Sexual dimorphism in the Harderian gland proteins of the golden hamster

SDS polyacrylamide gel electrophoresis of male and female hamster Harderian gland homogenates has shown a clear-cut sexual dimorphism. At least three major proteins present in the male gland are missing from the female gland. Two of the above are associated with the tubular clusters of the male gland while the third seems to be a structural component.     

Sex differences in haem biosynthesis and porphyrin content in the Harderian gland of the golden hamster

Methods are described for the measurement of seven haem biosynthetic enzymes in Harderian gland tissue from male and female golden hamsters. Sex differences were found in five of the seven enzymes. In each case, female tissue exhibited higher activity than male tissue. These differences in enzyme activity are sufficient to account for the major sex difference in porphyrin content in the Harderian gland of this species.     

Dimorphic expression of uncoupling protein-3 in golden hamster harderian gland: effects of castration and testosterone administration

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50 to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained alpha-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids.     

Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs

A series of periodic increases in intracellular free calcium concentration ([Ca2+]i) occurred upon fertilization in golden hamster eggs. The spatial distribution of the Ca2+ transients was investigated in single zona-free, aequorin-injected eggs, inseminated by single sperm. A supersensitive TV camera system for recording Ca2+-aequorin luminescence enabled us to observe the spatial distribution of the Ca2+ rise. In the first response, which usually occurred 10-30 sec after the sperm attachment, the increase in [Ca2+]i began near the sperm attachment site, and the Ca2+ rise spread over the entire egg within 4-7 sec. The Ca2+ rise attained its peak in 5-8 sec, declined with almost even distribution, and ceased in 12-17 sec. The spreading Ca2+ rise was repeated in the second and sometimes the third response, starting from the same focus, but spreading more rapidly (approximately 2 sec). In succeeding responses [Ca2+]i increased synchronously in the whole cytoplasm within 1 sec. When additional sperm attached to the egg after the occurrence of the first response by the first sperm, the spread of the Ca2+ rise could take place from near the site of additional sperm attachment but only in the second or third response.     

Effect of castration and testosterone therapy on harderian gland protein patterns of the golden hamster (Mesocricetus auratus).

1. Sodium dodecyl sulphate 7-12% gradient polyacrylamide gel electrophoresis of male and female hamster Harderian gland whole homogenate shows a clear-cut sexual dimorphism, which consists of the presence of two male-specific glycoproteins (168 and 116 kDa) and two specific female proteins (210 and 190 kDa). 2. In the male, castration causes a significant decrease in the concentration of the two glycoprotein fractions. 3. Replacement therapy with testosterone propionate (T) restores the intact male pattern.     

The effects of bromocriptine and prolactin on porphyrin biosynthesis and morphology in the female hamster Harderian gland

Porphyrin biosynthesis was examined in the Harderian gland of the female golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, -aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are high in normal female glands. Enzyme activity was lowered in females ovariectomised for 6 weeks, and both enzyme activity and porphyrin content were greatly lowered in ovariectomised females given the dopamine agonist bromocriptine; this suppression could be prevented by simultaneous prolactin administration. Bromocriptine (but not ovariectomy alone) also masculinised the morphology of the Harderian gland, resulting in the appearance of type II cells and polytubular complexes; again, the simultaneous administration of prolactin prevented masculinisation. The results support the hypothesis that while androgens have an inhibitory effect on porphyrin synthesis within this model, prolactin may have a major facilitatory role.Abbreviations ALA -aminolaevulinic acid - ALA-s -aminolaevulinate synthase     

Changes in Harderian gland activity in the female golden hamster during the oestrous cycle, pregnancy and lactation.

The Harderian gland, which is situated within the bony orbit, is usually thought of as a source of lubrication for the eye. However, recent studies have suggested links with reproductive function. In the male golden hamster, both gland histology and activity are known to be under hormonal influence, and the present experiment was undertaken to examine gland weight and activity (as measured by the production of porphyrins) over the oestrous cycle and during pregnancy and early lactation in the female hamster. Gland weight, the number of solid intraluminal porphyrin accretions, and concentrations of copro- and proto-porphyrin were all maximal on day 1 of the cycle (oestrous) and at their lowest on day 2 (or jointly on days 2 and 3), rising gradually thereafter. Porphyrin concentrations are considerably higher during pregnancy and early lactation than during the cycle, and the solid porphyrin accretions, although diminished in number, are larger. Although there is no indication of either the function or the physiological basis of these changes during the cycle or pregnancy, these findings do suggest that in the female golden hamster, as in the male, there is a link between Harderian gland activity and reproductive 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 antiadrogen 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. The possible implication of 5-reductase in the regulation of secretory cell types in the Harderian glands of hamsters is discussed.     

Ultrastructural study on the effects of hypophysectomy on the golden hamster parathyroid gland.

