The Pineal Gland,but Not Melatonin,Is Associated with the Termination of Seasonal Testicular Activity in an Annual Reproductive Cycle in Roseringed Parakeet Psittacula krameri

The role of the pineal gland and its hormone melatonin in the regulation of annual testicular events was investigated for the first time in a psittacine bird, the roseringed parakeet (Psittacula krameri). Accordingly, the testicular responsiveness of the birds was evaluated following surgical pinealectomy with or without the exogenous administration of melatonin and the experimental manipulations of the endogenous levels of melatonin through exposing the birds to continuous illumination. An identical schedule was followed during the four reproductive phases, each characterizing a distinct testicular status in the annual cycle, namely, the phases of gametogenic quiescence (preparatory phase), seasonal recovery of gametogenesis (progressive phase), seasonal initiation of sperm formation (pre‐breeding phase), and peak gametogenic activity (breeding phase). In each reproductive phase, the birds were subjected to various experimental conditions, and the effects were studied comparing the testicular conditions in the respective control birds. The study included germ cell profiles of the seminiferous tubules, the activities of steroidogenic enzymes 17β‐hydroxysteroid dehydrogenase (17β‐HSD), and Δ⁵3β‐hydroxysteroid dehydrogenase (Δ⁵3β‐ HSD) in the testis, and the serum levels of testosterone and melatonin. An analysis of the data reveals that the pineal gland and its hormone melatonin may play an inhibitory role in the development of the testis until the attainment of the seasonal peak in the annual reproductive cycle. However, in all probability, the termination of the seasonal activity of the testis or the initiation of testicular regression in the annual reproductive cycle appears to be the function of the pineal gland, but not of melatonin.     

Cytologic variation in the pineal organ of roseringed parakeets (Psittacula krameri) with respect to artificial photoperiods and annual cyclicity in testicular functions

Abstract

Adult male roseringed parakeets were transferred to 16L: 8D (LP), or 8L: 16D (SP) for 45 or 90 days on four particular dates corresponding to the different phases of an annual testicular cycle and the cytological responses of the pineal were studied comparing them with the features in respective natural photoperiodic (NP) birds. Different cytological characteristics including the values of nuclear diameter in pinealocytes indicated that LP for 45 days during the pre‐breeding phase and for 90 days during each phase resulted in an increased pineal activity, while SP for 45 and 90 days induced inhibitory responses of the pineal during each, but not the progressive phase of the annual testicular cycle. During the latter phase, none of the artificial photoperiodic schedules, other than LP for 90 days, influenced the cytological features of the pineal. The results suggest that the photosensitivity of the pineal in these parakeets varies in relation to the testicular functions in an annual testicular cycle, but the seasonal pattern of photoperiodic response of the pineal and that of the previously studied testes in the same birds does not seem to be identical.     

Tentative immunohistochemical demonstration of melatonin in the rat pineal gland

Summary In the present study an attempt was made to demonstrate melatonin in the rat pineal gland by means of immunohistochemistry. The anti-body used was raised against 5-methoxy-N-acetyltryptophan which is chemically similar to melatonin. Specific fluorescence was demonstrable only in pineals from rats killed during the night, when melatonin formation is high. It was restricted to parenchymal cells lying in a marginal zone of the organ. These results are discussed in relation to a subdivision of the pineal parenchyma into cortical and medullary areas.Supported by a grant of the Deutsche Forschungsgemeinschaft (VO 135/4) within the Schwerpunktprogramm Neuroendokrinologie     

Immunohistochemical evidence for the presence of melatonin in the pineal gland,the retina and the Harderian gland

Summary The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20–25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10–16, 1980 (Vivien-Roels et al. 1980)The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates     

Vasotocin, melatonin and narcolepsy: Possible involvement of the pineal gland in its patho-physiological mechanism

Both the pineal nonapeptide hormone arginine vasotocin (AVT) (2.5 μg) administered intra-nasally and the pineal indole melatonin (50 mg) administered intravenously to three male narcoleptics (two with auxiliary symptoms and one with sleep attacks only), dramatically increased the amount of REM sleep and decreased REM sleep latency. The duration of the sleep onset REM periods in the two narcoleptics with auxiliary symptoms increased by more than 100 percent after AVT and melatonin administration. In the narcoleptic with sleep attacks only both AVT and melatonin induced REM periods at sleep onset. The hypothesis is advanced that narcolepsy represents an impairment of the melatonin-AVT control in the induction and circadian organization of REM sleep associated with an immaturity of REM triggering centers.     

