Physiological oxidative stress model: Syrian hamster Harderian gland-sex differences in antioxidant enzymes.

The Syrian hamster Harderian gland, a juxtaorbital organ exhibiting marked gender-associated differences in contents of porphyrins and melatonin, was used as a model system for comparing strong (in females) and moderate (in males) physiological oxidative stress. Histological differences showing much higher cell damage in females were studied in conjunction with lipid peroxidation and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Lipid peroxidation and enzyme activities were measured throughout the circadian cycle, revealing the importance of dynamical processes in oxidative stress. Especially in lipid peroxidation and in catalase, short-lasting rises exhibited strongest gender differences. Peaks of lipid peroxidation were about three times higher in females, compared to males. Catalase peaks of females exceeded those in males by several hundred-fold. Average levels of superoxide dismutase and glutathione peroxidase were about three or two times higher in females, respectively. A clear-cut diurnally peaking rhythm was found in glutathione peroxidase of females, which was not apparent in males. Glutathione reductase showed differences in time patterns, but less in average activities. The time courses of lipid peroxidation and of protective enzymes are not explained by circulating melatonin, whereas melatonin formed in the Harderian gland should contribute to differences in average levels. Neither damage nor antioxidative defense simply reflect the illumination cycle and are, therefore, not only a consequence of photoreactions.

Mast cells in the Harderian gland of female Syrian hamsters during the estrous cycle and pregnancy: effects of the light/dark cycle.

The number of identifiable mast cells and the intraluminal area occupied by porphyrin deposits was studied on semithin sections from female hamster Harderian glands during the estrous cycle and pregnancy. Although the serum levels of estradiol, progesterone, luteinizing hormone and follicle stimulating hormone exhibited significant changes throughout the cycle, no correlation between these changes and the variations in the number of recognizable mast cells was observed. However both during diestrous 1 and proestrous cycles, the number of identifiable mast cells was higher at midnight than at noon (in 14 h light:10 h dark photoperiod with lights on at 07:00 h). A more exhaustive study revealed the presence of 'degranulated mast cells' which were not stained with toluidine blue. Thus, a diurnal cycle in degranulation might occur in the Harderian glands from female hamsters. No significant variations were observed in the area occupied by intraluminal porphyrin deposits during the estrous cycle. However, both the relative number of mast cells and the area occupied by intraluminal porphyrins decreased from day 4 of pregnancy to day 14 showing a strong correlation. The Harderian glands from female Syrian hamsters might provide a useful model for the study of mast cell degranulation during porphyria.

Invasive processes in the normal Harderian gland of Syrian hamster.

In this contribution we will pay special attention to several morphological findings that we can observe, under some circumstances, in the normal Harderian gland of the Syrian hamster. The accumulation of porphyrins in this gland results in mitochondrial damage and extensive cell death. Many damaged cells are secreted into the lumen of the tubule-alveoli, but most of them seem to produce an invasive process that even affects the vascular components of the gland. In this way, many blood vessels are invaded and appear partially filled with the invasive mass, which sometimes totally occludes the lumen of the vessels. We have also observed other surprising features related to a special kind of activity in certain secretory cells. Such activity results in a peculiar "segregation" of a cytoplasmic fragment, containing the nucleus. The affected cells seem to gather up their cytoplasm and nucleus towards the basal zone, while the rest of the cell, including practically the whole amount of lipid droplets, is relegated to the vicinity of the lumen. All these phenomena seem finally to result in the detachment of some clusters, composed of a limited number of cells, which display a basophilic cytoplasm practically free of lipid droplets.

Histopathological features of the Harderian glands in transgenic mice carrying MMTV/N-ras protooncogene.

