse. The results in quail are consistent with the photoperiodic time measurement system being based on a weakly self-sustaining (rapidly damping) circadian system that is invariably reset by 6 h of light, whereas in starlings the pacemakers are strongly self-sustaining. The results support the notion that hourglass pacemakers can be highly damped circadian pacemakers.

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups.

Three experimental protocols were employed to clarify whether the circadian system is involved in photoperiodic time-measurement in the blackheaded bunting, Emberiza melanocephala. In a single-pulse paradigm, one 8-h light pulse was delivered at different times to groups of birds across three days of constant darkness (DD). Photoperiodic induction, as measured by a rise in plasma luteinizing hormone (LH), showed clear circadian rhythmicity. The second experiment examined the LH responses in birds exposed to lighting cycles using a Nanda-Hamner type of protocol and confirmed full photostimulation under 6L:30D. The third experiment measured the time of the first photo-induced rise in LH in birds subjected to 30 h of continuous light following entrainment under short days (6L:18D). This experiment aimed to identify the position of the photoinducible phase (Φi). LH first rose at hour 18 following dawn indicating that Φi lies in the middle of the day. Plasma concentrations of melatonin were also measured under 6L:18D and 6L:30D light cycles as another physiological marker of the circadian system in buntings. The pattern of melatonin secretion under these LD cycles showed properties consistent with the driving oscillator being circadian in nature. It is concluded that the circadian pacemaker driving the photoinducible rhythm in blackheaded bunting is strongly self-sustaining and free-runs under constant conditions.

In photoperiodic species, short daylength resonance cycles of modulo t + ½t (t = 24 h) behave like long days because they entrain the circadian system so that alternate light pulses coincide with the photoinducible phase (ø1) in castrated quail. However, while a long-day response after exposure to a single long daylength is readily detected by a rise in plasma LH (photoinduction), long-term exposure to LD 6:30 is ineffective in this respect. To discover whether this occurs because of unusual entrainment, circadian rhythms in quail and starlings were investigated. Whereas starlings entrained in the expected way with alternate pulses falling at different circadian phases, activity bouts in quail appeared to follow 24 h after successive light pulses. Because of this, activity was examined in free-running conditions to confirm that the pacemaker in quail was indeed being reset to a constant phase (reset to circadian time [CT] 0) by successive pulses. Examination of the circadian rhythms of plasma melatonin secretion under LD 6:30 also showed a resetting to CT 0. The positioning of all light pulses at the same circadian phase in the early subjective day explains the lack of photoinduction in quail since ø1in the early subjective night phase remains unilluminated. A second feature in quail is that when the length of the photophase is gradually increased within T36h cycles, there is a progressive increase in the degree of photoinduction although the photophase length remains well below the critical daylength for induction in normal T24h cycles. We therefore tested whether ø1is reset to a constant phase by successive pulses in LD 6:30, and that this phase is also advanced relative to light onset so that photophases shorter than the critical daylength can interact with ø1to cause induction. Such a reduction in critical daylength relative to successive LD 6:30 pulses was confirmed by transferring quail to various types of long day and measuring the change in LH secretion. When the long-day test was replaced with continuous light, stimulation of LH secretion occurred 5-7 h earlier in quail pretreated with LD 6:30 and LD 6:54 compared to quail pretreated with LD 6:18 or LD 6:42, implying that ø1had been markedly phase advanced under resonance cycles.

Photoperiodic stimulation of quail (Coturnix coturnix japonica) resulted in the appearance of a nuclear fos-like protein within neurones of the basal tuberal hypothalamus. On transfer to long days the number of neurones containing this fos-like immunoreactivity increased from about 150 to 700, the neurones being scattered throughout the length of the tubero-infundibular complex. This activation had occurred by early in the second long day and was maintained for at least three long days. Over this period circulating levels of LH increased seven-fold, indicating that photoperiodic induction had taken place in the birds. A similar time-course of fos-like induction occurred in castrated quail exposed to a single long day and then returned to short days. Activation mirrored the long-term changes in LH secretion found in this paradigm and fos-like immunoreactivity showed the same “carry-over” characteristics of photoperiodic induction, being maximal two days after the quail had been exposed to the single long day (and were again on short days) and when LH secretion was at its maximum. Activation of fos-like immunoreactive cells did not take place when long-day quail were transferred to short photoperiods. The evidence supports the view that the neurones being activated are involved in a specific fashion in the avian photoperiodic response.

