Syrian Hamster Harderian Gland Research Articles

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).

Invasive processes in the normal Harderian gland of Syrian hamster

Invasive processes in the normal Harderian gland of Syrian hamster

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

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. 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. 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. 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.

The Harderian gland: perspectives.

This is the three-hundred and second anniversary of the first description of Harderian glands by Johann Jakob Harder. Despite more than three centuries of study, many features of these glands, including their definition and function, are not yet established to everyone's satisfaction. Some topics of Harderian biology will be reviewed. Morphology, ontogeny: Mammalian Harderian glands secrete largely by exocytosis, which is unusual in a lipid-secreting exocrine gland. Polytubular complexes are features of male hamster glands; their significance is unknown. Porphyrins: High levels of porphyrin in rodent Harderian glands have made this an attractive organ for research on the control of porphyrin synthesis. Attempts to use it as a model of human porphyria, however, have been disappointing. We do not know the function of Harderian porphyrin. The golden (hamster) age: Syrian (golden) hamster Harderian glands show marked sexual differences, including porphyrin levels. This has made them a very useful experimental animal for endocrine studies. The pineal connection: Rodent Harderian glands contain melatonin. Pinealologists often also study Harderian glands, and Harderianologists also often study the pineal. This relationship has contributed valuable insights to our knowledge of both systems. Lipids: Rodent Harderian glands are about 20% lipid by wet weight, and the main secretory product is lipid. The main lipid in many animals is 1-alkyl-2, 3-diacylglycerol. Harderian gland lipid is extensively studied for insights into lipid biochemistry; less has been done on understanding the function of lipids in the physiology of the glands. Behavior: Lipids may function as vehicles for pheromone secretion. In addition, the possible use of Harderian secretion during cold temperatures by gerbils has been noted. Immunology: Bird Harderian glands are probably lymphoid organs. This does not seem to be true of mammals, though the female Syrian hamster shows large numbers of mast cells. Harderian glands of amphibians and reptiles: Research on nonmammalian Harderian glands has been limited, though important insights continue to emerge from these studies.

Cloning of a Syrian hamster cDNA related to sexual dimorphism: establishment of a new family of proteins

The clone FHG22, isolated from a female minus male subtracted cDNA library obtained from the sexually dimorphic Syrian hamster Harderian glands (HG) is 440 bp long with a 95 amino acids ORF, and hybridizes to a female HG-specific 0.6 kb mRNA. The FHG22 nucleotide and amino acid sequences are similar to the subunits from prostatein, uteroglobin, major cat allergen Fel dI (chain 1) and mouse salivary androgen binding proteins (subunit α). Therefore I propose that all those polypeptides belong to a common new family. The hamster genome has a single copy of the FHG22 gene, without homologous genes. FHG22 mRNA is also found in male and female parotid (higher levels in females) and submandibular glands, indicating a tissue and sex-dependent control of expression.

Androgen regulation of gene expression in the Syrian hamster Harderian gland

The androgenic control of sexual dimorphism has been studied in the Harderian gland from Syrian hamster and compared to rat Harderian gland, a system without dimorphism. Hybridization in situ with a rat cDNA clone has revealed the presence of androgen receptor mRNA in all secretory cells from male and female hamster glands. Testosterone or 5-α-dihydrotestosterone administration to females both caused a 60% decrease in the levels of 5-aminolevulinate synthase mRNA after 1 day of treatment, but the resulting patterns of in vitro translation using RNA from glands treated with the two androgens are different. Testosterone alters the mRNA levels for androgen receptor and 5-aminolevulinate synthase in the glands only 6 h after its implantation in females, and the action is maintained up to 10days of treatment. Finally, androgen administration to females or deprivation in males alter androgen receptor but not 5-aminolevulinate synthase mRNA levels in rat Harderian glands. Our results suggest that the androgen receptor from Harderian glands is responsible for the sexual dimorphism found in Syrian hamsters, whereas the lack of sexual dimorphism in rat seems to be due to a restricted effect of androgens in the glands.