The Harderian gland is a tubule-alveolar gland found within the orbit, on the posterior aspect of the eyeball. In mice, it is composed almost exclusively of secretory tubules and alveoli. The Harderian glands of transgenic mice, carrying the activated N-ras oncogene under the transcriptional control of the mouse mammary tumor virus long terminal repeat promoter (MMTV-LTR), were examined and compared to those of normal mice. Thirty transgenic mice provided by A. Pellicer (NYU) and 30 normal mice were examined in this study. Harderian glands were dissected, immersed in a formalin-based fixative, and embedded in paraffin. The sections of these glands were studied via histological techniques. Our results show that proliferative alterations in the Harderian glands of these transgenic mice are present even in the youngest animals. Such alterations correspond to different tumoral evolution stages, ranging from hyperplasia to wide tissue destruction. In the most advanced situations, these changes are accompanied by a glandular hypertrophy. Our results suggest a very high tumoral incidence in the Harderian glands of transgenic mice compared to normal mice. Tumors appear spontaneously in some areas, but not at the same time in the whole gland.

Porphyric enzymes in hamster Harderian gland, a model of damage by porphyrins and their precursors. A chronobiological study on the role of sex differences.

The Syrian hamster Harderian gland (HG), representing a highly porphyrogenic organ, was used as a model system for studying physiologically occurring damage of biomolecules by porphyrins and their precursors, phenomena associated with from the pathological situation of porphyrias. The species used exhibits the peculiarity of much higher porphyrogenesis in females than in males, offering possibilities for comparison of effects by different porphyrin levels in one species. Since concentrations of free, and therefore, radical-generating porphyric metabolites are difficult to determine in the presence of high amounts of secreted and crystallizing porphyrins, which are, moreover, mainly surface-reactive, and since indications existed for temporal changes in the oxidative stress caused by these molecules, the following approach was chosen: in HGs of both females and males, activities of the relevant porphyric enzymes, delta-aminolevulinate synthase (ALA-S), delta-aminolevulinate dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), were determined throughout the circadian cycle. Results were compared with the temporal patterns of lipid peroxidation and protein damage in the same glands. In females, a strong correspondence was observed between protein carbonyl and lipid peroxidation, peaking at the end of both photophase and scotophase; maximal activities of the three porphyric enzymes ALA-S, ALA-D, and PBG-D either coincided or slightly preceded the peaks of oxidative damage. In males, lower enzyme activities, especially in PBG-D, were associated with weakly expressed rhythmicity. Correspondingly, lipid peroxidation was lower and exhibited a smaller rhythm amplitude; protein carbonyl of males showed a temporal pattern differing from that of females, with regard to amplitude and phasing. These data are in agreement with morphological observations demonstrating particularly severe cell damage in the female HG under normal conditions.

Ultrastructure of the blood vessels in the Harderian gland of the hamster (Mesocricetus auratus): existence of sinusoids.

The Harderian gland blood supply of female and male hamsters was studied using light and electron microscopy. A profuse vascularization surrounding secretory acini was observed. Among the blood vessels, the existence of large and irregular sinusoidal capillaries was apparent. These sinusoids appeared in close association to the basal aspect of the secretory cells. Typical, small, fenestrated capillaries were also observed within the connective tissue. The existence of this particular vascularization together with other morphological features of the secretory cell basal pole suggest a possible endocrine function of these orbital glands.

Eating quality of beef from biotypes included in the PGI "Ternera Asturiana" showing distinct physicochemical characteristics and tenderization pattern.

Different biotypes of the Protected Geographical Indication (PGI) "Ternera Asturiana" were studied to determine if their differences in physicochemical characteristics and tenderization pattern during maturation (3 to 21 days) had an effect on the consumer evaluation of beef palatability. Biotype affected significantly pH, water holding capacity, chemical composition (P < 0.001) and meat lightness (P < 0.05). Ageing time affected significantly (P < 0.05) colour, meat toughness and sensory attributes in a different way within each biotype. Multivariate analysis showed two different meat groups: 1) meat from mh-genotypes, characterized by high juice losses, lightness (L*), protein content and high sensory acceptability at intermediate (7 and 14 days) ageing times; 2) meat from rustic (AM) breed and biotypes free of myostatin mutation (AV (+/+) and AV x AM), showing higher intramuscular fat, myoglobin content, and instrumental toughness and requiring longer storage times (21 days). This should be taken into account for the proper post-mortem management and commercialization of each product to achieve its best sensory quality.

The pineal gland of the trumpet-tailed rat (Octodon degus).