The photoperiodic clock in quail (Coturnix colurnix japonica) is based upon a rhythm of photoinducibility (Oi) but the extent to which this rhythm is circadian remains unclear. Two types of experiment investigated this situation. In the first, gonadectomized quail were adapted to live in periods of darkness by training them on a schedule containing one short day and 3 days of darkness (SD/DD/DD/DD). They were then exposed to a single pulse of 6 or 10 h of light at different times across 3 days of darkness. The photoperiodic response, measured by the increase in LH secretion, showed clear rhythmicity, demonstrating unequivocally the circadian nature of Oi. The second set of experiments employed Nanda-Hamner cycles and varied the length of the photoperiod from 6 to 11 h. Responsiveness in a 36 h or a 60 h cycle was highly dependent upon the length of the photoperiod, something not predicted from theory. For instance, LD 6:30 was not photoperiodically inductive but LD 10:26 was clearly inductive. Close analysis of patterns of LH secretion indicated an unexpected delay before induction occurred and then a rapid rise to a stable level of induction. When LH was measured in every pulse under LD 10:26 there was no evidence that LH levels alternately increased and decreased. This is not consistent with the simplest interpretation of Nanda-Hamner experiments where alternate pulses of light are thought to “entrain” the rhythm or “induce” a photoperiodic response by coinciding with Oi. It is concluded that the quail's photoinducible rhythm is indeed based on a circadian rhythm but one that is only weakly self-sustaining. Possibly as a consequence of this, the rhythm's behaviour under abnormal photoperiodic cycles may be rather different from that found in other species and from other circadian rhythms in quail.

The neuroendocrine mechanisms regulating FSH secretion in birds are very similar to those in mammals, and the 400–600 GnRH cell bodies lie in the anterior hypothalamus, passing their axons in a broad band to end on the hypophysial portal vessels. Birds possess two forms of GnRH, but it is probable that only one of these (8-Gln-GnRH) is secreted from the median eminence: Like its mammalian counterpart, the bird stimulates the release of both FSH and LH. In male birds, FSH in conjunction with androgens causes spermatogenesis, while in females it triggers oogenesis and also aids yolk uptake from the circulation. It is still unknown what role the gonadotropins play in establishing and maintaining the characteristic hierarchy of oocytes within the avian ovary that allows for daily ovulations.

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 (Φ1) in quail appear very different from those of the locomotor circadian rhythm, and raise doubts as to whether Φ1 is a primary circadian oscillator.

Typically, birds come into breeding in the spring as a response to long days, and end reproduction some weeks later by becoming refractory to those long days. The refractory state is subsequently dissipated by the short days of autumn and winter, so producing once again a bird that can respond to long days. Bird species differ in the extent to which refractoriness is developed; the present experiments took advantage of the relative, rather than the absolute, refractoriness in quail to measure quantitatively the dissipation process. Quail were made refractory by exposure to long days, then transferred to short days and at various times thereafter photostimulated with longer daylengths, the degree of photoresponsiveness being assessed by measuring changes in luteinizing hormone (LH) secretion or cloacal gland size or both. The most clearcut results came from using 13L:11D as the test stimulus to measure photoresponsiveness, and this indicated, in both intact and castrated quail, no response to 13L:11D after one week of short days, a minor response after two weeks, a strong response after three weeks and a full response after five weeks. Thus refractoriness appears to be dissipated gradually under short days, and not in an all-or-none fashion. Confirmation of this conclusion came from experiments in which refractory quail were moved to short days and after one or two weeks transferred to a range of long daylengths. After one week of short days no responses were obtained to 13L:11D or 14L:10D and moderate responses only to 16L:8D, but after two weeks of short days the magnitudes of all the responses were increased. Other experiments showed that the depth of refractoriness (as assessed by transferring to short days and then to 13L:11D) was dependent upon the previous duration of exposure to long days and to the length of that long day. Finally, the development of refractoriness over a three month period on long days was also investigated.

The ontogenesis of sexual hormones and the responsiveness of both ovaries to LH were studied during the last third of incubation of chick embryos. Pituitary LH and FSH and serum 17 beta-estradiol increase through development to reach a plateau near hatching. Serum LH and the ovarian progesterone content show maxima on Day 19 of incubation. Serum progesterone levels are highly variable, with apparently unchanged values during development. Ovine LH was able to stimulate progesterone secretion in vitro in the left ovary at all times during development. LH stimulation of 17 beta-estradiol secretion in vitro is high on Day 13 in both ovaries but lower at later stages.