The ultrastructure of the parathyroid glands of hypophysectomized golden hamsters was studied. In the parathyroid glands of hypophysectomized animals the Golgi complexes and secretory granules were significantly decreased and large vacuolar bodies were significantly increased compared with those of the control animals. In addition, the chief cells contained a few prosecretory granules in the Golgi areas and a few secretory granules were present in the peripheral cytoplasm. These results suggest that the synthesis and release of parathyroid hormone may be suppressed in the parathyroid glands of the hypophysectomized animals.     

Electron-microscopic studies on the maturation of secretory cells in the mouse Harderian gland

The porphyrins in the Harderian glands of mice are first detectable at 7-8 days of age in both sexes. Thereafter, the levels show a marked rise during the closed-eye period, reaching a peak around the time of eyelid disjunction and then decrease gradually until day 25. At onset of puberty, the level rises again and exhibits a sexual dimorphism. The development of the Harderian gland was examined by light and electron microscopy in the mouse. Although two types of secretory cells, designated as type A and type B, comprise the glandular epithelium in fully developed glands, the time of neonatal appearance is different between the two. Type A cells first appear on the 5th day of age, while type B cells appear around the 7th day corresponding to the time at which porphyrins are first detected. Results of the investigations suggest that the porphyrins in the Harderian gland of mice may be synthesized mainly by type B cells.     

Hormonal regulation and characterisation of the aldehyde oxidase-like gene of hamster Harderian gland

The HG is a compound tubulo-alveolar gland located in the orbital cavity of the majority of vertebrates. In the golden hamster it shows a clear cut sexual dimorphism in both morphological and biochemical parameters such as cell types, protein pattern, lipid metabolism, porphyrin content, steroid hormone receptor expression. In a previous study we found that in primary culture of male hamster Harderian gland (HG), androgens (A) increase the MHG07 (male Harderian gland) expression and this effect is abrogated by both flutamide and cycloheximide. The present study represents a deeper analysis on MHG07 regulation by other members of steroid/thyroid hormone superfamily. Estrogens (E) impair the stimulatory effect of A and after the addition of a pure anti-estrogen, ICI 164,384, the negative effect of E is abrogated. Dexamethasone (Dex), used alone or in combination with A negatively affect the MHG07 expression. Also T(3) increases the expression of MHG07 mRNA. Progesterone (P) does not affect the expression of MHG07 mRNA. The use of cycloheximide abrogates the effect of steroids, suggesting that the latter act through their own receptors. Dose-response experiments show that low steroid concentrations (10(-12)M) are sufficient to affect the MHG07 expression. It is argued that the expression of MHG07 is under a highly coordinate relationship between androgen, estrogen, glucocorticoid, retinoic acid and thyroid hormones.     

The effects of bromocriptine and prolactin on porphyrin biosynthesis in the Harderian gland of the male hamster,Mesocritecus auratus

Porphyrin biosynthesis was examined in the Harderian gland of the male golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, 5-aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are low in normal male glands but were greatly raised in males castrated for 6 weeks. However, porphyrin synthesis remained at basal levels in castrates given the dopamine agonist bromocriptine; this suppression could be reversed by simultaneous prolactin administration, and castrated males receiving prolactin alone exhibited very high enzyme activity and porphyrin content. Bromocriptine also prevents the morphological feminisation of the Harderian gland which would normally occur after castration; again, the simultaneous administration of prolactin permits feminisation to occur. The results support the hypothesis that, while androgens have an inhibitory effect on porphyrin synthesis within this model, other factors, including prolactin, are permissive.Abbreviations ALA 5-aminolaevulinic acid - ALA-s 5-aminolaevulinate synthase - GH growth hormone     

Further studies on the regulation of the Harderian glands of golden hamsters by the thyroid gland

Summary Long-term increased or decreased circulating levels of thyroid hormones significantly modify porphyrin concentrations and morphology in the Harderian glands of male and female hamsters. Administration of T₃ reduced porphyrin concentrations in females; this treatment or decreasing thyroid hormone levels with KClO₄ suppressed the post-castration rise of porphyrins in males. Hypophysectomy led to increased porphyrins in the Harderian glands of males; this rise was suppressed in hypophysectomized males by T₃ or T₄. In females, hypophysectomy reduced porphyrins which were further reduced by daily administration of T₃ or T₄. These modifications in the normal females were identical in castrated males. Mitotic activity in the Harderian glands of females was stimulated by KClO₄ and by hypophysectomy with or without exogenous T₃. In males, castration increased mitotic activity which was suppressed by T₃ and exacerbated by KClO₄. Increased mitotic activity seemingly follows loss of tissue mass. The data show that thyroid hormones act directly on the Harderian glands rather than indirectly through modification of TSH synthesis/release. Female type glands in males are a consequence of loss of gonadal androgens by castration, or by suppression or loss of thyroid hormones by hypophysectomy or by treatment with KClO₄. However, male type glands in females are the result of androgen treatment, and/or increased levels of thyroid hormones via reduced ambient temperatures or of photic input. We conclude that regulation of the Harderian gland appears to be different in the two sexes.Abbreviations T ₃ Triiodothyronine - T ₄ Thyroxine - TSH Thyroid Stimulating Hormone - KClO ₄ Potassium Perchlorate - h hours - ml milliliter - mg milligram - g gram - male - female - castrated male - AP hypophysectomized - CON Control - ALA delta aminole-vulenic acid - HG Harderian Gland     

