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The harderian gland: Its biology and role in porphyrin synthesis
Molec. Aspects Med. Vol. 11, pp. 145-152, 1990 Printed in Great Britain. All rights reserved.
0098-2997/90 $0.00 + .50 (~) 1989 Pergamon Press plc.
THE HARDERIAN GLAND: ITS BIOLOGY AND ROLE IN PORPHYRIN SYNTHESIS Anthony P. Payne University Department of Anatomy, Glasgow University, G12 8QQ, U.K.
The Harderian gland has been known for nearly 300 years, yet its functions remain largely speculative. In one group, the rodents, it is a major site for the synthesis and storage of porphyrins. In the most useful laboratory species i) porphyrin content and enzyme activities are much higher than in other tissues; ii) gland structure is inherently linked to synthetic capacity and iii) the gland may exhibit extraordinary lability of both structure and porphyrin synthesis in response to a variety of manipulations. It deserves to be far more widely used in porphyrin research than it is at present. 1) ~-IE NATURAL HISTOI~Y OF THE HARDERIAN GLAND The Harderian gland (first described in deer by Harder in 1694) occurs in most terrestrial vertebrates, that is the anuran amphibia, reptiles, birds and mammals. The known exceptions are mammalian carnivores (which possess a-different gland system), bats and the higher primates. It also occurs in species which are believed to be secondarily aquatic, such as crocodiles and cetaceans. Early reports suggested that the gland occurs in man as a transient embryological feature which is normally lost but may persist in some instances (Fleiseher, 1907), though this requires modern confirmation. The gland is located within the bony orbit and is frequently larger than the eye itself. Its chief characteristic (distinguishing it from other orbital glands) is that its duet opens onto the surface of the nictitating membrane but its glandular part (which is massive) is outside the nictitating membrane. Rather, the glandular portion surrounds much of the eyeball and optic nerve on its nasal or medial side; in species such as the rat, it even interdigitates with other orbital contents such as the extraocular muscles (Grafflint 1942). The secretions produeed by the gland vary greatly and may be serous (as in reptiles, where it may function as an accessory salivary gland), mucous (as in birds and ar~hibia) or lipid (as in most man~nals); for a review, see Sakai (1981). Histologically, the secretory part of the rodent gland consists of tubules formed from a single layer of epithelial cells surrounded by a network of myoepithelial cells. The epithelial cells are usually colt~nnar with basal nuclei, apical microvilli and large numbers of lipid vacuoles. There are many speeialisations and differences between species. In rodents, there is no morphologically identifiable duct system within the gland itself and the epithelial cell types (which vary from 1-3 depending on the species) seem uniformly distributed. The simple description of the rodent gland as compound 'tubular' may, therefore, be more useful than the widespread assertion that the gland is compound 'tubulo-alveolar' in strue~re.
145
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A.P. Payne
2) FUNCTIONS OF THE HARDERIAN GLAND It is widely assumed that the original or primary function of the gland is to provide a lubricating secretion for the front of the eye. W h e r e the duct opens onto the posterior surface of the nictitating membrane the value is clear, though there are many cases where it opens onto the front. However, there are many alternative functions which have been proposed. These include:a) That the gland is a site of immune response for the conjunctival sac. In many species, particularly birds, the gland contains immunocompetent cells such as B-lymphoeytes (Burns, 1975) which respond to both topic and systemic application of bovine serum albumen (Burns, 1976). In rabbits and guinea pigs, much of the lipid secretion is in the form of alkyldiacylglycerols,which may be bactericidal (Jest, 1974). b) That the gland may be a source of thermoregulatory lip ids. In species such as the gerbil, it has been suggested that up to 40% of pelage lipids (necessary for thermal insulation and waterproofing) may be derived from the Harderian gland and distributed to the fur by grooming patterns which commence at the head and progress backwards. When gerbils are immersed for short periods in ice cold saline, core temperature only drops slowly. In Harderianectemised gerbils temperature drop is rapid, as it is in gerbils which have been shampooed to remove pelage lipids (Thiessen and Kittrell, 1980). c) That the gland is a source of pheromones. In mammals, pheromones are often produced caudally, e.g. urine, faeces and the secretions of preputial glands, vagina and anal glands. However,it is likely that facial cues, emanating from salivary or orbital glands, are also important. In gerbils, Harderian secretions are released and investigated during social interactions (Thiessen et al, 1976); m a l e hamsters actively investigate smears