This study was undertaken to examine the effect of electrical stimulation of the hypothalamus at different times of day on luteinizing hormone (LH) secretion in male castrated quail on short days (8L:16D). The posterior hypothalamus was stimulated with square-wave pulses of 80 microA for 2 min through chronically-implanted platinum microelectrodes. Stimulation was carried out on each quail at 4 (treatment A), 10 (B), or 14 h (C) after dawn. Plasma LH levels were increased markedly within 2 min of ending the stimulation but reached basal levels again over the next 20 min or so. The absolute increase was significantly greater in treatment B (10 h after lights on) than at the other times tested. This is consistent with a rhythm in hypothalamic responsivity. The results are discussed in the context of the rhythm of photoinducibility which occurs early in the night and which is used by quail as a photoperiodic clock to regulate seasonal reproduction.

When starlings (Sturnus vulgaris) are transferred from short to long days, hypothalamic content of gonadotrophin-releasing hormone (GnRH) and pituitary gonadotrophin content eventually decrease, as birds become photorefractory, to values lower than they were on short days. This implies that both should increase as photorefractoriness is terminated some time after transfer from long to short days. Further, since thyroidectomy causes termination of photorefractoriness in birds held on long days, this should also result in an increase in hypothalamic GnRH and pituitary gonadotrophin contents. This study tests these hypotheses. Male starlings were transferred from short to long days for 10 weeks, by which time they should have become photorefractory. One group of birds was then killed; blood was collected, hypothalami and pituitaries were excised, and the stage of moult and testicular weights recorded. The remaining birds were then kept on long days, transferred to short days or thyroidectomized and kept on long days. Groups of birds in each treatment group were killed and sampled 2, 8 and 14 weeks later. Hypothalamic content of GnRH, and pituitary and plasma FSH and prolactin concentrations, were measured by radioimmunoassay. In birds sampled after 10 weeks of long days, hypothalamic content of GnRH was quite low, pituitary and plasma levels of FSH were very low, testes were small and pituitary and plasma prolactin levels were high. In intact birds kept on long days, hypothalamic GnRH content decreased further and remained low. Pituitary and plasma FSH levels remained low, testes remained small and pituitary and plasma prolactin levels decreased slowly. In intact birds transferred to short days, hypothalamic GnRH content increased to values significantly (PP These results demonstrate that short days activate the hypothalamus to a state of 'readiness to function' and confirm that the thyroid glands are involved in this response. J. Endocr. (1986) 110, 73–79

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured in the plasma of male white-crowned sparrows (Zonotrichia leucophrys gambelii) using heterologous radioimmunoassay systems. Levels were determined in intact birds during a photo-periodically induced (20L:4D) gonadal cycle and compared with those in castrates on the same photoregime. In the intact birds testicular weight increased rapidly between Days 10 and 55 while plasma LH reached a maximum level by Day 6 and remained high until Day 50. Between Days 50 and 75 there was a decline in circulating LH to basal levels, coincident with the onset of photorefractoriness and spontaneous gonadal regression. Control birds on short days (8L:16D) showed no changes in plasma levels of LH. In contrast, levels of FSH increased gradually on 20L:4D to reach a maximum by Day 36 when the testes and almost attained maximum size. Thereafter, they steadily declined to basal levels by Day 75. Control birds on 8L:16D had plasma FSH levels below the detectable limits of the assay. During photostimulation (20L:4D) of castrated sparrows, plasma levels of FSH and LH reached levels up to tenfold those in intact birds. The temporal patterns in both gonadotropins were similar with maxima at Day 16, and declining to basal levels by Day 75. Short-day castrates sampled over the same time period showed a slight increase in plasma LH levels, whereas plasma FSH remained undectable.

Intact and castrated male canaries were maintained under 8-hr day lengths until fully photosensitive and then subjected to either 20 or 11 hr daily photoperiods. Plasma LH and FSH were monitored in the birds. Under 20-hr day lengths the castrates became photorefractory, as judged by a marked decline in plasma LH levels, more quickly than the intact birds. Plasma FSH levels decreased at the same time as LH. Under 11-hr day lengths the castrates became photorefractory while the intacts still had not reached this state by the end of the experiment. In a further experiment, one group of birds was castrated and a second was left intact. They were all immediately transferred from 8- to 16-hr lengths. Again the castrates became photorefractory more rapidly. Castration accelerates the rate of onset of a photorefractory state in the canary by mechanisms as yet unknown.