Effects of pinealectomy on the ultrastructure of the golden hamster parathyroid gland

Ultrastructural changes of the parathyroid glands of pinealectomized golden hamsters were investigated. The main changes in the parathyroid glands 1 hour and 1 day after pinealectomy compared with the control and sham-operated groups were an increase of the Golgi complexes, cisternae of the granular endoplasmic reticulum and large vacuolar bodies. In addition, many chief cells contained numerous prosecretory granules in the Golgi areas and many secretory granules in the peripheral cytoplasm. The morphology of the parathyroid glands 7 and 30 days after pinealectomy resembled that of the control parathyroid glands. These results suggest that pinealectomy affects the secretory activity of the parathyroid gland.     

Application of real-time confocal microscopy to intracellular calcium ion dynamics in rat arterioles

The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Confocal microscopy was employed to study the alteration in intracellular calcium ion concentration ([Ca(2+)](i)) in arterioles (external diameters <100 microm) with respect to selected modifying reagents. 5-Hydroxytryptamine (1 microM), ATP (10 microM), and endothelin 1-3 (5 nM) elicited an increase in [Ca(2+)](i) in most arteriole smooth muscle cells. The [Ca(2+)](i) increase sometimes propagated in an intercellular manner. When noradrenaline (10 microM) was used as a stimulant, [Ca(2+)](i) increase was observed only in a portion of the smooth muscle cells. It was also noted that the reaction of these cells with respect to ATP is different between testis and brain arterioles; the [Ca(2+)](i) increase in testicular arterioles is dependent on Ca(2+) influx from extracellular space, whereas in cerebral arterioles it plays a role in both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores (i.e., sarco/endoplasmic reticulum). These results indicate that arterioles in different tissues may differ greatly in their responses. Real-time confocal microscopy was found to be a useful tool for investigating the structural and functional changes in living tissues.     

Interstitial and parenchymal cells in the pineal gland of the golden hamster

Summary A combined thin-section/freeze-fracture study was performed on the superficial pineal gland of the golden hamster, comparing the parenchymal and interstitial cells of this animal with those previously investigated in rats. In contrast to rats, no gap junctions and gap/tight junction combinations could be found between pineal parenchymal cells of the hamster. Furthermore, the interstitial cells of the hamster pineal gland were found to have large flat cytoplasmic processes, which abut over large areas equipped with tight junctions. In thin sections, profiles of interstitial cell processes were seen to surround groups of pinealocytes. Interstitial cells and their sheet-like, tight junction-sealed processes thus appear to delimit lobule-like compartments of the hamster pineal gland. Because the classification of the interstitial cells is uncertain, the expression of several markers characteristic of mature and immature astrocytes and astrocyte subpopulations has been investigated by indirect immunohistology. Many of the non-neuronal elements in the pineal gland are vimentin-positive glial cells, subpopulations of which express glial fibrillary acidic protein (GFA) and C1 antigen. The astroglial character of these cells is supported by the lack of expression of markers for neuronal, meningeal and endothelial cells. M1 antigen-positive cells have not been detected.Supported by a grant from Deutsche Forschungsgemeinschaft (Scha 185/9-2)     

Effects of melatonin on the ultrastructure of the golden hamster parathyroid gland.

Ultrastructural changes of the parathyroid glands of melatonin-treated golden hamsters were studied. Many chief cells in the parathyroid glands after 1 hour of administration of melatonin contained poorly-developed Golgi complexes associated with a few prosecretory granules and numerous lipid droplets as compared with those of the control animals. The morphology of the parathyroid glands after 5 hours of administration resembled that of the control animals. Many chief cells in the parathyroid glands after 24 hours of administration had well-developed Golgi complexes and cisternae of the granular endoplasmic reticulum, numerous prosecretory granules, a few lipid droplets and many secretory granules in the peripheral cytoplasm as compared with those of the control animals. The ultrastructure of the parathyroid glands after 48 hours of administration was almost similar to that of the control animals. It is considered that melatonin affects the secretory activity of the parathyroid gland.     

Effects of hypergravity environment on the parathyroid gland of the propranolol-treated golden hamster

The fine structure of the parathyroid glands of propranolol-treated hamsters subjected to 5 x gravity environment was studied. In the parathyroid glands of the propranolol-treated hamsters exposed to hypergravity environment, the volume density occupied by the Golgi complexes and cisternae of the granular endoplasmic reticulum was increased as compared to that of propranolol-treated hamsters and was decreased as compared to that of hamsters exposed to a hypergravity environment but was almost similar to that of control hamsters. In addition, many chief cells contained rich free ribosomes, abundant mitochondria and some secretory granules located in the peripheral cytoplasm. These findings suggest that the parathyroid gland which may be suppressed by treatment of propranolol and stimulated in response to a hypergravity environment indicates the secretory activity of the control parathyroid gland.