The structure and ultrastructure of the pineal gland of the degu or trumpet-tailed rat (Octodon degus), a rodent inhabiting tropical-equatorial areas, was examined under light and electron microscopy. On the basis of its form, size, and location, the pineal gland of the degu is classified as a proximal or "A" type. The connective tissue appeared poorly developed and the gland contained non-fenestrated capillaries. A single population of typical pinealocytes was found. In addition, a small number of glial cells and cells with electron dense bodies appeared scattered throughout the gland. Cells with dense granules were found isolated or forming small groups always in close proximity to blood vessels. Numerous sympathetic nerve fibers with small dense-core vesicles were found. Also, some myelinated nerve fibers were observed. The physiological significance of the presence of large electron-dense granules in some pineal cells and their particular location around the blood vessels in discussed.

Lymphoid cells in the harderian gland of the rodent Octodon degus.

The Harderian gland of the degu (Octodon degus) is composed of tubulo-alveolar secretory units that share most of morphological features found in the Harderian glands of other rodents. However, a peculiar characteristic observed in the glands of female degus is the existence of lymphoid cell clusters within the connective tissue surrounding the secretory adenomeres. Lymphocytes and lymphoblasts are found associated with blood vessels and especially with nerve bundles in the medullary region of the gland. Occasionally, macrophages and plasma cells are also observed. Although the Golgi apparatus appears well developed, the ultrastructural characteristics of most of these lymphoid elements correspond to those of inactive lymphocytes. Unmyelinated fibers containing clear and dense-core vesicles are found closely related to lymphocytes. On some occasions, lymphocytes present extensive areas of apposition with structures resembling intercellular junctions. The analogy of the lymphoid clusters reported in this study with those described in the avian Harderian gland is discussed.

Effects of human chorionic gonadotropin and progesterone administration on porphyrin biosynthesis and histology of the Harderian glands in male and female Syrian hamsters.

We investigated the influence of hCG and progesterone on the control of porphyrin biosynthesis and histology in the Syrian hamster Harderian glands. Castration of male hamsters caused a marked elevation in porphyrin biosynthesis as revealed by the concentrations of porphyrins and the mRNA levels of the porphyrin pathway rate-limiting enzyme, 5-aminolevulinate synthase (ALV-S). Injection of hCG into castrated male hamsters also resulted in a significant increase in both porphyrin concentrations and levels of ALV-S mRNA compared with those in saline-injected castrated hamsters. Type II cells, which are filled with large lipid vacuoles and are characteristic of male phenotype, disappeared after castration, but administration of hCG partially prevented this change. On the other hand, neither administration of hCG nor progesterone implants could increase the very high porphyrin concentrations and ALV-S mRNA levels characteristic of female Syrian hamsters. As in the case of castrated male hamsters, injections of 20 IU hCG to female Syrian hamsters increased the relative number of Type II cells per square millimeter, whereas progesterone administration did not modify the relative number of Type II cells. These results indicate that hCG can modify Harderian gland morphology in both male and female hamsters and can exert a positive control in the expression of ALV-S gene in castrated male hamsters.

Ultrastructural study of the cellular types in the pineal organ of Gambusia affinis (teleost).

The pineal organ of Gambusia affinis was studied via light and electron microscopy. The cell types studied included photoreceptor cells, supporting cells, and a third cell type. The photoreceptor cells, which appear to form clusters, are divided into four regions: outer segment, inner segment, cell soma, and synaptic pedicle. Synaptic ribbons are commonly observed in the synaptic pedicle. The supporting cells separate the photoreceptor cells from the thick basal lamina that surrounds the entire pineal organ. The supporting cells show highly organized membrane formations, some lipid-like inclusions, and a diplosome. One of the centrioles gives rise to an invaginated cilium. The third cell type is observed infrequently and appears to be located mainly in the vicinity of the outer segments. The morphological characteristics of this cell type are similar to those of phagocytic cells. The ultrastructural features of the pineal organ of G. affinis are compared with those of other teleosts.

Membrane formations in the pineal cells of the teleost Gambusia affinis.

Highly organized membrane formations in the cytoplasm of photoreceptor and supporting cells of the pineal organ of Gambusia affinis are described. These membranous structures show different characteristics depending on the cell type where they are located. Some of the morphological characteristics of these membranous formations are similar to those of myeloid bodies, but their presence in the photoreceptor cells raises some doubt about their being myeloid bodies. The three-dimensional interpretation of these structures is discussed.

