Immunocytochemical evidence for the presence of an albumin-like substance in the rat pineal gland

Summary Two rabbits and two guinea pigs were immunized with arginine vasotocin (AVT) conjugated to bovine albumin with glutaraldehyde. Only one preparation of antiserum (anti-G 7) was of value. Anti-G 7 immunochemically defined the same rat pineal cells previously reported as presumptive AVT cells. However, absorption of anti-G 7 with bovine albumin inhibited the staining of the pineal cells demonstrating that they contained an albumin-like substance. Positive immunochemical staining of the rat pars nervosa suggested that anti-G 7 contained antibodies able to react with arginine vasopressin (AVP). Loss of a positive reaction in the posterior lobe on absorption of anti-G 7 with AVT or AVP confirmed this. However, the addition of AVT to anti-G 7 failed to inhibit the immunochemical staining of the pineal cells. This study reports the presence of an albumin-like substance in pineal cells previously described as presumptive AVT cells, and discusses possible explanations for the inability of anti-G 7 to recognize immunocytochemically the native AVT molecule. Confirmation of AVT in the pineal gland by immunocytochemistry must await the availability of more specific antisera.     

Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland

The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA-positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11- and 46-fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.     

Distribution of hydroxyindole-O-methyltransferase mRNA in the rat brain: an in situ hybridisation study

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes. Received: 26 June 1997 / Accepted: 15 September 1997     

Presence of neurophysins I and II in the human pineal gland: Comparison with the content of neurohypophyseal hormones

We have previously measured the individual content of immunoreactive vasopressin (AVP), oxytocin (OT) and vasotocin (AVT) in 155 human pineal glands, and report here identification and measurement of the neurophysin (Np) content of the same glands, using specific homologous human neurophysin I (HNp I) and neurophysin II (HNp II) radioimmunoassays. Median values for HNp I were for men 47 ng/gland (range, 5 to 1360) and for women 24 ng/gland (range, 5 to 1000); median values for HNp II were respectively 7 ng/gland (range, 2 to 191) and 15 ng/gland (range, 2 to 356) with no significant difference between men and women for HNp I and HNp II but a significant difference (p<0.001) between HNp I and HNp II for both sexes. Gel filtration showed that pineal neurophysins were eluted at the same volume as both standard Np and Np from human posthypophyses used as controls. HNp I correlated both with AVP (r_s=0.54 for men and 0.55 for women) and OT (r_s=0.86 for men and 0.57 for women) but not with AVT, while HNp II correlated with AVP (r_s=0.52 for men and 0.53 for women) and OT (r_s=0.92 for men and 0.50 for women) but not with AVT. This study thus confirms the presence of two neurophysins in the human pineal gland and further indicates that they are related to AVP and OT concentrations in the same gland. The results also imply, however, that the presence of immunoreactive AVT (more probably a closely related peptide) is independent of the neurophysins.     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.     

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (Mr approximately 10,000-15,000) and low (Mr approximately 500-1000) Mr species from Sephadex G-25 chromatography of 0.2 N acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration Mr 500-1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.     

Arginine vasotocin neuronal phenotypes, telencephalic fiber varicosities, and social behavior in butterflyfishes (Chaetodontidae): potential similarities to birds and mammals

The neuropeptide arginine vasopressin (AVP) influences many social behaviors through its action in the forebrain of mammals. However, the function of the homologous arginine vasotocin (AVT) in the forebrain of fishes, specifically the telencephalon remains unresolved. We tested whether the density of AVT-immunoreactive (-ir) fiber varicosities, somata size or number of AVT-ir neuronal phenotypes within the forebrain were predictive of social behavior in reproductive males of seven species of butterflyfishes (family Chaetodontidae) in four phylogenetic clades. Similar to other fishes, the aggressive (often territorial) species in most cases had larger AVT-ir cells within the gigantocellular preoptic cell group. Linear discriminant function analyses demonstrated that the density of AVT-ir varicosities within homologous telencephalic nuclei to those important for social behavior in mammals and birds were predictive of aggressive behavior, social affiliations, and mating system. Of note, the density of AVT-ir varicosities within the ventral nucleus of the ventral telencephalon, thought to be homologous to the septum of other vertebrates, was the strongest predictor of aggressive behavior, social affiliation, and mating system. These results are consistent with the postulate that AVT within the telencephalon of fishes plays an important role in social behavior and may function in a similar manner to that of AVT / AVP in birds and mammals despite having cell populations solely within the preoptic area.     

