Inhibitory action of melatonin on a pineal antigonadotropin.

An acidic extract of bovine pineal glands, partially purified by gel chromatography, yielded two distinct fractions which inhibited compensatory ovarian hypertrophy in mice. When the fractions were tested for antigonadotropic properties in the presence of melatonin only one fraction was found to be effective. Melatonin by itself and no stimulatory effect on COH, suggesting that melatonin blocked the antigonadotropic activity of the one fraction.     

Chemical differences between bovine pineal antigonadotropin and arginine vasotocin

Studies were conducted in order to characterize chemically a partially purified antigonadotropic factor extracted from bovine pineal glands (PAG). Because several reports have appeared recently suggesting a role for arginine vasotocin (AVT) as a pineal antigonadotropin, our experiments were designed to determine the presence or absence of this nonapeptide in our material. A comparison of biologically active PAG and synthetic AVT revealed dissimilar UV absorption and fluorescence maxima, different mobilities on thin layer chromatography and paper electrophoresis, as well as different elution patterns on DEAE Sephadex ion-exchange chromatography. Amino acid analysis using 2 different methods revealed dissimilar amino acid compositions. On the basis of these and other chemical data, it is concluded that our preparations of PAG do not contain AVT.     

Presence of a pineal nerve in sheep and rabbit fetuses

The presence of a nerve located just caudal to the pineal gland in the midsagittal plane is demonstrated in sheep and rabbit fetuses. This nerve lies freely in the subarachnoid space and extends from the pineal gland to a region of the CNS located dorsal to the rostralmost part of the subcommissural organ (SCO). In rabbit fetuses the nerve is obsered on days 23 and 24 of gestation; we suggest that it is an ontogenetic equivalent to the pineal nerve of anuran amphibians. The developmental fate of the mammalian fetal pineal nerve is dicussed.     

Localization of kallikrein in rat pineal glands

The presence of kallikrein mRNA has been reported in the pineal gland of rats. Using an antibody to rat tissue kallikrein, we immunohistochemically examined the localization of cell components producing tissue kallikrein in this gland. The kallikrein immunoreactive cells were scattered in the parenchyma of the pineal gland. Their cell bodies were polymorphic with cell processes and a large nucleus similar to that of the pinealocyte. Frequently immunoreactive materials were seen to be localized in the perivascular areas.     

Radioimmunoassay of 5-methoxy tryptophol in sheep plasma and pineal glands

No 5-methoxytryptophol (ML) could be detected in sheep plasma using a specific, sensitive radioimmunoassay developed for the purpose. Blood samples were collected from sheep during darkness and daylight and during various stages of the estrous cycle, but in no sample was the ML content above the detection limit of the method. Addition of 1 mM pargyline and neostigmine to blood immediately after collection to block metabolism did not result in a detectable ML concentration. The failure to detect ML was not due to degradation since added ML was not degraded by blood enzymes even after 16 hours incubation at 37 degrees C. Injection of 100 microgram and 1 mg ML sc in sheep resulted in a rapid rise of ML to 95-130pg/ml and 560-1000pg/ml respectively and disappearing with a half life of approximately 15-20 minutes. Sheep pineal glands collected during the light phase contained ML (51 +/- 5pg/mg tissue. X +/- SE, n = 7) which represents less than 6% of the melatonin content. It is concluded that if ML is present in sheep blood it is present at very low levels. It is thus unlikely to be a major circulating pineal hormone in this species, however, its role within the CNS as a local hormone cannot be excluded.     

Localization of NADPH-diaphorase activity in the pineal gland of the domestic pig

The study showed the presence of NADPH-diaphorase containing structures in the pineal gland of the domestic pig. NADPH-diaphorase activity was found in the nerve fibers and in the endothelial cells of the vasculature. The nerve fibers were localized in the capsule, around the blood vessels as well as in the parenchyma. The positive staining was not observed in the pig pinealocytes.     

Localization and genomic organization of sheep antimicrobial peptide genes

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two β-defensins and eight cathelicidins. We mapped the two-exon β-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep–hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.     

'Synaptic' bodies in the pineal glands of the cow, sheep and pig

The present results show that 'synaptic' bodies (SB) are a heterogeneous group of organelles in the pineal glands of Artiodactyla. Basically, rod-like (ribbons) and sphere-like (spherules) SB can be distinguished. In the pig small numbers of both 'synaptic' ribbons (SR) and 'synaptic' spherules (SS) are found. In bovine and ovine pineal glands few SS but no SR were seen. The same results were obtained from animals of the same species but from different continents. This suggests that the numbers of SB are species specific rather than influenced by the different lighting conditions in Europe and in equatorial Africa.     

Localization and hormonal regulation of ovarian production of histamine in the sheep

Concentrations of histamine were measured within the follicular wall, follicular fluid and ovarian interstitium throughout the periovulatory period in sheep. Histamine within follicular tissue declined after the onset of the preovulatory surge of luteinizing hormone (LH) and remained low until after ovulation, when levels then increased markedly. Alterations in histamine within the follicular wall were not reflected by corresponding changes within follicular fluid or ovarian interstitium. Release of histamine from tissue during short-term incubation was greatest for follicles obtained after ovulation, which was not influenced by presence of LH in the incubation medium. Luteinizing hormone caused depletion of stores of histamine from the wall of follicles collected before the preovulatory surge of LH. Histamine could act as a paracrine mediator in the follicular mechanisms of ovulation and(or) luteinization.     

Ultrastructural and immunocytochemical characterization of interstitial cells in pre- and postnatal developing sheep pineal gland.

Pineal gland interstitial cells from 32 sheep embryos (from day 54 of gestation until birth) and 18 sheep (from 1 month to >2 years) were analysed using ultrastructural and immunohistochemical techniques. From day 98 of gestation and throughout postnatal development, a second cell type was observed in addition to pinealocytes; these cells displayed uniform ultrastructural features similar to those of CNS astrocytes. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis. Expression of phosphotungstic acid hematoxylin, glial fibrillary acidic protein and vimentin indicates that the second cell population in the developing ovine pineal gland is, in fact, a combination of glial-astrocyte cells at varying stages of maturity. Pineal interstitial cells started to show signs of functional activity evident in vascular tropism; such activity, evident from around day 98 of gestation, appeared to relate to the exchange of substances between the pineal parenchyma and blood vessels and, though it continued throughout postnatal development, was most evident in animals slaughtered between 9 months and 2 years of age (group II). Morphologically, functional activity in interstitial cells in this age-group was apparent in: 1, formation of specific contact sites between interstitial cells and nerve fibres in the perivascular space; and 2, the presence of numerous gap junctions between the bulbous endings of cytoplasmic processes.     

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries)

Summary Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called interstitial cells or supporting cells. In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.     

Characterization of vasopressin and oxytocin immunoreactivity in the sheep and rat pineal gland: absence of vasotocin and detection of a vasopressin-like peptide

The presence of arginine-vasopressin (VP), arginine-vasotocin (VT) and oxytocin (OT) were studied in sheep and rat pineal gland by the combination of HPLC and radioimmunoassays. Three immunoreactive substances, which had the same retention time as OT, VP and VT, were detected in sheep and rat pineal extracts after HPLC separation. Parallelism of dilution curve and standard curve demonstrated that two of them were identical to OT and VP respectively. The third component was distinct from VT on basis of its immunological reaction with two different antibodies; it resembled the immunoreactivity of VP. This substance was also detected in the hypothalamus, brain cortex and pituitary gland, but not in the hippocampus and adrenal gland. The results support the notion that VT is absent in the adult mammalian pineal gland and point to the existence of another, possibly related peptide.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.