Vasotocin, melatonin and narcolepsy: Possible involvement of the pineal gland in its patho-physiological mechanism

Both the pineal nonapeptide hormone arginine vasotocin (AVT) (2.5 μg) administered intra-nasally and the pineal indole melatonin (50 mg) administered intravenously to three male narcoleptics (two with auxiliary symptoms and one with sleep attacks only), dramatically increased the amount of REM sleep and decreased REM sleep latency. The duration of the sleep onset REM periods in the two narcoleptics with auxiliary symptoms increased by more than 100 percent after AVT and melatonin administration. In the narcoleptic with sleep attacks only both AVT and melatonin induced REM periods at sleep onset. The hypothesis is advanced that narcolepsy represents an impairment of the melatonin-AVT control in the induction and circadian organization of REM sleep associated with an immaturity of REM triggering centers.     

哺乳动物昼夜节律组构中的下丘脑视交叉上核和松果腺

哺乳动物下丘脑视交叉上核（SCN）是昼夜节律最主要的起搏器,控制着机体的生理和行为的节律。它具有自身内在的节律性,同时也受光照周期信号和一些内源性化学物质的调节。检查腺分泌裉黑素（MEL）受SCN的调控,MEL通过作用于SCN上高亲和性MEL受体,启动第二、第三信使系统,调整SCN的昼夜节律活动。这种调整具有时间敏感性。     

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.Abbreviations CT circadian time - LD light-dark - LL constant light - SCN suprachiasmatic nucleus - LLdim constant dim light - NE norepinephrine - SCG superior cervical ganglia - WB whole body - E eye - EX extraretina - C control     

Determination of endogenous melatonin in the individual pineal glands of inbred mice using precolumn oxidation reversed-phase micro-high-performance liquid chromatography

The amount of endogenous melatonin in the individual pineal glands of inbred mice has been determined using reversed-phase micro-high-performance liquid chromatography after precolumn oxidation of melatonin to a compound having strong fluorescence. The fluorescent compound was identified as N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide. The excitation and emission wavelengths of this compound are 245 and 380 nm, respectively, and the fluorescence intensity is 6.8 times greater than that of melatonin. Molar absorptivity and fluorescence quantum yield of this compound are 46,300[L mol(-1)cm(-1)] and 0.31 (245 nm), respectively. The lower quantification limit of melatonin in biological samples using this precolumn oxidation method is 200 amol, and the calibration curve of spiked melatonin is linear from 200 amol to 50 fmol (r>0.999). The sensitivity of the present method is almost 10 times higher than that of the previous method. The values of endogenous melatonin obtained for ICR, C57BL, BALB/c, and AKR mice are 4.7, 6.1, 7.4, and 18.8 fmol/pineal gland, respectively. The amounts of endogenous pineal melatonin of these strains had not been clearly reported due to the poor enzymatic activities for melatonin biosynthesis; this is the first report that clearly demonstrates the existence of endogenous melatonin in these inbred mice.     

Seasonal changes in adrenal and gonadal activity in the quail, Perdicula asiatica: involvement of the pineal gland

The present study assessed annual adrenal gland activity in the Indian tropical Jungle bush quail, Perdicula asiatica. We also elucidated the role of the annual variations in gonadal steroids and melatonin in the regulation of its activity. Increasing day length (photoperiod), ambient temperature and rainfall are positively correlated with adrenal and gonadal functions, and inversely related to pineal gland activity. Pineal, adrenal and gonadal weights showed cyclical patterns relative to environmental factors, which were also correlated with plasma melatonin, corticosterone and gonadal steroids, respectively. In both sexes of P. asiatica, pineal gland weight and/or plasma melatonin levels were inversely related to adrenal lipids, (e.g. phospholipids, free and esterified cholesterol) and plasma corticosterone levels. Melatonin levels also showed an inverse relationship with plasma testosterone and estradiol levels. These studies indicate that changes in environmental factors promote annual variations in adrenal and gonadal activity probably by modulating the pineal gland. Melatonin receptors have been localized in the pars tuberalis, adrenal gland and gonads of birds, the pineal gland may, therefore, mediate environmental stimuli indirectly and directly to down regulate adrenal and gonadal activity, which run in parallel in this species.     