Plasma levels of LH were analyzed in male ring doves throughout a breeding cycle. LH levels were low in visually isolated animals [(8.0 ± 1.6 ng/ml) (X ± SE)], peaked the day after pairing with a female (14.3 ± 2.52 ng/ml), and declined to baseline levels during incubation and brooding. If eggs or squab were removed during the period of parental care, LH levels rose. The increase in LH following removal of eggs was more prolonged in late incubation than in early incubation. We conclude that the female enhances while the young (eggs or squab) suppress LH in male ring doves.

Photosensitive White-crowned Sparrows ( Zonotrichia leucophrys gambelii ) previously maintained on short day lengths (8-hr light; 16-hr darkness) were transferred to complete darkness. At various intervals thereafter (2-100 hr) they were exposed to a single 8-hr light period. To determine if the light period had been inductive, plasma luteinizing-hormone concentration was measured by radioimmunoassay in blood samples taken before and after the light period. Inductive light periods recurred at approximately daily intervals. The data provide further evidence for the involvement of a circadian rhythm in time measurement in the photoperiodic responses of birds.

A technique has been developed for the investigation of the photopigment involved in the photoperiodic control of reproduction in Japanese quail,Coturnix coturnix. When these photoreceptors were exposed to white or monochromatic light a clear relationship was found between light intensity and the extent of photo-induced luteinizing hormone (LH) secretion. A spectroradiometric investigation of the passage of light through the skull and brain enabled us to illuminate the hypothalamic region with equal numbers of photons at a range of wavelengths. Action spectra were then conducted and showed a photopigment with a peak sensitivity at wavelengths near 500 nm. An excellent match was obtained when the standard absorption spectrum for a rhodopsin was fitted to the action spectrum, suggesting a rhodopsin maximally sensitive at 492 nm. The absolute sensitivity of the photoreceptors was calculated at a range of wavelengths: with light at 500 nm, 2.85×10−12 μE·cm−2·s−1 triggered the photoperiodic response. This level of sensitivity is matched only by the rhodopsin visual pigments.

To explore the relationship between the testes and the hypothalamo-hypophyseal axis in sexually quiescent tree sparrows, we measured the concentration of immunoreactive luteinizing hormone (LH) in serial plasma samples taken from photosensitive castrates, hemicastrates, and laparotomized controls held on a winter-like (8-hr) day length. Postcastration concentrations of LH, measured between Days 3 and 38, always exceeded (P < 0.05) the precastration concentration. In hemicastrates, post-treatment concentrations exceeded the pretreatment concentration only at Day 38. Post-treatment concentrations in hemicastrates and controls were lower than those in castrates through Day 17 but not reliably different from each other through Day 38. We concluded that the small testes of nonphoto-stimulated tree sparrows inhibit gonadotropin (LH) secretion by a feedback effect of testicular hormones on the hypothalamo-hypophyseal axis. LH concentrations in castrates exposed to an 8-hr day length always were lower (P < 0.05) than those in castrates transferred to a day length (20 hr) known to be gonadostimulatory. This finding suggests that, in intact males, an independent stimulatory effect of long days on gonadotropin secretion in superimposed upon any stimulatory effect caused by a photoinduced reduction in sensitivity of the hypothalamo-hypophyseal axis to androgen.

Plasma and pituitary levels of immunoreactive luteinizing hormone (LH) were measured in female, male, and castrated male tree sparrows during a photoinduced gonadal cycle. Our aims were to compare patterns of LH secretion, particularly during spontaneous gonadal regression, and to determine the testes' influence on the secretory pattern of males. Seven days after females were transferred from 8- to 20-hr daily photoperiods, the concentration of plasma LH had increased 11-fold. Two weeks later, it had declined to a level only 3-fold greater than that in short-day controls, where it remained until day 56, when ovarian growth culminated. The onset of ovarian regression between days 56 and 77 was accompanied by a reduction in plasma LH to the prestimulation level (ca. 0.5 ng/ml) and by a similar reduction in pituitary LH content, which, by contrast, had increased steadily through day 28. The plasma cycle of males was quantitatively similar, but it reflected the transition from photosensitivity to photorefractoriness less incisively. Pituitary cycles of males and females were qualitatively similar. The concentration of plasma LH in castrates, compared with that in intact males, was elevated through day 56. A 4-fold increase in pituitary LH preceded a reduction in the circulating level that paralleled the onset of testicular and ovarian regression. Qualitative similarity among plasma cycles plus the eventual reduction of plasma LH in castrates to the low level characteristic of chronically photostimulated birds of both sexes argues that the LH-release mechanism becomes refractory in castrates as in males and females. We, therefore, conclude that testicular steroids are not obligatory determinants of the temporal pattern of circulating LH in photostimulated males, yet they apparently inhibit the photoresponsive hypothalamohypophysial axis. An elevated plasma level in short-day castrates suggests that a functional relationship between the hypothalamohypophysial axis and the testes exists also in nonphotostimulated males.

