Pineal melatonin secretion,but not ocular melatonin secretion,is sufficient to maintain normal immune responses in Japanese quail (Coturnix coturnix japonica)

Reports that plasma melatonin is an important immune regulator in avian species have been rather sparse and contradictory. Also, the primary source of immune-modulating melatonin has yet to be determined in birds. In Japanese quail (Coturnix coturnix japonica), the pineal gland and eyes contribute roughly two thirds and one third of the melatonin found in the blood, respectively. Two experiments were conducted to evaluate melatonin as an immune modulator in Japanese quail and to determine the primary source of immune-modulating melatonin in this species. Experiment 1 was designed to evaluate the involvement of the pineal gland and the eyes in immunocompetence. Each of three groups of quail was assigned a surgical treatment and the cellular and humoral immune responses were determined 8 weeks following surgery. The surgical treatments were pinealectomy (Px), sham pinealectomy (SH-Px), and ocular enucleation (eye removal (Ex)). Experiment 2 utilized exogenous melatonin as a replacement to reconstitute immune responses in surgically immunocompromised birds. In this experiment, 50.0 microg/ml of melatonin, or diluent only, was provided to Px and SH-Px birds in the drinking water ad libitum. The cellular and humoral immune responses were determined after 8 weeks of melatonin treatment. In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days postintravenous injection with a Chukar red blood cell suspension. Flow cytometric analysis of peripheral blood lymphocytes was performed to determine the relative percentage of CD4(+) and CD8(+) T- and B-lymphocytes in all treatments of Experiment 2. In Experiment 1, both the SH-Px and Ex surgical treatments produced similar cellular and humoral immune responses, and these responses were significantly greater than those in Px-treated birds. Pinealectomy significantly reduced the cellular and humoral immune responses from SH-Px by 25.8% and 41.3%, respectively. In Experiment 2, Px again resulted in depressed cellular and humoral immune responses. In addition, Px significantly reduced CD8(+) T-lymphocyte numbers compared to SH-Px, while B-lymphocytes remained unchanged. Melatonin administration to Px birds increased the cellular (32.9%) and humoral (30.6%) immune responses to the level of control (SH-Px) birds, although this reconstitution was not due to increased CD8(+) T- or B-lymphocytes. From these data, it was clear that removal of the pineal gland, but not the eyes, reduced cellular and humoral immune responses, which were reconstituted to normal levels by exogenous melatonin. These data suggest that immunodepression is only observed in birds with two thirds of the plasma melatonin removed by pinealectomy. Removal of one third of the plasma melatonin (by ocular enucleation) is not sufficient to reduce cellular and humoral responses in the Japanese quail.     

Rapid Adjustment of the Pineal N-Acetyltransferase Rhythm to Change From Long to Short Photoperiod in the Japanese Quail (Coturnix coturnix japonica)

The dynamics of adjusting the pineal N-acetyltransferase rhythm from long to short photoperiod was assessed in the Japanese quail (Coturnix coturnix japonica). The transition from LD 16:8 to LD 8:16 was accomplished by symmetrical prolongation of the dark period. In LD 16:8, the period of elevated nocturnal activity lasted approximately 7 hours. During the first prolonged night, the evening N-acetyltransferase rise advanced by almost 3 hours relative to the rise in LD 16:8 and occurred at the same time as during the 3rd, 7th, and 14th day after the transition. The morning N-acetyltransferase decline did not shift during the first long night; during the third night it was delayed relative to the decline in LD 16:8 by more than 2 hours and occurred at the same time as during the 7th and 14th night following the LD 16:8 to LD 8:16 transition. Three, 7, and 14 days after the transition, the period of elevated N-acetyltransferase activity lasted approximately 12 hours. Hence extension of the N-acetyltransferase rhythm profile proceeded first into the evening and then only into the morning hours, and it was accomplished within 2 to 3 days.     

