Syrian Hamster Harderian Gland Research Articles

Autophagic and proteolytic processes in the Harderian gland are modulated during the estrous cycle

The Syrian hamster Harderian gland (HG) is an organ that undergoes physiological autophagy in response to oxidative stress induced by porphyrin production. Porphyrin production in the HG has marked sex differences and is closely linked to reproductive function. In the present study, we observed that the estrous cycle and associated estrogen variations may affect oxidative-stress-induced proteolytic processes. In particular, significant changes in autophagic activity were detected during the estrous cycle. Notably, increased activation of macroautophagy as well as chaperone-mediated autophagy in the estrus phase coincided with a minimal antioxidant capability and the highest protein damage levels. By contrast, autophagic machinery was found to be blocked in the diestrus phase, likely due to mammalian target of rapamycin activation, which could be corroborated by the subsequent pS6K activation. Analogous results were observed regarding proteasome activity, which also showed maximal activity in the estrus phase. Interestingly, all these mechanisms were associated with important morphological changes in the HG during the estrous cycle. We observed statistically significant increases in Type II cells, which may be related to extensive autophagy in the estrus phase. Physiologically, this would result in a significant release of porphyrins specifically when females are more receptive. These data support the role of porphyrins as pheromones, as other authors have previously suggested, thus making the HG a scent organ. In addition, these results suggest a porphyrin-based approach to the treatment of porphyria during pregnancy, a condition for which no treatment is currently known.

Analysis of constant tissue remodeling in Syrian hamster Harderian gland: intra-tubular and inter-tubular syncytial masses.

The Syrian hamster Harderian gland (HG) has a marked sexual dimorphism and exhibits an extraordinary rate of porphyrinogenesis. The physiological oxidative stress, derived from constant porphyrin production, is so high that the HG needs additional survival autophagic mechanisms to fight against this chronic exposure, provoking the triggering of a holocrine secretion in female glands that forms two types of secretory masses: intra-tubular-syncytial and inter-tubular-syncytial masses. The aim of this work was to study the development of this inter-tubular holocrine secretion. To approach this task, we have considered that the steps developed during the formation of the so-called invasive masses consist of the growth of epithelial cells, cell detachment from the basal lamina and invasion of surrounding tissues. The presence of these masses, particularly in the female HG, are closely linked to sexual dimorphism in redox balance and to alterations in the expression of certain factors such as cytokeratins, P-cadherin, matrix metalloproteinases, cathepsin H, proliferating cell nuclear antigen, p53, CD-31 and vascular endothelial growth factor, which seem to be involved in tissue remodeling. The results document unusual mechanisms of secretion in Syrian hamster HG: an extraordinary system of massive secretion through the conjunctive tissue, disrupting the branched structure of the gland.

Melatonin modulates autophagy through a redox‐mediated action in female Syrian hamster Harderian gland controlling cell types and gland activity

The Syrian hamster Harderian gland exhibits sexually dimorphic porphyrin biosynthesis, wherein the female glands display an extraordinarily high concentration of porphyrins. Damage derived from this production of porphyrins, mediated by reactive oxygen species, causes the glands to develop autophagic processes, which culminate in detachment-derived cell death; these cells normally play a central role in the secretory activity of the gland. The main aim of this study was to analyze how a change in the redox state impacts autophagy. Female Syrian hamsters were treated daily with melatonin (25 μg, subcutaneously) at ZT 10 for 1-2 months (N-acetyl-5-methoxytryptamine), an endogenous antioxidant that ameliorates the deleterious effects of free radicals via a variety of mechanisms. The length of treatment affected the redox balance, the autophagy machinery, and the activation of p53 and NF-κB. One-month treatment displaces redox balance to the antioxidant side, promotes autophagy through a p53-mediated mechanism, and increases cell detachment. Meanwhile, 2-month treatment restores redox balance to the oxidant side, activates NF-κB reducing autophagy to basal levels, increases number of type II cells, and reduces number of detached cells. Our results conclude that the redox state can modulate autophagy through redox-sensitive transcriptions factors. Additionally, these findings support a hypothesis that ascribes differences in the autophagic-lysosomal pathway to epithelial cell types, thereby restricting detachment-induced autophagic cell death to epithelial cell type I.