The development of the reproductive system was studied in juvenile starlings during the acquisition of photosensitivity, the attainment of sexual maturation after photostimulation and the subsequent onset of photorefractoriness, using immunohistochemistry for LHRH and radioimmunoassay measurements of hypothalamic, pituitary and plasma hormone concentrations. The first stage of sexual development induced by exposure of photorefractory immature starlings to short days (8 h light:16 h darkness; 8L:16D) was characterized by a decrease in pituitary prolactin content within 1 week and an increase in hypothalamic LHRH content, in the size of the LHRH perikarya and in the intensity of immunostaining in the median eminence in 4–6 weeks. Sexual maturation occurring after exposure to long days (18L:6D) was associated with further increases in LHRH content and cell size, and increases in LH and prolactin concentrations. During testicular regression, LHRH perikarya were reduced in size and staining intensity but LHRH immunostaining in the median eminence and content in the hypothalamus remained high until gonadal regression was almost complete. Prolactin levels were maximal during testicular regression. These results suggest that gonadal regression is initiated by a reduction in LHRH synthesis and possibly, in addition, an external inhibitory influence on LHRH release. Hypothalamic LHRH content eventually declined and LHRH immunostaining in the median eminence was much reduced in fully photorefractory starlings maintained under long days. Journal of Endocrinology (1989) 122, 255–268

Changes in concentrations of hypothalamic gonadotrophin-releasing hormone (GnRH) and pituitary and plasma FSH and prolactin were measured in intact and thyroidectomized female starlings (Sturnus vulgaris) after transfer from short to long photoperiods. In intact birds, hypothalamic GnRH did not increase significantly during the first 6 weeks of photostimulation, but by 12 weeks, as birds became photorefractory, it had decreased to levels significantly lower than those before photostimulation. In thyroidectomized birds, which did not become photorefractory, hypothalamic GnRH remained high after 12 weeks of photostimulation. Pituitary FSH increased in both intact and thyroidectomized birds; it then decreased to low levels in intact photorefractory birds, but remained high in thyroidectomized birds. Plasma FSH increased to a peak after 2 weeks, but by 6 weeks it had decreased to low levels in both groups. In intact birds there was a 70-fold increase in pituitary prolactin during the first 6 weeks, and levels were still high after 12 weeks of photostimulation. In thyroidectomized birds, pituitary prolactin remained low. The results suggest that while the initial effect of long daylengths is to cause gonadal maturation, the ultimate effect is to switch off the reproductive system. J. Endocr. (1985) 105, 71–77

To explore the possibility that adrenal hormones play a role in the natural termination of testicular function in chronically photo-stimulated tree sparrows, corticosterone, the principal adrenal steroid of birds, was implanted intracerebrally in photoresponsive tree sparrows 3 weeks after they were transferred to a gonadostimulatory photoregime. Three weeks later, plasmas were assayed for immunoreactive luteinizing hormone (LH), testes were weighed, and implantation sites were determined. When implanted in a region of the basal hypothalamus known to be androgen sensitive, corticosterone reduced the concentration of plasma LH and blocked testicular growth. Control implants in the optic chiasma or tracts were not gonadoinhibitory. These results confirm the potential for inhibition of gonadotropin secretion by an effect of corticosterone on the hypothalamohypophyseal axis, but the physiological significance and mechanism of corticosterone's antigonadotropic effect remain to be established.

The photoperiodic control of gonadal development was examined in the Japanese quail (Coturnix coturnix japonica). Long (20 hours) daily photoperiods caused a quantitatively uniform growth of testes and ovaries to full reproductive condition, whereas no development occurred in birds held on 6-hour daily photoperiods. Arginine vasotocin and oxytocin were identified by pharmacological and chromatographic methods in both the pars nervosa and the median eminence. The concentrations of these peptides showed no significant correlations with the rate of gonadal development. Similarly, no significant changes were detected in the hypothalamic neurosecretory system under different photoperiodic regimes, although there appeared to be a trent towards a greater accumulation of stainable material in the reticular layer of the median eminence of birds held on short daily photoperiods. Dehydration resulted in a correlated depletion of neurosecretory material and neurohypophysial hormones from the median eminence and pars nervosa with a greater loss of arginine vasotocin than oxytocin from the pars nervosa.