Female Syrian hamster Harderian gland: development and effects of high environmental temperature and melatonin injections on histology and porphyrin deposits.

We have investigated the development of the Harderian glands of female Syrian hamsters from birth to 8 months of age. The effects of melatonin injections on Harderian gland histology and porphyrin deposits of female hamsters exposed to two different temperatures were also studied. The morphology of the Harderian glands from 30-day-old female hamsters resembled those of sexually mature adult animals. The intraluminal area occupied by porphyrins increased significantly between 20 to 30 days of age. However, the relative number of mast cells per mm2 rose between 30 to 90 days. Both porphyrins and mast cell numbers appeared clearly reduced in the 8-month-old group. Secretory cells characterized by large lipid droplets (type II cells) were not observed. Daily afternoon injections of 25 micrograms of melatonin to female hamsters exposed to 22 degrees C for 14 weeks resulted in the discontinuity of estrous cyclicity, a marked decrease in the intraluminal area occupied by porphyrins, a reduction of the number of mast cells per mm2, and in a marked augmentation of the number of type II cells per mm2. Although the administration of similar dosages of melatonin to hamsters exposed to an environmental temperature of 32 degrees C did not interrupt estrous cycles, a clear reduction of the area occupied by porphyrins was observed. However, the number of mast cells and type II cells per mm2 was unaffected in these animals. Our results suggest that hormones other than ovarian steroids are involved in the regulation of the female hamster Harderian glands. The possible role of melatonin on Harderian gland metabolism is discussed.

Immunocytochemical localization of melatonin in the harderian gland of Syrian hamster.

BACKGROUND: The Harderian gland (HG) is a tubulo-alveolar gland found within the ocular orbit of animals which present a nictitating membrane. The Harderian gland is regarded as an extrapineal melatonin producing organ and both, photoperiod and melatonin have been shown to exert an important role in the metabolism and morphological features of such a gland. Our results seem to support the presence of melatonin in the nuclei of the HG cells, although our studies have not definitively proved such presence. METHODS: An Immunocytochemical anti-melatonin technique was done over free sections of Bouin fixed material obtained from Syrian hamsters. Some of the sections were embedded in an epoxy resin and studied under electron microscope. RESULTS: The presence of positive immunoreaction was observed at the level of the nuclear membranes and in close relation to chromatin. No differences were observed between males and females nor between pinealectomized animals and control ones. CONCLUSIONS: Our results suggest the binding of melatonin to the cell nucleus in all cell types of the gland. These observations are in accord with the binding studies performed by Acuña-Castroviejo in purified cell nuclei of rat liver (Acuña-Castroviejo et al., 1994. J. Pineal Res., 16:100-112) and the earlier one by Menéndez-Peláez et al. (1993a,b, J. Pineal Res., 15:59-69; J. Cell Biochem., 53:373-3*2) using the light microscope. Our results seem to support the idea of a nuclear action of melatonin and they agree with the observations of Carlberg and Wiesenberg (1995, J. Pineal Res., 18:171-178) about the activation of some orphan receptors by melatonin.

Chronic administration of melatonin induces changes in porphyrins and in the histology of male and female hamster harderian gland: interrelation with the gonadal status.

In this paper, we have investigated the influence of melatonin on the histology and porphyrin content of the Syrian hamster Harderian glands. Daily afternoon injections of 25 micrograms of melatonin to female hamsters for 12 weeks resulted in the discontinuity of estrous cyclicity, a marked decrease in the Harderian gland intraluminal area occupied by porphyrins, and in a significant rise in the number of Type II cells. A similar decrease in porphyrins was observed after 8 weeks of ovariectomy. However, if the melatonin injections were given for only 8 weeks (without inducing gonadal atrophy), no changes were observed in the area occupied by intraluminal porphyrins, suggesting that the effects of melatonin in female Syrian hamsters might be associated with the subsequent gonadal atrophy. Castration of male hamsters induced a significant increase in porphyrins and a clear drop in the number of Type II cells. These changes were totally prevented when melatonin was administered daily from the day of castration. Our results suggest that melatonin, at least in male Syrian hamsters, plays a role in Harderian metabolism, acting directly on the Harderian secretory cells or indirectly through pituitary hormones.