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine-hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP), PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation. Furthermore, the staining of pinealocytes for PGP 9.5 may be exploited to study the three-dimensional morphology and the architecture of pinealocytes and their processes under various experimental conditions.     

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP)

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.     

Molecular cloning, mRNA expression, and immunocytochemical localization of a putative blue-light photoreceptor CRY4 in the chicken pineal gland

In non-mammalian vertebrates, the pineal gland contains an endogenous circadian oscillator and serves as a photosensitive neuroendocrinal organ. To better understand the pineal phototransduction mechanism, we focused on the chicken putative blue-light photoreceptive molecule, Cryptochrome4 (cCRY4). Here we report the molecular cloning of pineal cCry4 cDNA, the in vivo expression of cCry4 mRNA, and the detection of cCRY4 protein. cCry4 is transcribed in a wide variety of chick tissues out of which the pineal gland and retina contain high levels of cCry4 mRNA. In the pineal gland, under 12 h light : 12 h dark cycles, the levels of both cCry4 mRNA and cCRY4 protein showed diurnal changes, and in cultured chick pineal cells, the cCry4 mRNA level was not only up-regulated by light but also controlled by circadian signals. Immunoblot analysis with a cCRY4-specific antibody detected cCRY4 in a soluble fraction of the pineal lysate. Immunocytochemistry revealed that cCRY4 was expressed in many parenchymal cells and a limited number of stromal cells. These cCRY4 features strikingly contrast with those of the chick pineal photoreceptor pinopsin, suggesting a possible temporal and/or spatial duplicity of the pineal photoreceptive system, the opsin- and CRY-based mechanisms.     

Seasonal Variations in the Expression of the mRNA Encoding ß1-Adrenoceptor and AA-NAT Enzyme,and in the AA-NAT Activity in the Pineal Gland of Vizcacha (Lagostomus maximus maximus) – Correlation With Serum Melatonin

The vizcacha is a photoperiodic rodent living in the southern hemisphere. The adult males exhibit an annual reproductive cycle characterized by a gonadal regression period during winter with, in some animals, an almost complete loss of spermatogenesis. In this study, we investigated whether biochemical parameters involved in melatonin synthesis in the vizcacha pineal gland exhibited an annual rhythm in parallel with the annual reproductive cycle. By use of in situ hybridization, an annual variation of mRNA encoding ß 1 -adrenoceptor was shown, with a maximum during autumn and winter. In situ hybridization for mRNA encoding AA-NAT enzyme also exhibited an annual rhythm with the lowest and highest levels in May and August, respectively. Likewise, in August the activity of arylalkylamine N-acetyltransferase enzyme also reached a maximum. Finally, dertermination of the serum concentrations of melatonin by use of radioimmunoassay showed an increase during winter. Moreover, our results are in concordance with several biochemical and morphological parameters of the reproductive axis of the male vizcacha, which support the reproductive rhythmicity of this rodent. Thus, our data suggest that the pineal gland and melatonin, which is activated via the sympathetic system, could be involved in the photoperiodically dependent annual reproductive behavior of the vizcacha.     

Ultrastructural observations on the central innervation of the guinea-pig pineal gland

Summary In the present study the central innervation of the guinea-pig pineal gland was investigated. The habenulae and the pineal stalk contain myelinated and non-myelinated nerve fibres with few dense-cored and electron-lucent vesicles. Some myelinated fibres leave the main nerve fibre bundles, lose their myelin-sheaths and terminate in the pineal gland. Although direct proof is lacking, the non-myelinated fibres appear to end near the site where the bulk of the myelinated fibres are located. Here a neuropil area exists where synapses between non-myelinated fibre elements are abundant. Neurosecretory fibres were also seen. The results support the concept of functional interrelationships between hypothalamus, epithalamus and the pineal gland.     