The pineal gland of the gerbil,Meriones unguiculatus

Summary By means of morphometric analytical procedures, a diurnal rhythm in the cellular volume of gerbil pinealocytes was determined. This rhythm has been attributed primarily to a change in the cytoplasmic volume of the pinealocytes which is low during the daylight hours and increases to reach a peak during the middle of the dark period. At the ultrastructural level, six cytoplasmic components of the pinealocytes were found to exhibit a rhythm: free cytoplasm, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER) and ribosomes, secretory vesicles, microtubules, and mitochondria. The presumptive secretory vesicles and the microtubules reached a peak in volume one hour before lights-off. It is suggested that lights-on and lights-off both signal a decrease in size and/or number of the secretory vesicles. The SER and RER/ribosomes reached their peak volume one hour after lights-off which is interpreted as indicating a peak in indoleamine synthesis and protein synthesis, respectively. The volume of free cytoplasm exhibits two peaks; one occurs one hour before lights-off while the second peak occurs in the middle of the dark phase. It is suggested that, although part of the secretory product of the pinealocyte may be present in dense-cored vesicles, other locations could include the free cytoplasm and clear secretory vesicles.Supported by NSF grant #PCM 77-05734     

Regulation of melatonin production by light,darkness, and temperature in the trout pineal

Summary The pineal gland of the rainbow trout, Salmo gairdneri, when kept under in vitro perifusion culture conditions, displays a consistently elevated level of melatonin production in darkness (Gern and Greenhouse 1988). Upon light exposure melatonin production falls and stabilizes at a new lower level that is dependent upon the irradiance of the stimulus. To achieve the maximal response for each irradiance, the duration of the stimulus must exceed 30 min. The response amplitude is maximally sensitive to photons presented over durations of 30–45 min; is very insensitive to shorter light exposures; and is maintained with no evidence of adaptation over longer exposures. Temperature plays a role in regulation of melatonin production both in darkness and during light exposure; increased temperature increases melatonin production in darkness and also increases the sensitivity of the response to light. The action spectrum for the response is best fit by the Dartnall nomogram for a vitamin A₁ based rhodopsin with peak sensitivity near 500 nm. The possible adaptive significance of control of melatonin synthesis by light and temperature is considered.Abbreviations LD lightdark cycle - RIA radioimmunoassay - I ¹²⁵ Iodine - HIOMT hydroxyindole-O-methyltransferase     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.     

Morphological changes in the clasper gland of the Atlantic stingray, Dasyatis sabina, associated with the seasonal reproductive cycle

The clasper gland of the Atlantic stingray, Dasyatis sabina, was examined over a 1-year period, covering an entire reproductive cycle. Changes in clasper gland tissue architecture, fluid production, and cell proliferation were assessed. No changes in tissue architecture were observed. Evidence of cell proliferation in the gland epithelium was not detected using immunocytochemistry for proliferating cell nuclear antigen, a cellular marker of mitosis. Epithelial cells were not observed to undergo mitosis, and cell membranes remained intact. The lack of structural changes and epithelial cell proliferation supports the proposed merocrinal mode of fluid secretion. Rays captured in nonbreeding months had clasper glands that exhibited tubules with reduced lumens. In contrast, rays caught during the breeding season had clasper gland tubules with enlarged lumens. Clasper gland fluid production was quantified through measurements of the fluid area and tubule area calculated from digital images. Clasper gland fluid production was significantly higher during the mating period than during months not associated with copulatory activity. These data support the notion that the clasper gland is involved in stingray copulatory activity. This study adds to the limited amount of literature focused on this poorly understood component of reproduction in skates and rays.     