Steroid secretion by ovarian cells of the Japanese quail (Coturnix coturnix japonica)

Mixed cell preparations (theca plus granulosa) prepared from the hierarchy of follicles of quails ovaries were incubated under defined conditions with or without the addition of ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), theophylline, cycloheximide, or dibutyryl cyclic adenine monophosphate (db cAMP); or in the presence of androstenedione or testosterone as aromatizable substrate. Steroids secreted into the medium during the 4-hr incubation period were assayed by radioimmunoassay. Cells from the largest follicles (F1) secreted predominantly progesterone, were stimulated by LH and db cAMP, and the response was potentiated by theophylline, but FSH had no stimulatory effect. The F1 cells showed increasing basal and LH-stimulated responses between 18 and 12 hr before the next expected oviposition. Cells from the smaller follicles (F3 and F4) secreted predominantly estrogens, and were stimulated by FSH but not by db cAMP and only to a small extent by theophylline. Addition of androstenedione (10(-7) M) or testosterone (10(-7) M) enhanced estrogen secretion, which was further raised by the simultaneous addition of FSH. These results confirm previous reports on the sites of steroid secretion within quail follicles and suggest that while the action of LH on the cells from F1 follicles may be mediated in part through the adenylate cyclase system, the action of FSH on the smaller follicles may be substantially independent of cAMP.     

Circadian rhythm of melatonin in the pineal gland of the Japanese quail (Coturnix coturnix japonica)

Melatonin was measured by radioimmunoassay in homogenates of pineal glands from quail (Coturnix coturnix japonica) kept under different photoperiods and in darkness. Under 8-, 12- and 16-h daylengths melatonin levels were increased during the dark period, the duration of the increase depending on the duration of the dark period. As the daylength was increased the peak occurred closer to lights-off, reflecting the more rapid melatonin rise under the longer photoperiods. The pineal melatonin rhythm continued in darkness with an amplitude relative to that seen under a light/dark cycle of slightly less than one-half after 2 days in darkness and one-third after 6 days in darkness. The corresponding average periods of the rhythm were 25.5 h and 25.7 h. These results show that there is a circadian rhythm of melatonin in the pineal gland of the quail which is entrained by light/dark cycles and which continues in darkness.     

Steroidogenesis in ovarian cells of the Japanese quail (Coturnix coturnix japonica)

The influence of follicular maturation on steroidogenesis and steroid metabolism by isolated Japanese quail granulosa and theca cells was examined. When stimulated with LH, granulosa cells of the largest follicle (F1) responded with a sixfold increase over unstimulated progesterone levels, whereas progesterone production in cells of F3 less than doubled even when maximally stimulated. Forskolin stimulated progesterone synthesis in both F1 and F3 granulosa cells, but its effect was less pronounced than that of LH. Furthermore, F1 cells metabolized 25-hydroxycholesterol to a greater extent than did F3 cells. There was no appreciable metabolism of [3H]progesterone by granulosa cells. Theca cells from the smaller follicles (F3-F5) responded to LH stimulation with greater estrogen and androstenedione production than theca cells from F1. [3H]Progesterone was metabolized mainly to androstenedione in theca cells. Thus, the overall pattern of in vitro steroidogenesis in quail granulosa cells is similar to that described for the chicken and turkey even though the quantitative differences in the steroidogenic capacity between developing and mature follicles are more striking in the quail. Furthermore, although the LH-stimulated androstenedione and estrogen production appears similar in developing quail and chicken theca cells, the profile of [3H]progesterone metabolism is different in quail theca cells from that found previously in chicken theca cells.     

Temperature modulation of photoperiodically induced LH secretion and its termination in Japanese quail (Coturnix coturnix japonica)

Long days induced LH release in photosensitive Japanese quail and ambient temperature did not affect this process. Temperature also did not affect the levels of circulating LH concentrations at the steady state of LH release on long days. On the other hand, low ambient temperature was required to reduce circulating LH to a nonbreeding level together with the change of the photoperiod from long to short days; the changes of the photoperiod without low ambient temperature induced a decrease of circulating LH only to a certain level (1-2 ng/ml) which could maintain reproductive activity. The results also indicated that there were three levels of circulating LH in quail which were tentatively designated as a nonbreeding level (less than 0.5 ng/ml), a basal breeding level (1-2 ng/ml), and a steady breeding level (ca. 4 ng/ml). By photostimulation, levels of circulating LH increased to the steady breeding level directly and gradually, or leveled off after overshooting it. Photoperiodic changes from long to short days under moderate temperature resulted in a decrease in circulating LH to the basal breeding level which, however, could keep the gonad and the accessory sex organs active. Photoperiodic changes under cold ambient temperature reduce circulating LH to the nonbreeding level at which the gonad and the accessory sex organs regressed completely. These results suggest that ambient temperature is involved in the mechanism controlling an annual reproductive cycle, especially at the termination of the reproductive activity, in Japanese quail.     