Androgenic control of porphyrin in the harderian glands of the male syrian hamster is modulated by the photoperiod, which suggests that the sexual differences in porphyrin concentrations in this gland are important functionally

The porphyrin concentrations of the Harderian glands of Syrian hamsters show marked sexual differences, with male levels being much lower than those of females. Porphyrinogenesis is inhibited by androgens, so orchidectomy leads to elevated male porphyrin concentrations; however, a number of other procedures (some of which also lower androgen levels) prevents this. We studied the effects of short-day photoperiods and melatonin on Harderian porphyrin concentrations. Intact, castrated, or pinealectomized hamsters of both sexes were exposed to long-day or short-day photoperiods. Intact or castrated hamsters were given melatonin injections in the morning or the afternoon, or were given beeswax pellets containing melatonin. After a variable period, Harderian glands were dissected and porphyrins were measured. Prolonged short-day exposure (13 weeks) led to increased Harderian porphyrin concentrations and this rise was prevented by pinealectomy. The rise in Harderian porphyrins following short-day exposure was small, compared with that following castration. Short-day photoperiods also prevented the rise in porphyrin levels associated with castration and this effect was prevented by removal of the pineal. Melatonin injections, whether given in the morning or in the afternoon, had no effect on Harderian porphyrin concentration of castrated male hamsters. Continuous release melatonin pellets reduced the postcastrational rise in porphyrin levels in one experiment, while having no effect in another. In female hamsters, neither short photoperiods nor melatonin pellets influenced Harderian porphyrin concentrations. These results suggested that a factor from the pineal gland helps maintain the low levels of porphyrin which are characteristic of male Harderian glands, despite the decrease in androgen levels which typically results from exposure to short days. Morning and afternoon injections of melatonin and continuous release melatonin pellets failed to resolve the question of whether this pineal factor is melatonin. Our results demonstrated that low male and high female porphyrin levels are maintained in Syrian hamsters, despite seasonal variations in the hormonal milieu, suggesting that these sexual differences are important for the (still unestablished) function of the Harderian glands in this species.

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.

Effects of ovariectomy and ageing on the structure and ultrastructure of the female Syrian hamster Harderian gland: a stereological analysis

The effects of ovariectomy and ageing on the structure and ultrastructure of the Syrian hamster Harderian gland were investigated by techniques of quantitative stereology. Tissues were obtained from intact 6-month-old, sham-operated 6-month-old, ovariectomized 6-month-old, intact 18-month-old and ovariectomized 18-month-old female hamsters. Glands from both ovariectomized and aged hamsters showed comparable qualitative and quantitative characteristics. They showed histological alterations that included thinning of the tubule walls, lowering of luminal porphyrins, invasion of lumina by neutrophils and the occurrence of interstitial porphyrins. Glands from both ovariectomized and aged hamsters showed statistically significant differences from control animals in relation to numerical density and cellular size. Finally, quantitative studies with the electron microscope revealed significant decreases in the volume densities of the cytoplasmic organelles concerned with secretion. These results support the hypotheses that the secretory activity of the female hamster Harderian gland is influenced, directly or indirectly, by ovarian hormones, and that many of the age-related modifications of the Harderian gland reflect alterations in ovarian function.

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)

Gender-associated differences in the development of 5-aminolevulinate synthase gene expression in the Harderian gland of Syrian hamsters

The mRNA levels for aminolevulinate synthase (ALV-S), the rate-limiting enzyme in porphyrin synthesis, were studied in male and female Syrian hamsters during postnatal development. Sex-associated differences in the expression of ALV-S gene were evident at the end of the third week of postnatal development. Serum levels of luteinizing hormone (LH), testosterone, cortisol, thyroid hormones and insulin-like growth factor were also studied in order to correlate their concentrations with the mRNA levels for ALV-S. Among these hormones, serum LH levels showed a positive correlation with the ALV-S mRNA levels. However, the expected negative correlation with testosterone levels was not clearly observed. Thus, in order to test the effects of testosterone on ALV-S gene expression, 11-day-old male and female Syrian hamsters and adult female hamsters were injected with 50 μg of testosterone for 4 days. Testosterone administration decreased the levels of ALV-S mRNA in the adult females but did not influence those of young females. The possible explanation for the insensitivity to testosterone during these postnatal stages might involve the maturational state of androgen receptors in the Harderian glands.

Melatonin binding sites in the Harderian gland of Syrian hamsters: sexual differences and effect of castration.

The presence of specific melatonin binding sites in the Harderian gland of Syrian hamsters was studied using [125I]melatonin. Saturation binding experiments conducted with [125I]melatonin at 37 degrees C using Harderian glands of both male and female Syrian hamsters revealed a single nanomolar-affinity site. The dissociation constants (Kd) were 6.47 and 6.94 nM for males and females, respectively. The concentration of the binding sites was 7.58 fmol/mg protein for males and 13.50 fmol/mg protein for females. Castration of male hamsters resulted in a significant increase in [125I]melatonin binding sites while chronic melatonin administration did not modify the binding properties. The results confirm the presence of melatonin binding sites in the Harderian glands of rodents. The gender-associated differences found together with the effects of castration in male hamsters suggest an androgenic control in [125I]melatonin binding sites of the Syrian hamster Harderian gland.

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.

Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters.