The gonadotropin content of the anterior pituitary in Japanese quail (Coturnix coturnix japonica) was studied in relation to photoperiodically induced gonadal development. The content was low in birds held on short daily photoperiods and in which there was no gonadal development. Long daily photoperiods caused complete gonadal development and a steady increase in gonadotropin content during the first 21 days with a slight decline thereafter. The anterior pituitary gland increased in weight under a long daily photoperiod. The results are discussed with respect to the balance between hormone synthesis and hormone release.

The essential role of the anterior pituitary gland in the development and the function of the avian gonad has been recognized for more than three decades. However, little is known concerning annual variation of gonadotropic function in species with marked gonadal cycles. The only extensive information available appears to be that of Greeley and Meyer (1953) who investigated the gonadotropic activity of the anterior pituitary in relationship to the annual testicular cycle in Ring-necked Pheasants (Phasianus colchicus) held in captivity under semi-natural conditions. In order to extend our understanding of the regulation of the hypothalamo-hypophysio-gonadal axis in birds under natural conditions, we have investigated the annual cycle of pituitary gonadotropic potency in relation to gonadal cycles in free populations of the White-crowned Sparrow, Zonotrichia leucophrys gambelii Nuttall. Such data are also indispensable in the interpretation of the results of experiments conducted under controlled conditions. The annual testicular cycle of Whitecrowned Sparrows has been investigated by Blanchard and Erickson (1949), by Oakeson (1954), and by Oakeson and Lilley (1960). Our data extend these investigations, and describe also the annual ovarian cycle.

1. Ovarian development in Zonotrichia leucophrys gambelii subjected to long daily photoperiods is a logarithmic function of time until a weight of approximately 50 mg. is attained. Although there is a slight non-photoperiodic ovarian growth especially in first-year birds, it is clear that long daily photoperiods are essential for normal ovarian growth and constitute the basic environmental information used in the control of the ovarian cycle.2. The photoperiodic control mechanism in the female differs from that of the male in that the logarithmic growth phase brings the ovary to only approximately one-tenth of its maximum weight, whereas the logarithmic growth phase of the testes in photostimulated males brings them to about one half of maximum (See TABLE II in Source Pdf) weight. In addition, the female differs from the male in that complete gonadal development can be induced only rarely in captivity with either artificial or natural photoperiodic stimulation, whereas sufficiently long daily photoperiods...

The characteristics of the gonadal photoperiodic responses in the White-crowned Sparrow, Zonotrichia leucophrys gambelii, have been examined by the use of cyclic photoperiodic regimes consisting of a day with a long photoperiod followed by n days with short, non-stimulatory daily photoperiod, n with the range 0-5; responses measured were the rates of gonadal growth and the concentration of pituitary gonadotropin. The gonadotropic effect of a single 20-hour photoperiod extends through at least seven days with short (8-hour) non-stimulatory photoperiods. The apparently simplest interpretation of the results of the experiments suggests that there must be separate, although perhaps not completely independent, control schemes for synthesis and release of pituitary gonadotropin.

Changes in the plasma titers of follicle-stimulating hormone were investigated in a free-living population of barheaded geese over the whole year. Adult males had pronounced seasonal changes from prebreeding levels of approximately equal to 20 ng/ml to a peak in the breeding season of about 300 ng/ml. Changes in females were only slight. An increase from nondetectable, approximately equal to 20 ng/ml to a mean of 32.2 ng/ml before egg-laying was found. The data are discussed with regard to the literature on free-living species and the previously published data on LH and testosterone levels in the same birds.

The concentrations of hormones in the plasma of male red-winged blackbirds caught at the height of an aggressive encounter are significantly different from those in males that have not recently engaged in aggressive behavior. Concentrations of luteinizing hormone in the plasma are decreased in the aggressive males, whereas androgen concentrations are affected in a more complex manner. Concentrations of corticoids do not appear to be affected by aggressive behavior.