Sexual dimorphism of autophagy in Syrian hamster Harderian gland culminates in a holocrine secretion in female glands

The Syrian hamster Harderian gland (HG) has a large porphyrin metabolism with a sexual dimorphism, showing male HGs much lower porphyrin concentrations than female glands. Damage derived from this production of porphyrins, displayed by reactive oxygen species, forces the gland to develop morphological changes that must have a physiological significance. Thus, oxidative stress is present in two states: mild oxidative stress in male HGs and extreme oxidative stress in female HGs. Cathepsins data gave indirect indications about the presence of programmed cell death affecting the lysosomal pathway, especially in female HGs, which showed an accumulation of autophagic bodies. Our results showed different degrees of autophagy in Syrian hamster HGs depending on sex and probably controlled by the redox-sensitive transcription factors: NF-kappaB and p53. The discovery of these sexual dimorphisms in redox signaling and in autophagy corroborates previous findings and underlines the key role of reactive oxygen species in the regulation of autophagy. In addition, in this paper we propose a physiological significance for these phenomena: male HGs develop a survival autophagy, while in female HGs, autophagy culminates in a detachment-derived cell death that plays a central role in its secretory activity, leading to a massive glandular secretion.

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus)

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

Survival mechanisms in a physiological oxidative stress model

The Syrian hamster Harderian gland has as the remarkable feature of an extraordinary rate of porphyrin production, even higher than the liver. The low activity of the last enzyme of the route gives rise to the accumulation of the uncomplex porphyrins in the female glands. Moreover, due to the localization of the Harderian gland, porphyrins exposed to light produce reactive oxygen species and, thus, the gland presents a physiological oxidative stress, with a great number of sings of degeneration, but without compromising the gland integrity. The appearance of abnormal features in this gland was largely described in the past, but the significance is interpreted for the first time in this study. We have found that autophagic processes are the first result of an elevated porphyrin metabolism, as it is observed in both sexes. This mechanism is considered, in this case, as a constant renovation system that allows the normal gland activity to be sustained. Furthermore, there is a second procedure, invasive processes toward connective tissue, which even occasionally reach blood vessels with intravasation of damaged gland components into the bloodstream. This effect is a consequence of a strong oxidative stress environment that is mainly observed in the female gland, resembling to tumoral progression. Both mechanisms, autophagy and invasive processes, have to be implied in the maintenance of the gland integrity.

Effects of continuous light exposure on antioxidant enzymes, porphyric enzymes and cellular damage in the Harderian gland of the Syrian hamster.

The Syrian hamster Harderian gland (HG), an organ present in the male two secretory cell types (type-I and type-II cells), is physiologically exposed to high oxidative stress because of high concentrations of porphyrins and their precursor, 5-aminolevulinic acid. Because of its juxtaorbital location, the HG is accessible to light, and subject to phototoxic effects of these substances. After having previously demonstrated circadian rhythms in antioxidant enzymes, porphyric enzymes and oxidative damage of proteins and lipids, as well as influences of melatonin on these parameters, we have now studied the effects of continuous light (LL), which suppresses melatonin secretion by the pineal gland. Measurements were performed in two different circadian phases, in order to detect the presence or absence of day/night differences. In LL, no differences between circadian phases of subjective day and subjective night were demonstrable for 5-aminolevulinate synthase, 5-aminolevulinate dehydratase, porphobilinogen deaminase, or superoxide dismutase; temporal differences in glutathione reductase and catalase were markedly diminished, whereas all these parameters showed marked day/night differences in the rats exposed to a light/dark cycle of 14:10. In LL, oxidative damage to lipids was minimally effected, while protein damage was enhanced. LL also caused a reduction in the percentage of type-II cells. Therefore, cell differentiation in the HG does not seem to be controlled only by the androgen, but, unexpectedly, also by melatonin.