Identification of cGnRH-II in the median eminence of Japanese quail (Coturnix coturnix japonica)

In a previous paper, we described the presence of cGnRH-II in the quail (Coturnix coturnix japonica) and chicken (Gallus gallus) median eminence using highly specific antibodies directed against a polypeptide corresponding to the C-terminal portion of cGnRH-II (van Gils et al., 1993). This finding remained very controversial, since no other study, with any other antibody, had ever reported the presence of cGnRH-II immunoreactive fibers in the median eminence of birds. In this study, the cGnRH-II immunoreactive substances in quail median eminence were isolated by RP-HPLC and identified by RIA. To eliminate the possibility that the cGnRH-II-like immunoreactivity in the median eminence was due to a cross-reaction of our anti-cGnRH-II antiserum with an unknown peptide, the cGnRH-II immunoreactive substances, present in a quail median eminence extract, were isolated by immunoaffinity chromatography using immunoaffinity-purified antibodies. In the eluate of the immunoaffinity column only one peptide could be detected by mass spectrometry. This peptide had a mass of 1235.56 Da, which is the same as synthetic cGnRH-II. In addition, MS/MS fragmentation generated an amino acid sequence corresponding to the sequence of cGnRH-II. The present study therefore identified indisputably cGnRH-II in the median eminence of the quail.     

Retinal melatonin is not involved in corneal mitotic rhythms in the Japanese quail: Effects of formoguanamine hydrochloride and eye-lid suture

Abstract: Relation between retinal melatonin and corneal mitotic rhythms in the Japanese quail was investigated in experiments manipulating the ocular physiology by treatments with formoguanamine hydrochloride (FG) and eye-lid suture. In experiment 1, we investigated the effects of FG, which is known to induce photoreceptor degeneration, on retinal melatonin and corneal mitotic rhythms. FG-treatment completely abolished the retinal melatonin rhythms in both LD 12: 12 and constant darkness (DD), but the corneal mitotic rhythm was maintained with high mitotic rate in darkness under a LD cycle and subjective night under DD. The result suggests that 1) the photoreceptor cells in the retina are the site for melatonin production and/or for the oscillator which drives the circadian rhythm in retinal melatonin, and 2) melatonin is not involved in generation of the corneal mitotic rhythm. In experiment 2, we investigated the effects of eye-lid suture, which is known to induce eye enlargement and bulgy cornea, on the retinal melatonin and corneal mitotic rhythms. Eye-lid suture abolished the corneal mitotic rhythm in both LD and DD, with a high mitotic rate being maintained throughout 24 hr. But retinal melatonin maintained its rhythm with high levels in darkness under a LD cycle and in subjective night under DD. The result suggests that 1) bulgy cornea in the sutured eye was induced by the increase in mitotic rate in the light period, and 2) disappearance of the corneal mitotic rhythm does not have a relation to retinal melatonin. These results suggest that retinal melatonin is not involved in generation of the corneal mitotic rhythm and that there are two circadian clock systems in the eye.     

Melatonin can produce immunoenhancement in Japanese quail (Coturnix coturnix japonica) without prior immunosuppression

In recent years, it has been determined that melatonin has important immunostimulatory properties in mammalian and avian species. Typically, this immunoenhancement has only been examined in immunosuppressed animals. The effect of melatonin on normal (unsuppressed) immune systems is yet to be evaluated in avian species. An experiment was performed to determine if transient and/or continuous melatonin treatments could enhance immune functions in Japanese quail without prior immunosuppression. All quail were kept on a short photoperiod (8:16LD) throughout the study. In this experiment, 50.0 microg/ml melatonin was provided ad libitum to adult Japanese quail in the drinking water either continuously or for 3h per day. Control birds received diluent continuously throughout the experiment. Both the cellular and humoral immune responses were determined immediately after 3 weeks treatment. A cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were calculated 7 days post-intravenous injection with a Chukar red blood cell (CRBC) suspension. The cellular and humoral immune responses were significantly elevated in the transient (3h) and continuous (24h) melatonin treatment groups as compared to the control group (0 h). As compared to the control group, the cellular immune response was increased 25% and 38% for the 3 and 24h melatonin treatments, respectively. The humoral immune response was increased 26% and 32% for the 3 and 24h melatonin treatments, respectively. Furthermore, continuous (24h) melatonin availability significantly increased the cellular, but not humoral immune responses as compared to the transient (3h) group, given melatonin for 3h prior to the scotophase (13:00-16:00 h). From these data, it was clear that transient and continuous administration of melatonin increased the cellular and humoral immune responses of Japanese quail without prior immunosuppression. These data suggest that the immunoenhancing effect of melatonin is not limited to reconstitution of weakened immune systems, but can be observed in normal, immunologically unsuppressed birds.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.     