The photoperiodic mechanisms underlying seasonal breeding in male Japanese quail change their characteristics annually, with the result that reproduction is terminated in the late summer rather than the autumn. The reproductive cycle was studied in 4 successive years to determine the critical daylengths for reproductive induction in spring, and for its termination in late summer. In spring luteinizing hormone levels first rise when the daylength has reached 11.9 h but in late summer the first signs of a decrease are detectable when the photoperiod has decreased to 14.7 h and levels are basal when it has reached about 14 h. Thus, quail appear to have evolved a refractoriness to daylengths in late summer that were maximally stimulatory earlier in the year. However, quail are not photorefractory in the ‘classic’ sense as they remain continuously in breeding if held on unchanging long days and can be photostimulated at any time of the year by daylengths in excess of 16 h light:8 h darkness. Their particular form of refractoriness seems to involve a seasonal shift in the critical daylength. Refractoriness can be induced in quail held in the laboratory under a simulated annual photocycle, and, more importantly, it develops in birds exposed to long daylengths of a fixed duration. For example, quail exposed to 20 h light:4 h darkness show a shift in the critical daylength from approximately 12 to 15 h over a period of about 2 months. The time course over which the shift occurs is intriguing and can explain the responses under natural photoperiods. Under fixed daylengths the critical daylength that induces regression depends upon the duration of the stimulatory photoperiod. Female quail also develop refractoriness. The evolution of refractoriness does not depend upon the activity of the hypothalamo-pituitary axis and develops in castrated quail as well as in birds implanted with androgens. Castrated quail show annual cycles in gonadotrophin secretion indistinguishable in timing from those in intact birds. Such results suggest that the physiological basis of refractoriness lies either in the neural circuits regulating neurohormone secretion or in the photoperiodic clock mechanism itself.

Experimental photoperiods, presented either once only or repeatedly, were used to assess the oscillatory and hourglass properties of the photoperiodic clock in Japanese quail. Gonadectomized quail on 8-hr daylengths respond to a single skeleton photoperiod consisting of two 8-hr light pulses separated by 2 hr of darkness (i.e., LDLD 8:2:8:6) with a marked increase in secretion rate of luteinizing hormone (LH). This response suggests that the second light pulse interacts with a "photoinducible phase" (Φi) lying some 10-16 hr from "dawn" (start of the first light pulse). If, however, groups of quail maintained on 8-hr daylengths are transferred to continuous darkness (DD), and the position of the Φiis sought by a single 8-hr light pulse applied at various times on the first or third day of DD, then an increase in circulating LH is, at best, barely detectable. It would appear that a strongly responsive Φidoes not recur rhythmically in DD. Instead, the light pulse apparently acts primarily as a "dawn" signal that triggers a single cycle of photoinducibility, since a second 8-hr light pulse, placed to begin 2 hr after the end of the first, induces a large increase in plasma LH. Similar results are obtained if any single 8-hr light pulse presented to animals held in darkness is preceded, 10 hr earlier, by a short "dawn" light signal. Such dawn signals can be effective when very short; a pulse of only 30 sec can cause a subsequent Φi. The dawn pulse is effective at any circadian phase and leads to a single cycle in photoinducibility. In contrast, a much longer light pulse (perhaps not less than 4 hr) is needed to interact with Φiif significant gonadotropin secretion is to be stimulated.In confirmation of the findings described above, we found that Nanda-Hamner lighting schedules have remarkably little effect in stimulating gonadotropin secretion in gonadectomized quail. There is, for example, a very marked difference between the effectiveness of "resonating" schedules such as LD 6:6, which stimulates a high LH secretion rate since each "inductive" light pulse is preceded by an appropriate "dawn" signal, and a theoretically effective schedule such as LD 6:30, which induces a very small response by comparison. Such schedules (even theoretically noninductive ones) can, however, be made very highly inductive if alternate light pulses are preceded by an appropriately positioned 15-min light pulse to act as "dawn."

Immunocytochemistry was used to determine the effects of photoperiod on the LHRH neurosecretory system in the brain of male European starlings. In this species, as in other birds, reproduction is triggered by long daylengths but continued exposure leads to photorefractoriness and to a complete shut-down of the reproductive system. These effects are thought to be mediated through changes in the secretion of LHRH. In starlings exposed to a photoperiod of 11 h light: 13 h darkness (11L: 13D) and with fully developed testes there was strong immunostaining of both LHRH perikarya (n = 522 ± 43 s.e.m.) and fibres. Photosensitive short-day (8L: 16D) starlings with undeveloped testes had an almost identical distribution of strongly immunoreactive perikarya (n = 523 ± 62) but there were fewer fibres. In the median eminence, fibre number was reduced significantly (P < 0·01) by some 30%. In long-day (18L: 6D) photorefractory starlings with fully regressed testes there was an even more obvious change in the LHRH system. Perikarya were only weakly immunoreactive and there was a significant (P < 0·01) reduction in mean diameter from 10 to 6·5 μm. In addition, there was a significant (P < 0·05) reduction in cell number (312 ± 62), although this may well result from the fact that some weakly stained cells fell below the limits of resolution and could not be counted. LHRH fibres disappeared almost entirely from the median eminence, and were not visible elsewhere in the brain. The higher neural pathways regulating photorefractoriness induced by long days are unknown but clearly both production of LHRH in the perikarya and release/storage of LHRH in the terminals is being profoundly modified. J. Endocr. (1987) 115, 211–220