Daily torpor alters multiple gene expression in the suprachiasmatic nucleus and pineal gland of the Djungarian hamster (Phodopus sungorus)

Circadian rhythms are still expressed in animals that display daily torpor, implying a temperature compensation of the pacemaker. Nevertheless, it remains unclear how the clock works in hypothermic states and whether torpor itself, as a temperature pulse, affects the circadian system. To reveal changes in the clockwork during torpor, we compared clock gene and neuropeptide expression by in situ hybridization in the suprachiasmatic nucleus (SCN) and pineal gland of normothermic and torpid Djungarian hamsters (Phodopus sungorus). Animals from light-dark (LD) 8ratio16 were sacrificed at 8 time points throughout 24 h. To investigate the effect of a previous torpor episode on the clock, we sacrificed a group of normothermic hamsters 1 day after torpor. In normothermic animals, Per1 peaked at zeitgeber time (ZT)4; whereas, Bmal1 reached maximal expression between ZT16 and ZT19. AVP mRNA in the SCN showed highest levels at ZT7. On the day of torpor, the levels of all mRNAs investigated, except for AVP mRNA, were increased during the torpor bout. Moreover, the Bmal1 rhythm was advanced. On the day after the hypothermia, Bmal1 and AVP rhythms showed severely depressed amplitude. Those distinct amplitude changes of Bmal1 and AVP on the day after a torpor episode expression suggests that torpor affects the circadian system, probably by altered translational processes that might lead to a modified protein feedback on gene expression. In the pineal gland, an important clock output, Aanat expression, peaked between ZT16 and ZT22 in normothermic animals. Aanat levels were significantly advanced on the day of hypothermia, an effect which was still visible 1 day afterward. In summary, this study showed that daily torpor affects the phase and amplitude of rhythmic clock gene and clock-controlled gene expression in the SCN. Furthermore, the rhythmic gene expression in a peripheral oscillator, the pineal gland, is also affected.     

Immunohistochemical localization of glial fibrillary acidic protein (GFAP) in rat pineal stalk astrocytes.

In the present work, the presence and distribution of astrocytes in the rat pineal stalk is investigated applying an immunohistochemical technique for the demonstration of glial fibrillary acidic protein (GFAP) on Epon-embedded semithin sections (0.5 micron thick). GFAP-immunoreactive cells are evenly and regularly distributed along the entire pineal stalk. The GFAP-immunoreactive cells display a stellate shape showing variable numbers of cell processes that are mainly oriented parallel to the longitudinal stalk axis. Astrocytic processes show a clear tendency to encircle the remaining elements of the pineal stalk; i.e., pinealocytes, nerve fibres and blood vessels. Furthermore, glial processes form a cover layer separating the stalk from surrounding anatomical structures.     

Vasopressin- and oxytocin-containing fibres in the pineal gland and subcommissural organ of the rat

Summary Vasopressin and oxytocin were specifically demonstrated in the rat brain using the unlabelled antibody-enzyme method and purification of the first antiserum. Vasopressin and oxytocin fibres extend via the subcommissural organ or habenular commissure into the pineal stalk and terminate in the anterior part of the pineal organ. In addition, immediately adjacent to the subsommissural organ many vasopressin-containing fibres run caudally toward the central grey. These results are discussed in relation to the proposed presence of vasotocin in the pineal gland.This study was supported by the Foundation for Medical Research, FUNGOThe authors wish to thank Dr. D.F. Swaab and Prof. J. Ariëns Kappers for their suggestions and critical remarks     

Human pineal luteinizing hormone receptors

The presence of luteinizing hormone receptors in human pineal glands from five females and three males, ranging in age from 61-89 yr, was examined by in situ hybridization and immunocytochemistry. The results demonstrated the presence of these receptors at the mRNA and protein levels in all the pineal glands examined. Pineal gland luteinizing hormone receptors could potentially be involved in the regulation of melatonin synthesis.