Effect of changing photoperiod on plasma thyroxine-binding prealbumin in Japanese quail (Coturnix coturnix japonica)

The concentration of thyroxine-binding prealbumin (TBPA) in blood plasma of Japanese quail is shown to be inversely related to changing photoperiod over the annual cycle and particularly from September to April. It is suggested that this protein has a key role among the various plasma factors undergoing change in conditioning animals for seasonal breeding. The changes in TBPA are opposite to, but not synchronous with, those of retinol-binding protein (RBP) and there is a wide variation in the $\text{TBPA}\text{RBP}$ molar ratio (0.6–1.6) during the year, which may indirectly influence the turnover rate of vitamin A.     

Effect of prolactin on thyroid function in Japanese quail (Coturnix coturnix japonica).

Ovine prolactin administration in doses of 10 and 50 IU given over periods of 10 or 20 days induced hypertrophy of the epithelial cells of the thyroid gland in Japanese quail. A decrease of serum thyroxine was observed in the birds that received prolactin (10 or 50 IU) for 10 days, but not in the birds injected with the same doses for 20 days. Prolactin seems to act at the thyroid level to inhibit thyroxine secretion, resulting in hypertrophy of the gland presumably through negative feedback. Prolactin also induced fat deposition.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): II. Effect of vitamin A deficiency

Levels of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A-free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A-deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.     

Seasonality of psychopathology and circannual melatonin rhythm

The association of seasonal changes in health and disease has been known for centuries. The prevalence of psychopathological symptoms with seasonal fluctuations and the use of melatonin as a biological marker of circadian and circannual rhythms is well documented. The aim of this work was to study the variability of melatonin secretion between summer and winter in our geographical area (28 degrees N, 16 degrees W) and relate the changes to the level of psychopathology. Ten drug-free, nonsmoker, healthy subjects were studied in summer (August) and winter (December). Blood samples for melatonin assays were collected every hour at night for 5 hr, from 22:00 to 02:00 hr, and next day at noon. Melatonin was assayed by an ELISA technique. Psychopathology was evaluated by means of the 28-item version of the General Health Questionnaire (GHQ-28). All subjects had a circadian rhythm of melatonin secretion in summer and winter. There was a seasonal rhythm with melatonin levels being significantly higher at night in winter than in summer. Melatonin levels at 22:00, 23:00, 24:00 and 01:00 hr and mean melatonin area under the curve (AUC) were significantly higher in winter than in summer. Melatonin AUC increased 80% in winter compared with summer. The GHQ-28 somatic and anxiety subscales and the total GHQ-28 score were significantly higher in winter than summer. Psychopathology scores were significantly and negatively correlated with melatonin production in summer and winter. Our data strongly suggest that melatonin production and psychopathology levels present seasonal fluctuations and these variations should be taken into account when conducting research in this field.     

Phytosterol additive boosts adrenal response to ACTH in male Japanese quail (Coturnix coturnix japonica)

To investigate the adrenal effect of a phytosterol (PS) additive, 80 male Japanese quail were divided into four sub-groups and fed 0, 40, 400, and 4,000?ppm of PS, respectively, for 21?days. Subsequently, 50% of the birds from each dosage group were subjected to a 6-day adrenal function test, whereby they were injected with long-lasting adrenocorticotropin (ACTH). The remaining quail in each PS dosage group were raised under normal conditions. The groups receiving 400 and 4000?ppm PS exhibited decreased serum levels of LDL-cholesterol with and without ACTH stimulation (P??0.05). Enhancement of CORT was observed on the 2nd and the 6th?days of the ACTH challenge in birds receiving 400?ppm (P?相似文献