This study tested the hypothesis that estradiol can enhance LH pulse frequency in the ewe by an action which does not depend on other ovarian hormones. Long-term ovariectomized ewes were treated with a small subcutaneous estradiol implant at a time equivalent to the early breeding season (October), and frequent blood samples (6-min intervals) were obtained during sequential 3-hour periods over the next 84 h. All ewes responded with an increase in frequency of LH pulses, a response evident by 60 h and maintained at 84 h after initiation of the estradiol stimulus. Mean (+/- SE) pretreatment frequency was one pulse every 41 +/- 2 min; that at the height of the response was one pulse every 34 +/- 2 min (p less than 0.01). This increased rate was equivalent to the annual mid-winter maximum observed in ovariectomized ewes not treated with estradiol. These findings are consistent with the hypothesis that estradiol can enhance LH pulse frequency by an action which does not depend on other ovarian steroids. It is suggested that this action contributes to the heightened pace of LH pulses during the follicular phase of the estrous cycle.

Plasma luteinizing hormone (LH) levels in serial blood samples of female ring doves (Streptopelia risoria) were measured by radioimmunoassay method. Our findings indicate the following pattern of LH levels: LH increases during early courtship which reaches a peak (5.43 ± 0.79 ng/ml) during the nesting period. LH declines following egg-laying (3.77 ± 0.33 ng/ml) and again after hatching (2.23 ± 0.28 ng/ml). It remains high in females which laid infertile eggs initially and subsequently showed a further laying. The results are compared with published data on plasma estrogens and progesterone in the dove's breeding cycle.

Reproduction in the Japanese quail (Coturnix coturnix japonica), as in many other bird species, is controlled by the daily photoperiod. The present experiments have investigated the role of the posterior or tuberal hypothalamus in the neuroendocrine regulation of this photoperiodic response. By using various types of small knives the tuberal hypothalamus was isolated surgically in male quail maintained on short daylengths. The birds were then transferred to long daily photoperiods and after fourteen days the effects on gonadotrophin secretion assessed by measuring testicular growth and the circulating level of luteinizing hormone (LH). Complete deafferentation of the tuberal hypothalamus blocked testicular growth and the plasma level of LH remained low. Partial cuts that severed the afferents entering from anterior and antero-lateral directions also blocked gonadotrophin secretion but cuts severing posterior connections were without effect. Following deafferentation of sexually mature males the LH level fell rapidly to reach a basal concentration within two days. The normal rise in LH that follows the castration of quail on long days was blocked by complete tuberal deafferentation. Electrolytic lesions were placed medially in the tuberal hypothalamus. Those in the postero-dorsal part of the infundibular nuclear complex (p.d.-i.n.c.) completely blocked testicular growth, while those in the ventral portion of the nucleus (v.-i.n.c.) only partially disrupted gonadotrophin secretion.

Injections of chicken or ovine luteinizing hormone (LH) into sexually mature male Japanese quail greatly increased plasma levels of testosterone. Maximal responses were obtained within 15 min of an iv injection and between 1 and 2 hr following sc or im injections. Saline treatment had no effect on plasma testosterone. In chronically castrated quail LH was not effective in altering androgen levels. The responses to LH were dose related, significant increases being obtained following sc injections of 5 μg of chicken LH (fraction AE1) or 10 μg of ovine LH (NIH-LH-S19). Chicken LH (AE1) was appropriately 1.8 times as potent as NIH-LH-S19. Ovine FSH (NIH-FSH-S10) stimulated testosterone release in very large doses (1 mg) but was at least 100 times less active than LH-S19. An iv injection of an antiserum raised against chicken LH into mature male quail caused a rapid decrease in plasma testosterone levels. Treatment with FSH-S-10 for up to 1 week failed to facilitate the subsequent response to an injection of LH. The responsiveness of the testis to exogenous LH was tested at various times during a photoinduced gonadal growth cycle. Sexually immature quail showed only a marginal response to an sc injection of 20 μg of NIH-LH-S19. A marked increase in responsiveness occurred after 6 long days. This coincides with the time when plasma testosterone levels increase naturally after transfer to long daylengths and with the period when Leydig cell maturation becomes complete. These in vivo results add further weight to the belief that, in birds, or at least in the quail, peripheral androgens are controlled by pituitary LH and that FSH plays no significant role in the acute release of testosterone from the mature testis.