Porphyrin accumulation in the harderian glands of female Syrian hamster results in mitochondrial damage and cell death.

BACKGROUND: The Harderian glands of female Syrian hamsters contain very high concentrations of protoporphyrin (in the range of micrograms per mg of tissue) which accumulate in the tubulo-alveoli of the gland. We have studied the process of synthesis, accumulation, and secretion of this cyclic compound by the secretory cells of the hamster Harderian glands. METHODS: The animals used were female Syrian hamster of 15, 35, 75, 180, and 360 days of age. Items first examined were (1) percentage of the "clear cells," (2) area occupied by intraluminal porphyrins, and (3) histological characteristics of "clear cells" by light and transmission electron microscopy (TEM). In a second study the total content of porphyrins was determined. Finally, the levels of mRNA for the enzyme aminolevulinate synthase (ALV-S) were measured. RESULTS: In the glands of female hamsters, both the tissue concentration and the intraluminal area occupied by protoporphyrin correlate with the appearance of a special type of cell (clear cells) which show signs of cell degeneration. In addition, the expression of the gene for ALV-S, which is the limiting enzyme in porphyrin production, also parallels the relative number of clear cells. Analyzed under TEM, these clear cells display dilated mitochondria and short and swollen endoplasmic reticulum cisternae. In a late phase of necrosis, the nuclear envelope appears disorganized with scarce chromatin. The mitochondria undergo complete destruction, resulting in electron-dense bacillary formations which progressively coalesce in large and dense areas of protoporphyrin. The cell dies after this accumulation, being secreted by a "cytogen" mechanism. CONCLUSIONS: In view of our results, the Harderian gland of female Syrian hamster may provide a useful model for the study of the mechanism by which the anomalous accumulation of protoporphyrin induces cell damage in human protoporphyria.

Development and androgen regulation of the secretory cell types of the Syrian hamster (Mesocricetus auratus) Harderian gland.

The secretory cell types of the hamster Harderian glands were studied in both male and female Syrian hamsters. As previously demonstrated, female hamsters showed a single secretory cell type (type I), while male hamsters displayed two secretory cell types (type I and type II). Type-II cells were observed after the first month of age correlating with the increase in testosterone levels. The administration of testosterone to adult female hamsters resulted in a marked increase in the percentage of type-II cells without a significant increase in the number of mitotic figures. Very low levels of serum testosterone were able to maintain the percentage of type-II cells. Castration of male hamsters produced a decrease in the percentage of type-II cells. This drop correlated with the reduction in serum testosterone levels. The chronic administration of a luteinizing hormone-releasing hormone agonist to male Syrian hamsters induced a significant reduction in both serum luteinizing hormone and testosterone. However, the percentage of type-II cells was similar to that of control hamsters suggesting that very low levels of circulating testosterone are able to maintain the percentage of type-II cells. In a final experiment male Syrian hamsters were treated with the antiandrogen cyproterone acetate. No changes were observed in the percentage of type-II cells, whereas serum luteinizing hormone and testosterone levels were significantly modified. We concluded that (1) type-II cells differentiate from type-I cells; (2) gonadal androgens are the major factor controlling this differentiation; and (3) the disappearance of type-II cells after androgen deprivation occurs through holocrine and apocrine mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the circadian mutation 'tau' on the Harderian glands of Syrian hamsters.