The morphological features and relative number of mast cells per mm2 were studied in the Harderian glands of male and female Syrian hamsters (Mesocricetus auratus) under different experimental conditions. The structural and ultrastructural characteristics of Harderian mast cells corresponded to those of connective tissue mast cells. The Harderian glands from female hamsters contained more mast cells than those of male hamsters. A subcutaneous implant of testosterone (2 mg/24 mg beeswax) resulted in a rapid decrease in the number of recognizable mast cells 6 h after the implantation. Neither orchidectomy nor ovariectomy significantly altered the relative number of mast cells. However, the daily subcutaneous injection of 20 IU of human chorionic gonadotropin during 20 days resulted in a significant decrease of identifiable mast cells. The administration of another steroid such as progesterone or the induction of states of hypo- and hyperthyroidism did not alter the distribution of mast cells in the Harderian glands of female Syrian hamsters.

Porphyrin metabolism in the harderian glands of Syrian hamsters: in vivo regulation by testicular hormones, lighting conditions, pineal gland, and pituitary hormones.

The porphyrin concentration in the harderian glands of male hamsters subjected to several endocrine manipulations was studied. Prolonged bilateral gonadectomy resulted in a marked increase in harderian porphyrin concentration. This change was not prevented by either pinealectomy or by constant white light exposure. Castrated hamsters exposed to constant red light showed higher porphyrin concentrations than castrated hamsters kept under white light. Among several hormones studied, serum luteinizing hormone and thyroid-stimulating hormone levels were unexpectedly higher in the constant red light exposed group than in the other groups. In order to test whether luteinizing hormone was involved in the postcastrational rise in harderian porphyrins, we administered a potent luteinizing hormone-releasing hormone (LHRH) agonist. The chronic administration of the LHRH agonist resulted in a decrease in serum luteinizing hormone (because it desensitized the LHRH receptors on the gonadotropes) and, consequently, in serum testosterone levels. However, no rise in harderian porphyrin was observed. It is concluded that the absence of testicular hormones might not be the triggering factor involved in harderian porphyrogenesis.

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.

Inhibition of Sα‐reductase does not simulate the effects of androgen deprivation on porphyrin metabolism by Harderian glands from the male Syrian hamster

As 5 alpha-reductase is involved in the metabolism of indolamines in the Harderian glands of Syrian hamsters, we have compared the effects of androgen deprivation and the administration of the potent 5 alpha-reductase inhibitor N,N-diethyl-4-methoxy-3-oxo-4-aza-5-androstene-17-carboxamida on Harderian porphyrin metabolism. Ten days after castration, porphyrin levels had increased to 40 times the values of intact hamsters. However, the inhibition of 5 alpha-reductase, which resulted in a significant decrease in the weight of accessory sex glands, did not modify porphyrin concentrations within the Harderian glands. It is concluded that, contrary to the situation for indolamine metabolism, testosterone per se is the main androgen involved in the sexual differences observed in Harderian porphyrin metabolism.

5-Aminolevulinate synthase mRNA levels in the Harderian gland of Syrian hamsters: Correlation with porphyrin concentrations and regulation by androgens and melatonin

The levels of 5-aminolevulinate synthase mRNA were investigated in the Harderian glands of male and female Syrian hamsters by using a cDNA clone from rat liver. Female hamsters showed higher levels of mRNA than those in males, while the administration of testosterone to female hamsters led to a reduction in mRNA levels. Castration of male hamsters caused a marked elevation of mRNA levels, whereas both the exposure to constant darkness or melatonin injections to castrated males partially prevented the effects of castration. Porphyrin concentration of Harderian glands showed a strong correlation with the mRNA levels of 5-aminolevulinate synthase in all the animals studied. These results lead to the conclusion that in this system, porphyrin metabolism is controlled through hormonal regulation of 5-aminolevulinate synthase gene expression.

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.

Indole and porphyrin content of the syrian hamster harderian glands during the proestrous and estrous phases of the estrous cycle

Porphyrin and indole metabolism was studied in the Harderian glands of Syrian hamsters during the proestrous and estrous stages of the estrous cycle. Porphyrins remained unaltered during these stages, but levels of different indoles (5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl-5-hydrpxytryptamine, and 5-hydroxyindole acetic acid) exhibited pronounced changes during the dark: light period in both proestrous and estrous. There was a strong parallelism between 5-hyroxytryptamine, N-acetyl-5-hydroxytryptamine and 5-hydroxyindole acetic acid levels. Hydroxytryptophan rhythms appeared slightly shifted from those of the other indoles. Immunoreactive melatonin present in the Harderian glands did not show a significant day-night change during the stages studied.