of whole female Harderian gland and, when these are placed on the face of an individual male, the aggressive responses of others towards it are reduced (Payne, 1977; 1979). d) That the gland may be part of a retinal-pineal axis. The Harderian gland shares a con~non biochemistry with the retina and pineal in the production of melatonin and other 5-methoxyindoles and in exhibiting diurnal and seasonal rhythms (Pevet, 1985; Menendez-Pelaez et al, 1987). In the hamster, there is far more melatonin in the Harderian gland than in the pineal (Reiter et al, 1983) and Harderianectomy decreases (or shifts) the rise in pineal melatonin which occurs during the dark period (Panke et ai, 1979). e) That the gland may act as a photoprotector. Considerable depletion of Harderian gland porphyrin content occurs when rats are subjected to sudden strong light (Hugo et al, 1987). When the secretory product from the conjunctival sac is spun, a sedimentary fraction is rich in porphyrin (Hugo et al these proceedings). This may absorb and hence protect the eye from excessive ultraviolet light. 3) THE HARDERIAN GLAND IN PORPHYRIN RESEARCH Although the functions of the Harderian gland remain obscure, its importance in the present context is that, in rodents, it is a major site for the synthesis and storage of porphyrins. Again, their function within this particular tissue is unknown. Porphyrins have been found in all rodent species so far essayed although the amount varies considerably (see Table 1). The major form is usually proteporphyrin. In hamsters, this represents up to 95% of gland porphyrins, w i t h minor traces of copro- and uroporphyrins, as well as penta-, hexa- and heptacarboxylic forms. In rats, Hardereporphyrin was initially thought to form up to 30% of total porphyrins (Kennedy, 1970), but recent studies have found only traces (J. Hugo, Personal Communication). In many species, the porphyrin content of the Harderian gland is considerably higher than
The Harderian Gland
147
in tissues such as the liver or kidney. In t h e female hamster (see below) this d i f f e r e n c e is of the order of a hundred fold. N o r is i t c l e a r t h a t the Harderian gland is controlled in a s~nilar way to other tissues. Thus, Margolis (1971) found that DDC and AIA would raise liver porphyrins in mice but not H a r d e r i a n porphyrins. Janousek and co-workers (see t h e s e Proceedings) found that griseofulvin, which elevates liver porphyrin t o t h e point of i n t r a c e l l u l a r c r y s t a l formation (Matilla and Molland, 1974) had no e f f e c t on t h e Herderian g l a n d . Nevertheless, t h e r e may be links between porphyrin
synthesis in the Harderian gland and in other tissues. For example, Harderianectomy resulted in unexpected decreases in porphyrin content and ALA-synthase activity in the liver but not in the kidney and increases in blood porphyrin levels (Spike et al - these Proceedings). TABLE 1 LEVELS OF PORPHYRIN IN ~-IE HARDERIAN GLANDS OF SEVERAL SPECIES OF RODENT (nrnol/g tissue) Male
Plains mouse (Pseudomys aus~alis)
Female
237 ~
83
1932 ±
772
Mongolian gerbil (Meriones unguiculatus)
89 ±
35
213 ±
69
Woodmouse (Apodemus sylvaticus)
61 ±
22
125 +
64
Golden hamster (Mesoericetus auratus)
42 ±
9
3300 +_ 302
The Harderian gland as a model for porph~rin biosynthesis The Harderian gland has been p o r t r a y e d as a l a r g e s t r u c t u r e whose functions are poorly understood; porphyrin synthesis~ which seems t o occur only in rodents, may b e c o n t r o l l e d d i f f e r e n t l y from other porphyrin-forming tissues. What then is the relevance of this gland t o porphyrin research? Firstly, the Harderian gland is of importance b e c a u s e of the amount of porphyrin i t produces. Initially thought t o b e purely a s t o r a g e o r g a n for bloed-borne porphyrins produced elsewhere, it is now known t h a t t h e gland is an active s i t e of synthesis. In the female golden hamster, for example, both the porphyrin c o n t e n t and the activity of ALA-synthase a r e g r e a t l y in excess of levels found in tissues such as liver or kidney ( Table 2). Secondly, because the gland exhibits considerable lability of s t r u c t u r e and synthesis, p a r t i c u l a r l y in response t o c h a n g e s in hormone levels and reproductive s t a t u s . Nowhere is this seen more c l e a r l y than in the gland of the golden hamster. Sex d i f f e r e n c e s in porphyrin c o n t e n t a r e commonly found in r o d e n t Harderian g l a n d s (see Table 1) but none a r e so marked as in the hamster. Again, t h e r e a r e major sex differences in t h e activity of the enzymes ALA-synthase, PBC_,-~leaminase, URO-d ac arb oxylase and COPRO-oxidase (see Thompson et al, 1984). Furthermore, t h e r e a r e morphological sex d i f f e r e n c e s in: i) epithelial ceil types; Type I iwhich has small lipid vacuoles) is the only cell type in the female gland, while the male also possesses Type II cells which have very l a r g e lipid vacuoles. I t is likely t h e s e a r e d i f f e r e n t forms of the same c e l l .