The Syrian hamster Harderian gland (HG) is an organ continually exposed to oxidative stress caused by high concentrations of porphyric metabolites. According to previous studies, melatonin, which is rhythmically secreted by the pineal gland and tonically produced by the HG, antagonizes the oxidative damage. HGs exhibit a strong gender-dependent correlation between porphyrins, melatonin, and histological appearance. In HGs of both sexes, we have investigated effects of a single gene defect in the circadian clock system (tau mutation) causing a shortened free-running period and an advanced maximum of circulating melatonin. Comparisons were made with wild-type animals, one group of which received daily pharmacological injections of melatonin in late photophase. Changes were observed in histological characteristics, porphyrin content, antioxidant enzyme activities, and damage of proteins and lipids. HGs of tau hamsters showed morphological changes which can be partially interpreted in terms of increased damage. Additionally, tau females exhibited a many-fold augmentation in the percentage of so-called type II cells, which are otherwise typical for the male glands. In tau hamsters of both sexes, major antioxidative enzyme activities (superoxide dismutase, glutathione reductase, and catalase) were markedly enhanced, a presumably compensatory response to increased oxidative stress. Higher oxidative damage in tau HGs was directly demonstrable by a many-fold increase in protein carbonyl. Rises in antioxidative enzymes were also observed upon injections of melatonin; this was, however, not accompanied by changes in protein carbonyl, so that enzyme inductions by the hormone should be understood as protective actions. Our data are not only in accordance with findings on protective effects by melatonin, but also with our earlier observation made in Drosophila that perturbations in the circadian system lead to increased oxidative stress.

Castration increases cell damage induced by porphyrins in the Harderian gland of male Syrian hamster. Necrosis and not apoptosis mediates the subsequent cell death.

It is known that the Harderian gland of male Syrian hamster synthesizes a much smaller amount of porphyrins than the gland of the female and that castration greatly increases this synthesis. We have studied in this experimental model the behavior of the different classes of secretory cells and their role in the synthesis of porphyrins, attempting to clarify the participation of these compounds in the cell damage leading to the formation of clear cells previously described in the gland of females. We have also investigated the mechanism underlying the death of these secretory cells after porphyrin accumulation (necrosis vs apoptosis). To achieve this, we have utilized the following techniques: (a) morphometrical; (b) ultrastructural; (c) biochemical (fluorescence spectrophotometry); and (d) molecular (DNA nick-end labeling in methacrylate sections and dot blot analysis). The glands from male hamsters (serving as control) present a very low rate of damaged cells that progressively rises after castration. This rise runs parallel to that of porphyrin synthesis, porphyrin deposits, and the decrease of Type II secretory cells. The damage and subsequent death of the secretory cells in the gland is produced by the deposit of porphyrins in the mitochondrial membrane. This porphyrin accumulation leads to a complete mitochondrial destruction that finally results in cell death and its secretion into the lumen. We finally conclude that this event is not a physiological cell death (apoptosis) but the consequence of the toxic accumulation of porphyrins (necrosis).

Photoperiod and the pineal gland regulate the male phenotype of the Harderian glands of male Syrian hamsters after androgen withdrawal.

The Harderian glands of Syrian hamsters exhibit a marked sexual dimorphism in cell types and porphyrin production. The glands of male hamsters have two secretory cell types (Type I and II) while the glands of females consist of a single secretory cell type (female Type I) and large intraluminal deposits of porphyrins. Besides androgens, there is evidence that the pineal gland, through the secretion of melatonin, contributes to the maintenance of the "male" and "female" phenotypes. In this study, we investigated the effects of castration, short photoperiods, and pinealectomy on the distribution of secretory cells and porphyrin deposits in the Harderian glands of male Syrian hamsters. Two groups of animals were maintained in long days (14 hr light/day). Hamsters in one group were left intact and those in the other were castrated. Another three groups were maintained in short days (8 hr light/day); these animals were either left intact, castrated, or both castrated and pinealectomized. The duration of the experiment was 5 weeks. Castration of long photoperiod-exposed animals resulted in a significant drop in the number of Type II cells and a large increase in the porphyrin deposits (P < 0.01). However, castrated animals exposed to short photoperiod showed a significant smaller change in both parameters compared with those exposed to long days (P < 0.05). Pinealectomy prevented the effects of short days in castrated animals. No significant changes were observed in the relative number of mitotic figures or in the number of cell nuclei, indicating that the changes observed were due in part to a transformation of Type II into Type I cells. In a second experiment, male hamsters were injected daily either with 25 micrograms of melatonin late in the afternoon or with the saline for 8 weeks. The administration of melatonin resulted in a significant (P < 0.05) increase in the percentage of Type II cells. We conclude that when circulating androgens are very low or absent, pineal melatonin maintains the male phenotype in the Syrian hamster Harderian gland.