High-performance liquid chromatographic analyses of porphyrins in hamster Harderian glands.

Porphyrin content and 5-aminolaevulinate synthase activity of the Harderian gland were measured in intact and gonadectomized male and female hamsters; porphyrin profiles were analysed by high-pressure liquid chromatography. The total porphyrin content of the two female groups was similar, but enzyme activity in females ovariectomised for 20 weeks significantly decreased. Intact males have low porphyrin content and enzyme activity, while in castrates (6 weeks) both increased to female levels. Protoporphyrin IX formed 93% of total porphyrins in intact females, compared with 70% of total porphyrins in intact males. The remainder in both sexes was chiefly penta- and hexacarboxylic porphyrins and coproporphyrin and (in females) Harderoporphyrin. Gonadectomy in either sex resulted in protoporphyrin levels intermediate between male and female values.

Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland.

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.

Purification and characterization of a growth factor from guinea pig harderian gland.

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.

Neuropeptides and the innervation of the avian lacrimal gland.

The chicken Harderian gland, the major lacrimal gland, has two major cell populations: a cortical secretory epithelium and a medullary interstitial cell population of lymphoid cells. There is an extensive acetylcholinesterase (AChE) network throughout the gland, as well as catecholamine positive fibers among the interstitial cells. There are substance P-like (SPLI) and vasoactive intestinal polypeptide-like (VIPLI) immunoreactive fibers throughout the gland. These fibers are particularly dense and varicose among the interstitial cells. The adjacent pterygopalatine ganglion complex has neuronal somata that exhibit VIPLI and were AChE-positive. This ganglion complex also contains SPLI and catecholamine-positive fibers. In regions of the ganglion, the somata appear surrounded by SPLI varicosities. Surgical ablation of the ganglion eliminated or reduced the VIPLI, AChE and catecholamine staining in the gland. The SPLI was reduced only in some regions. Ablation of the superior cervical ganglion or severance of the radix autonomica resulted in the loss of catecholamine staining in the pterygopalatine ganglion and the gland. Severance of the ophthalmic or infraorbital nerves had no effect on the VIPLI or the SPLI staining pattern in the gland.

Identification of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice.

A new class of alkyl glycerolipids, 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, was identified in lipid extracts prepared from harderian gland tumors of mice. After saponification, this lipid class yielded 1-alkyl-3-(1'-glycerol)glycerols. Identification was based on mass spectrometry, proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, and chromatography of various derivatives and appropriate standards that were synthesized. The alkyl moieties of this unique lipid class consisted of saturated aliphatic chains with chain lengths of 14 to 20 carbon atoms. The acyl moieties were mostly saturated and monounsaturated aliphatic chains ranging from 14 to 24 carbon atoms. The alkyl and acyl moieties of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols were similar to those of alkyldiacylglycerols present in the same tissue, except for the presence of monounsaturated alkyl moieties in the latter. 1-Alkyl-2-acyl-3-(2', 3'-diacylglycerol)glycerols were only found in trace amounts in the normal harderian glands of mice. The total quantity of the alkyl and acyl moieties with a chain length greater than 20 carbon atoms in the alkyldiacylglycerols from tumors were considerably lower than those found in normal harderian glands of mice. This is the first report of the presence of bisglyceryl ether lipids in mammalian tissue; its unique chemical structure is consistent with the type of ether-linked lipid products that could be synthesized in the reaction catalyzed by alkyldihydroxyacetone-P synthase.

Steroid hormone receptors and the sexual phenotype of the Harderian gland in hamsters.

To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The invitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [3H]7 alpha,17 alpha-dimethyl-17 beta-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (Kd) of 0.7 nmol/l and maximal saturation binding capacity of 84.0 +/- 3.0 (S.D.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5 alpha-dihydrotestosterone, testosterone and 3 alpha-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17 beta, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8-9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species.

B cell maturation in the chicken Harderian gland.

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.

Harderian glands of golden hamsters: morphological and biochemical responses to thyroid hormones.

Manipulation of circulating levels of thyroid hormones modifies Harderian gland structure and porphyrin concentrations in male and female golden hamsters. Specifically, thyroxine (T4) and triiodothyronine (T3) induce the morphological conversion of the Harderian glands of females to approximate those of the male. Further, porphyrin concentrations are markedly decreased by this treatment. This effect occurs in ovariectomized animals as well, indicating that the gonads are not involved. Suppression of thyroid function by potassium perchlorate (KClO4) drastically reduces Harderian gland weight in both males and females. However, KClO4 decreases porphyrin levels in the Harderian glands of females and increases it in the male. Concurrently, KClO4 also induces a morphological conversion of the Harderian glands of males to the female type. This effect is evident in photoperiods of either 14:10 (h) or 8:16 (h).

First demonstration of porphyrins in the lacrimal glands: similarity to the Harderian gland.

The Harderian gland of rodents is the only known tissue with physiological occurrence of high concentrations of porphyrins. In this report we describe the occurrence of considerable concentrations of porphyrins in the extra-orbital and intra-orbital lacrimal glands of the male rat. Similarly as in the Harderian gland, HPLC analysis revealed protoporphyrin as being the prevalent porphyrin homologue in the lacrimal glands. The results may contribute to elucidation of the yet unknown function of the Harderian gland and its porphyrins.

Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry.

Lipids of Harderian ophthalmic gland were separated by means of thin-layer chromatography with flame ionization detection in an latroscan apparatus. Wax ester and polar lipids (phosphatidylethanolamine and phosphatidylcholine) were detected as the main lipids in rats and glyceryl ether diester and both polar lipids were the main lipids in mice. Fatty acids were determined in individual lipid classes by means of gas chromatography and gas chromatography-mass spectrometry on capillary columns. The content of fatty acids, the positional isomers of monoenoic acids being predominantly C18, C20 and C22, is most interesting. Very-long-chain fatty acids, saturated fatty acids up to C30 and even monoenoic acids up to C28 were detected. Branched-chain fatty acids, predominantly iso and anteiso, are minority components, although their chain length distribution (C15-C27) is broad.

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland.

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.

Branched long chain bases in cerebrosides of the guinea pig Harderian gland.

Cerebrosides obtained from the guinea pig Harderian gland were analyzed. The purified cerebrosides gave a single spot on thin-layer chromatography, the Rf value being similar to that of phrenosine obtained from whale brain. The cerebrosides consisted of 74.7% of glucosylceramide and 25.3% of galactosylceramide. The fatty acid composition of these cerebrosides was 0.7% of non-hydroxy fatty acids and 99.3% of alpha-hydroxy fatty acids. Among these alpha-hydroxy fatty acids, a small amount of methyl branched acids was detected. The substituted position of methyl branching of alpha-hydroxy fatty acids was the 16th carbon atom from the carboxyl end irrespective of the carbon chain length. The long chain bases were composed of sphinganine (78%) and sphingenine (22%). 4-D-Hydroxysphinganine was not found. The most remarkable feature of the long chain bases of cerebrosides in the Harderian gland was the presence of a large amount of methyl branched sphinganine. The cerebrosides obtained from the cerebrum and cerebellum of the same animal were also analyzed. The sugar, fatty acid, and long chain base compositions of these cerebrosides were similar to those of whale brain cerebrosides. Methyl branched sphinganine was not found in guinea pig brain.

The prevention of porphyrin loss from tissues during routine histological processing: quantitative studies on the Harderian gland.

The rodent Harderian gland is an important site of porphyrin biosynthesis and storage. Porphyrins are visible at the light and electron microscope level as large intraluminal accretions, as large interstitial accretions surrounded by foreign body giant cells, or as small interstitial deposits within free macrophages. Since porphyrins are soluble in a wide range of solvents, it is important to employ histological routines which minimize porphyrin loss during processing in addition to giving good tissue preservation. In this quantitative investigation, these requirements were optimally achieved with fixation in 3% buffered glutaraldehyde and the use of amyl acetate as a clearing agent.

Stereochemistry of 2,3-alkanediols obtained from the Harderian gland of Mongolian gerbil (Meriones unguiculatus).

The stereochemistry of the alcohol moieties of 2,3-alkanediol diacyl esters obtained from the Harderian gland of the Mongolian gerbil was investigated. There were five major 2,3-alkanediols, C14-C22 (even carbon numbers), all having the erythro configuration as determined by GC-MS analysis of their isopropylidene derivatives in comparison with synthetic erythro- and threo-2,3-hexadecanediols. 13C-NMR spectroscopy of the synthetic materials showed distinct differences of chemical shift at the C-1, C-3, and C-4 carbons, from which the native 2,3-alkanediols were definitely determined to be in the erythro series. The absolute configurations of the C-2 and C-3 asymmetric centers were assigned as 2S and 3R, respectively, based on known 2S,3R-octanediol.

The Harderian gland, its secretory duct and porphyrin content in the Plains mouse (Pseudomys australis).

The Harderian gland of the Australian Plains mouse (Pseudomys australis) consists of tubules lined by a single layer of epithelial cells with a surrounding meshwork of myoepithelial cells. The epithelium contains three types of cell. Type I cells, characterised by their many large apical lipid vacuoles, are comparable with cells reported in other rodents. Secretion is exocytotic and, to a lesser extent, apocrine. Type II cells are highly distinctive with extremely large mitochondria arranged in stacks and whorls. Unusually for Harderian glands, these cells possess prominent conventional Golgi complexes and lysosomes. Cytoplasmic 'slashes', possible vacuole-precursors, are also present. Type III cells have few distinguishing features and may be resting cells. They are occasionally binucleate and mitotic figures occur. The gland contains stored porphyrins, chiefly protoporphyrin, in the form of solid intraluminal accretions. There is a marked sex difference, with females having higher levels of porphyrin than males. Among the components of the interstitial tissue are mast cells, plasma cells and porphyrin-containing macrophages. The secretory duct contains large quantities of cell debris, including nuclei, and is lined by columnar cells with single large apical vacuoles. The outer opening of the duct is lined by mucus-secreting cells and stratified squamous epithelium.

Harderian glands of golden hamsters: temporal and sexual differences in immunoreactive melatonin.

Throughout a 24-h period, immunoreactive melatonin concentrations in Harderian glands of female golden hamsters were approximately 200 pg/mg protein with a significant decline to 80 pg/mg protein only at about 0600, 2 h after light on. Concentrations in glands of males are diurnally constant and low (ca. 20 pg/mg protein). Castration increases immunoreactive melatonin in glands of males to female levels. While blinding alone had no effect, it did prevent the castration-induced increase. Lower concentrations were measured in glands of blinded or blinded ovariectomized females but this decrease was not significant. These data suggest that immunoreactive melatonin concentrations in the Harderian glands of hamsters are controlled by testosterone or its derivatives; these same factors also control the male or female character of these glands.

The effects of spectral power distribution and illuminance levels on key parameters in the male golden hamster and rat with preliminary observations on the effects of pinealectomy.

Three different light sources were used to determine the effects of spectral power distribution (SPD) and illuminance levels on growth and organ weights of male golden hamsters and rats. SPD had little effect on organ weights or measurements of either rats or hamsters. However, responses to illuminance levels were quite apparent, provided they were equalized for the scotopic eye sensitivity curve characteristic of nocturnal animals. Under seven illuminance levels from 0 to 3.9 scotopic fc, hamsters demonstrated graded responses in gonadal weights and presumed function from 0 to 0.02 scotopic fc. Above this level, photopic saturation was apparent. The neuroendocrine system of pinealectomized animals failed to show sensitivity to illuminance levels. The suggestion is made that the pineal gland acts to monitor illuminance levels (below about 0.02 scotopic fc) as well as photic duration. While the latter appears to be an "all or none" effect, the former appears to be graded.

Fatty acid composition of cardiolipin from guinea pig Harderian gland.

The fatty acid composition of cardiolipin from the Harderian gland of guinea pig was examined by gas chromatography and gas chromatography-mass spectrometry. At least 33 kinds of fatty acids were detected. Oleic acid was the most prominent component, accounting for 18.2 mol% of the total fatty acids. About 70.2 mol% of fatty acids had methyl branches. Ethyl branches were also detected (1.3 mol%). Straight chain saturated acids comprised only 10.3 mol%. On the other hand, linoleic, linolenic, and arachidonic acids were not found in this lipid. The 2-(2'-) acyl moieties contained larger amounts of oleic acid and smaller amounts of branched chain acids than the 1-(1'-)acyl moieties, but the saturated straight chain acids showed even distribution between the 1-(1'-) and 2-(2'-)positions. The fatty acids of cardiolipin from the liver of the same animal were also examined. Linoleic acid was the most abundant component (66.9 mol%), and saturated straight chain acids occupied 21.9 mol%. Branched chain acids were detected but comprised only 11.2 mol%.

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.