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A. P. Payne ii) UItrastructural features such as the polytubular complexes (prabably derived from SER) occur only in male cells. iii) interstitial mast ceils are some 40 times more numerous in female glands than in
male ones, TABLE 2 THE PORPHYRIN ~ T E N T AND ALA SYNTHASE ACrlVrIY OF LIVER, KIDNEY AND HARDERIAN GLAND IN THE FEMALE GOLDEN HAMSTER. These data are representative means taken from Spike et al these Proceedings.
P orphyrin content
(nmol/g)
ALA-S Activity (nmol ALA/b/g protein)
Liver
25
•
2
180
+
29
Kidney
27
=~
5
380
+
75
539
1800
Harderian gland
3400 +
_+ 511
Castration changes all male characteristics to the female pattern, including porphyrin content, enzyme activities and all morphological indice~ androgen replacement restores them (Payne et al, 1977; Sun and Nadakavukaren, 1980). As such, it has been argued that androgens constitute a 'eoarse tuning' mechanism by which porphyrin synthesis is allowed or inhibited (Payne and Moore, 1987). In each kind of menipulation, it is noteworthy that changes in porphyrin synthesis are invariably aceompanied by changes in gland structure. The relationship of the ovary to porphyrin synthesis in the female gland is more complex (Spike et al~ 1986). Ovariectomy lowers ALA-synthase activity and appears to be accompanied by degenerative changes in the gland, including attenuation of the epithelial cells, invasion of the tubules by neutrephils, the appearance of large porphyrin accretions in the interstitial tissue (presumably where tubule walls have disintegrated) and the appearance of maerophages with crystalline porphyrin deposits. These events have been interpreted as an escalating series of degenerative changes (Payne et al, 1985). Agairb therefore, changes in porphyrin synthesis are accompanied by changes in gland structure. The role of the ovary in controlling porphyrin synthesis is clearly less dr~natic than that of testicular androgens and has been interpreted as a 'fine tuning' mechanism (Payne and Moore, 1987). Nevertheless, there are other sorts of data which support the notion of ovarian control:i) Changes over pregnancy; and lactation Levels of ALA-synthase in the Harderian gland rise during a first pregnancy in the hamster and reach a peak just after birth. Similar changes also occur in ALA-synthase levels in liver and kidney which reach a peak just before birth. These changes do not occur during a third or subsequent pregnancy (Spike et al - these Proceedings).
2) Changes with age The female hamster ceases to be fertile somewhere between 9 and 15 months of age. Harderian gland porphyrin and ALA-synthase are significantly reduced in females at 18 and 24 months compared with ones at 6 months. Furthermore, the degenerative changes in gland morphology found after ovariectomy also occur in post-reproductive senescence (Spike et al, 1988). Conclusions- The Harderian gland may prove a particularly useful model of porphyrin biosynthesis. Its porphyrin content and enzyme activity are very high compared with some other tissues. Also, it has particular attributes in some species of laboratory rodent (conspicuous sex differences and marked hormonal co0trol) which may make it a
The Harderian Gland
149
suitable model for human porphyrias such as acute intermittent porphyria which are ecarnoner after puberty than before, which occur more frequently in women and which are aggravated by hormone disturbances such as the menstrual cycle, pregnancy or the contraceptive pill (Brodie et al, 1977; MeCOll et al, 1982). References Brodie, M.J., Moore, M.R., Thompson, G.G., Goldberg, A., and Low, R.A.L. (1977). Obstet. Gynaeeol. 84- 726-731.
Br. &
Burns, R.B. (1975) Canad. J. Zool. 53- 1258-1269 Burns, R.B. (1976) Clin. exp. Immunol. 26- 371-374. Fleiseher, D. (1907) Anat. Anz. 30: 465-470. Grafflin, A.C. (1942) Am. J. Anat. 71-* 43-64. Hugo, J., Krijt, J., Vokurka, M, and Janousak, V. (1987) Gen. Physiol. Biophys. 8: 401-40. Jost, U. (1974) Hoppe Seyler's Z. fur Physiol. Chem. 355: 422-426 Kennedy, G.Y. (1970) Comp. Bioeham. Physiol. 36- 21-36. McCoU, K.E.L., Wallace, A.M., