Effects of prepubertal manipulation with androgens on the development of sexual differences in the harderian glands of Syrian hamsters.

The onset of sexual differences in the metabolism of porphyrins and melatonin in the Harderian glands of Syrian hamsters was studied. Three weeks after birth, the porphyrin concentrations were already higher in glands of females than in those of males. Castration of 22-day-old male hamsters led to an increase in Harderian porphyrin concentrations, although the levels of intact females were not reached. The administration of testosterone to 22-day-old female hamsters resulted in a marked decrease in porphyrin concentrations. Study of the development of sexual differences in the enzymes involved in melatonin synthesis, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) indicated that not all the sexual differences observed in these glands begin at the same time. Thus, while differences in NAT activity were detected after the age of 3 weeks, male-female differences in HIOMT activity were only observed after 7 weeks. Castration of prepubertal male hamsters lowered NAT but not HIOMT activities. The administration of testosterone to prepubertal female hamsters led to male activity levels in both enzymes. Although circulating androgens seem to have a crucial role in maintaining sexual differences, other hormones including those from the pituitary and thyroid glands are probably also important for generating these sexual differences.

Testosterone sensitive dihydropyridine binding in the Harderian gland of the male hamster.

It is well known that in different tissues, dihydropyridines bind at nanomolar concentrations to a receptor and block voltage-operated Ca2+ channels. In studies reported here, Harderian gland tissue homogenates from intact male hamsters exhibited significant dihydropyridine binding (Bmax = 1700 fmoles/mg protein) of high affinity (Kd = 1.1 nM). Tissue homogenates from female animals exhibited a similar Kd value (1.35 nM) but receptor density per mg protein was significantly reduced (Bmax = 270 fmoles). Dihydropyridine binding of Harderian gland tissue homogenates from castrated males was reduced greater than 80% (Bmax = 225 fmoles/mg protein). Treatment of castrated males with subcutaneous testosterone pellets resulted in significant restoration of dihydropyridine binding activity (approximately 80%, Bmax = 1630 fmoles/mg protein) with a comparable binding constant (Kd = 1.50 nM) as observed for noncastrated, control animals. Addition of testosterone (ex vivo) to homogenates from castrated hamsters did not restore dihydropyridine binding to control levels. These data indicate: (a) the Harderian gland from male hamsters exhibits significant dihydropyridine binding; (b) ligand binding is abolished following castration; and (c) significant restoration of dihydropyridine binding occurs following in vivo testosterone treatment. The dependence of dihydropyridine binding restoration upon in vivo steroid hormone administration suggests probable involvement of the steroid at the transcriptional level although non-genomic mechanisms such as the binding of testosterone to a receptor resident in the plasma membrane and subsequent activation of Ca2+ channels can not be ruled out.

N-acetyltransferase activity in the Harderian glands of the Syrian hamster, Mesocricetus auratus, is regulated by androgens and by hormones of the pituitary-thyroid axis.

This study tested the hypothesis that activity of the enzyme N-acetyltransferase (NAT) in the Harderian gland of the Syrian hamster is regulated both by androgens and by hormones of the pituitary-thyroid axis. To test the effects of castration and hypothyroidism, intact or castrated male hamsters were given either tap water or methimazole in their drinking water for 3 weeks. Methimazole suppresses iodination of thyroglobulin, thereby decreasing circulating levels of thyroid hormones and increasing TSH levels. Hypothyroidism or castration caused elevated or depressed Harderian gland NAT activities respectively, compared with euthyroid controls. When castration and hypothyroidism were combined, the animals exhibited high NAT activity compared with castrated euthyroid males. To test the effects of castration and hyperthyroidism, male hamsters were given daily injections of thyroxine (T4) or diluent and were either castrated or left intact for 4 weeks. Intact animals given T4 had depressed Harderian NAT activity; serum thyroid hormone levels were elevated and TSH levels were depressed compared with those of intact controls. Castrated animals had depressed NAT activity below that of intact controls; serum thyroid hormone levels were normal but TSH levels were depressed. Castrated animals given T4 injections had NAT activity similar to that of euthyroid castrated hamsters; thyroid hormone levels were elevated but TSH levels were similar to those seen in euthyroid castrated hamsters. In another experiment, both T4 and tri-iodothyronine (T3) were equally effective in decreasing NAT activity in intact males. To determine the effects of the removal of pituitary influences, male hamsters were hypophysectomized. NAT activity in the Harderian glands of these animals was reduced compared with intact controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin specifically stimulates type-II thyroxine 5'-deiodination in brown adipose tissue of Syrian hamsters.

The response of type-II thyroxine 5'-deiodinase (5'-DII) to melatonin treatment was studied in the Syrian hamster. Male hamsters were treated for 15 days with a s.c. pellet containing melatonin, and 5'-DII activity in brown adipose tissue, anterior pituitary gland, Harderian gland and pineal gland was measured using a radioenzymatic technique. Melatonin-treated animals exhibited enhanced 5'-DII activity restricted to brown adipose tissue; the increase was threefold above the values measured in the control group. Serum concentrations of thyroid hormones were unaffected by melatonin treatment. We conclude that the stimulatory effect of melatonin on type-II thyroxine 5'-deiodination is specifically directed to the isoenzyme located in brown adipose tissue and is not accompanied by changes in serum thyroid hormones.

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.

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 micrograms 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.

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-alpha-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 10 days 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.

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.

Ultrastructural study of neuromuscular junction in rectus femoris muscle of streptozotocin-diabetic rats.

The neuromuscular junctions (NMJ) from rectus femoris muscle in streptozotocin (STZ)-induced diabetic rats were examined by electron microscopy eight weeks after the STZ injection. When compared to controls and vehicle-injected groups, both the axon terminal and the junctional sarcoplasm showed serious alterations including mitochondrial degeneration, presence of myeloid bodies, breakdown of presynaptic membrane and changes in the form of the synaptic vesicles. The results suggest that NMJ can contribute to the pathogenesis of diabetic proximal myopathy.

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-hydroxytryptamine, 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-hydroxytryptamine, 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.

Melatonin stimulates brain glutathione peroxidase activity.

Exogenously administered melatonin causes a 2-fold rise in glutathione peroxidase activity within 30 min in the brain of the rat. Furthermore, brain glutathione peroxidase activity is higher at night than during the day and is correlated with high night-time tissue melatonin levels. Glutathione peroxidase is thought to be the principal enzyme eliminating peroxides in the brain. This antioxidative enzyme reduces the formation of hydroxyl radicals formed via iron-catalyzed Fenton-type reactions from hydrogen peroxide by reducing this oxidant to water. Since the hydroxyl radical is the most noxious oxygen radical known, induction of brain glutathione peroxidase might be an important mechanism by which melatonin exerts its potent neuroprotective effects.

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.

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.

Beta-adrenergic stimulation prior to darkness advances the nocturnal increase of Syrian hamster pineal melatonin synthesis.

Pineal glands of male Syrian hamsters stimulated in vivo with isoproterenol (ISO) for 4 h before the onset of darkness showed a 4-h advance in the timing of the nighttime increases in both N-acetyltransferase activity and melatonin levels. When ISO (1 mg/kg) was administered every 2 h to animals kept in light during the night, a significant increase in melatonin synthesis was observed after 4-6 h. The results suggest that the Syrian hamster pineal gland can respond in vivo to continuous beta-adrenergic stimulation, but a lag period of 4-6 h is required before there is an increase in melatonin synthesis.

In vivo administration of isoproterenol or forskolin during the light phase induces increases in the melatonin content of the Syrian hamster pineal gland without a rise in N-acetyltransferase activity.

In vivo melatonin production was stimulated during the daytime in pineal glands of female Syrian hamsters following the administration of several injections of either isoproterenol, a beta-receptor agonist, or forskolin, an adenylate cyclase stimulator. The large increase in melatonin following either isoproterenol or forskolin administration was not accompanied by significant changes in N-acetyltransferase (NAT) activity. The results suggest that the Syrian hamster pineal gland, as in other species, responds by producing melatonin during the light phase if the stimulus is adjusted to its particular and specific regulatory mechanisms, i.e., if beta-adrenergic stimulation is continued for 4-8 h. The lack of a commensurate increase in NAT activity raises the question of the need of maximal enzymatic activity for a significant rise in melatonin production in the Syrian hamster pineal gland.

Age and food restriction alter the porphyrin concentration and mRNA levels for 5-aminolevulinate synthase in rat Harderian gland.

The effects of age and food restriction on the porphyrin concentration in Harderian glands were studied in male Fisher 344 rats. Harderian gland porphyrin concentrations increased with age; this was statistically significant in 20 month old animals compared with 3 month old animals. Food restriction (by 40%) prevented the age-associated rise in porphyrins; thus, in 20 month old food restricted rats had porphyrin concentrations similar to those found in young animals. In a second experiment, we correlated the age-associated rise in Harderian gland porphyrin concentrations with an increase in mRNA levels for 5-aminolevulinate synthase (ALV-S). Both the porphyrin concentration and ALV-S mRNA rose at 12 and 18 months of age, but decreased by 24 months of age. It is concluded that, a) porphyrin biosynthesis in the Harderian glands increases up to 20 months of age but decreases in rats that are 24 months old, and b) food restriction prevents the porphyrin rise associated with age in the Harderian gland of male Fisher 344 rats.

Darkness-induced changes in noradrenergic input determine the 24 hour variation in beta-adrenergic receptor density in the rat pineal gland: in vivo physiological and pharmacological evidence.

In male rats housed under a 14:10 LD cycle (lights on at 0600 h), pineal beta-adrenergic receptors, assessed as 125Iodopindolol (IPIN) binding to membrane preparations, showed a 24 hour variation characterized by a nocturnal increase that peaked around middark (2300 h-0200 h) and a decrease during the latter half of the dark period. Animals exposed to light for 3 hours into the normal dark period showed a similar increase in IPIN binding that was prevented by a single sc injection (0.5 mg/kg) of isoproterenol (ISO). The decrease in IPIN binding observed after middark was prevented both by moving the animals to light at 0200 h and by propranolol administration (20 mg/kg). Likewise, the reduction in IPIN binding was induced in light exposed animals both by ISO administration (in a dose dependent manner) and by injection of norepinephrine (NE) plus the catecholamine uptake blocker desmethylimipramine (DMI). DMI alone was without effect. Chronic denervation of the pineal gland by superior cervical ganglionectomy (SCGx) increased IPIN binding to levels not higher than those observed at middark. The results suggest that rat pineal beta-adrenergic receptors are regulated in a rhythmic 24 hour pattern. A decrease in density (downregulation) induced by a darkness-associated increase in NE release, occurs late in the night before lights on; recovery from the down regulated state (upregulation) occurs during the light and early dark phase, reaching a maximum density of beta-adrenergic receptors at middark not different from that observed in chronically denervated pineal glands.

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.

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.