Effects of δ-aminolevulinic acid and melatonin in the harderian gland of female syrian hamsters

Effects of δ-aminolevulinic acid (ALA) and melatonin were investigated in the female Syrian hamster Harderian gland. This is an organ physiologically exposed to strong oxidative stress due to the highest porphyrinogenic rates known in nature. Enzyme activities of porphyrin biosynthesis and of antioxidative protection, oxidative protein modification, and histological integrity were studied. In the porphyrin biosynthetic pathway, ALA and melatonin acted synergistically by downregulating ALA synthase (ALA-S) and stimulating product formation from ALA; the combination of ALA and melatonin suppressed ALA-S activity, down to about 1% of that in controls. While ALA effects on porphyrinogenesis can be interpreted in terms of homeostasis, melatonin’s actions may be seen in relation to seasonality and/or reduction of oxidative stress. Among antioxidant enzymes, superoxide dismutase (SOD) and glutathione reductase (GR) activities were diminished by ALA, presumably due to the vulnerability of their active centers to free radicals, whereas melatonin moderately increased SOD. Both ALA and melatonin strongly stimulated catalase (CAT), thereby counteracting the oxidative stress induced by ALA and its metabolites. Nevertheless, exogenous ALA caused a strong net rise in protein carbonyl and considerable damage of tissue. When given together with ALA, melatonin antagonized these effects and largely protected the integrity of glandular structures.

Melatonin protects against δ-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands

Effects of the prooxidant δ-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by δ-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of δ-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes δ-aminolevulinate synthase (ALA-S) and δ-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in δ-aminolevulinate synthase, δ-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of δ-aminolevulinic acid and melatonin led to approximately normal levels of δ-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian 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, δ-aminolevulinate synthase (ALA-S), δ-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.

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.

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.

Effects of the circadian mutation ‘tau’ on the Harderian glands of Syrian hamsters

Effects of the circadian mutation ‘tau’ on the Harderian glands of Syrian hamsters

Hormonal regulation of FHG22 mRNA in Syrian hamster Harderian glands: role of estradiol

The regulation of the FHG22 gene by sex steroids has been studied in Syrian hamster Harderian gland, an organ with sexual dimorphism in which the FHG22 mRNA is female-specific. Testosterone treatment of females caused irregular inhibitory effects on the FHG22 mRNA levels, whereas male castration originated transitory increases during less than 2 weeks. Treatment of 15 day-castrated males for 1 or 2 days with β-estradiol-3-benzoate caused a marked stimulation in the FHG22 mRNA levels. The results found in vivo may be explained considering those found in female Harderian gland serum-free primary cell cultures. In the absence of hormones, the FHG22 mRNA levels decreased along the time and neither progesterone, testosterone or 5α-dihydrotestosterone affected the expression. However, estradiol stimulated the FHG22 mRNA expression in a time and dose-dependent manner: increasing effects were detected between 8–96 h of treatment and the EC 50 was about 10 −9 M. The estradiol effect was reverted by the antiestrogen ICI 164 384 or by cycloheximide. We conclude that estradiol stimulates FHG22 mRNA expression in Harderian gland, although other agents may also control the expression in vivo.

Sexual differences in 5′-deiodinase activity in the harderian gland of Syrian hamsters and the effect of pinealectomy: Regulation by androgens

Sexual differences on thyroxine 5′-deiodinase (5′-D) in the Harderian gland of Syrian hamsters were investigated. We compared the 24-h profile of 5′-D activity in male and female hamsters, observing a clear rhythm in males but not in females. Female values were always significantly higher than male ones. After pinealectomy day/night variations in male 5′-D activity at the time points studies were abolished, results that are in correlation with serum thyroid hormones. We also studied the regulation by androgen of the enzyme activity. Basal 5′-D activity increased in castrated males and levels fell when animals were implanted with testosterone or its product 5α-dihydrotestosterone (DHT). Female 5′-D activity was also inhibited by androgens. As only the addition of DHT in the presence of epitestosterone, an inhibitor of the conversion of testosterone on DHT, in castrated males was able to decrease 5′-D activity to control animal levels, we suggest a probable direct effect of DHT by itself. © 1996 Wiley-Liss, Inc.

Sexual differences in 5′‐deiodinase activity in the harderian gland of Syrian hamsters and the effect of pinealectomy: Regulation by androgens

Sexual differences on thyroxine 5′-deiodinase (5′-D) in the Harderian gland of Syrian hamsters were investigated. We compared the 24-h profile of 5′-D activity in male and female hamsters, observing a clear rhythm in males but not in females. Female values were always significantly higher than male ones. After pinealectomy day/night variations in male 5′-D activity at the time points studies were abolished, results that are in correlation with serum thyroid hormones. We also studied the regulation by androgen of the enzyme activity. Basal 5′-D activity increased in castrated males and levels fell when animals were implanted with testosterone or its product 5α-dihydrotestosterone (DHT). Female 5′-D activity was also inhibited by androgens. As only the addition of DHT in the presence of epitestosterone, an inhibitor of the conversion of testosterone on DHT, in castrated males was able to decrease 5′-D activity to control animal levels, we suggest a probable direct effect of DHT by itself. © 1996 Wiley-Liss, Inc.

Isolation and identification of sex-specific cDNA clones from the Syrian hamster harderian gland.

Syrian hamster Harderian glands show a typical sexual dimorphism, with males having two secretory cell types and females having one cell type and intraluminal porphyrin accretions, among other differences. Since these differences may be due to the expression of specific genes, our interest is to identify those genes and their role on the development and control of the sexual dimorphism. The experimental approach was to construct cDNA libraries for male and female Syrian hamster Harderian glands and then subtracted libraries for male vs. female and for female vs. male. By this method, cDNA libraries enriched either in male-specific or in female-specific clones were obtained. Clones from those libraries were checked for differential expression by using double colony hybridization with [32P]-cDNA from male and female glands. Then, the selected clones were checked again for expression in Harderian glands by Northern hybridization, using poly(A+) RNA from males, castrated males, and females. Finally, the clones were sequenced and compared to search for significant homologies. One of the male-specific clones showed strong homology with rat cytochrome p450b/e. Among the female-specific clones, homologies were found to the complement C3 fragment from several species, to sequences from the mouse mammary tumor virus, and to the subunits C1 and C2 of the rat prostatic steroid binding protein. Several other clones showed no significant homologies and need further characterization.

Isolation and identification of sex‐specific cDNA clones from the Syrian hamster Harderian gland

Isolation and identification of sex‐specific cDNA clones from the Syrian hamster Harderian gland

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.

Regulation of the aminolevulinate synthase gene in the Syrian hamster Harderian gland: changes during development and circadian rhythm and role of some hormones.

The Syrian hamster Harderian gland has been advocated as a model to study the porphyrin biosynthetic pathway, since it shows by far the highest porphyrin concentration known to date. Another particular characteristic is the sexual dimorphism at both the morphological and the biochemical levels. We found a variation in the ALV-S (aminolevulinate synthase) gene expression according to sex, with females exhibiting much higher mRNA levels than do males. After castration, ALV-S mRNA rose considerably in males, this increase being inhibited by darkness or treatment with melatonin. Treatment with hCG or progesterone did not vary the ALV-S mRNA levels in females. Castrated males, however, showed a much larger increase when they were treated with hCG. No variations have been found in the expression of the ALV-S gene in female HG throughout the estrous cycle. During development, males and females showed similar ALV-S mRNA levels until they were 20 days old. Afterwards, they started showing gender-associated differences. In females, ALV-S mRNA levels rose during the first 3 months of life, and thereafter they decreased progressively with aging. A circadian rhythm has been found in the gene expression of ALV-S mRNA in females, showing very low levels in the morning and reaching a peak during the first hours of darkness. It was an endogenous rhythm, probably regulated at the transcriptional level. It is proposed that the light-dark period duration modulates this rhythm through the suprachiasmatic nucleus which in turn acts on the pineal secretion of melatonin that regulates ALV-S gene expression.