Cold prevents the light induced inactivation of pineal N-acetyltransferase in the Djungarian hamster,Phodopus sungorus

Summary In the Djungarian hamster seasonal acclimatization is primarily controlled by photoperiod, but exposure to low ambient temperature amplifies the intensity and duration of short day-induced winter adaptations. The aim of this study was to test, whether the pineal gland is involved in integrating both environmental cues. Exposure of hamsters to cold (0 °C) reduces the sensitivity of the pineal gland to light at night and prevents inactivation of N-acetyltransferase (NAT). The parallel time course of NAT activity and plasma norepinephrine content suggests that circulating catecholamines may stimulate melatonin synthesis under cold load.Abbreviations NAT N-acetyltransferase - NE norepinephrine - T _a ambient temperature     

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 µg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 µg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.     

Intraventricular blood vessels associated with the deep pineal gland of the Mongolian gerbil,Meriones unguiculatus

Summary Intraventricular blood vessels and choroidal-like cells were studied using scanning electron microscopy and correlative light microscopy. The intraventricular blood vessels were covered on their ependymal surface with a layer of cells essentially identical to the ependyma of the choroid plexus in the gerbil. Similar choroidal-like cells were seen either singly or in clusters associated with the cerebrospinal fluid-contacting pinealocytes of the suprapineal recess. Processes of the cerebrospinal fluid-contacting pinealocytes were seen extending to and making contact with the choroidal-like cells. The intraventricular blood vessels appeared to be derived from the choroid plexus, and typically took one of three courses in and around the surface of the deep pineal: (1) the vessels or their equivalent were located in the suprapineal recess with no indication of penetration into the substance of the deep pineal; (2) the vessels coursed from the suprapineal recess around the anterior surface of the habenular commissure to enter the ventral surface of the deep pineal; or (3) the vessels entered the parenchyma of the deep pineal from its dorsal surface and could be seen coursing through the substance of the gland. The close association between the choroidal-like cells and the intraventricular blood vessels with the deep pineal gland add morphological support for the possibility of interaction between the cerebrospinal fluid, or perhaps the choroid plexus, and the deep pineal gland.     

The pineal complex and melatonin affect the expression of the daily rhythm of behavioral thermoregulation in the green iguana

Daily variation in the body temperature of the green iguana (Iguana iguana) was studied by telemetry in laboratory photo-thermal enclosures under a 12Light12Dark (LD) photoperiod. The lizards showed robust daily rhythms of thermoregulation maintaining their body temperatures (T_b) at higher levels during the day than during the night. Some animals maintained rhythmicity when kept in constant darkness. On lightdark cycles parietalectomy produced only a transient increase of median T_b in the first or second night following the operation. Pinealectomized lizards on the other hand maintained their body temperatures at significantly lower levels during the day and at significantly higher levels during the night than did sham-operated or intact lizards. This effect was apparently permanent, since one month after pinealectomy lizards still displayed the altered pattern. Plasma melatonin levels in intact animals were high during the night and low during the day and were unaffected by parietalectomy. Pinealectomized lizards showed low levels of plasma melatonin during both the day and the night. A daily intraperitoneal injection of melatonin in pinealectomized animals given a few minutes after the light to dark transition decreased the body temperatures selected by the lizards during the night and increased the body temperatures selected during the following day. Control injections of saline solution had no effect. The significance of these results is discussed in relation to the role of the pineal complex and melatonin in the mediation of thermoregulatory behavior.Abbreviations LD LightDark - T _b body temperature - PAR-X parietalectomy - PIN-X pinealectomy     

Environmental correlate of the testicular events in a major carp Catla catla in an annual reproductive cycle

Current communication describes annual testicular events in free-living Indian major carp Catla catla and their probable environmental synchronizer(s). The study was initiated with month-wise evaluation of gametogenic and steroidogenic status of the testis, and thus dividing the annual testicular cycle into the preparatory spawning (November to March), the pre-spawning (April to June), the spawning (July to August) and the post-spawning (September to October) phases. An exhaustive statistical analysis of the data on the studied variables of testicular functions and various components of the environment indicated seasonal fluctuations of photoperiod as the major environmental factor associated with the seasonal reproductive activity of this carp. Ambient temperature appeared as a dependent variable of photoperiod, and thereby, may have substantial influences on the development of testis in Catla catla. Rainfall, on the other hand, showed significant correlation only with the peak reproductive activity, i.e. the act of spawning. Collectively, it appears logical to surmise that photo-thermal conditions may act as proximate and rainfall may play a role of ultimate environmental factor in the regulation of annual testicular events in Indian major carp Catla catla.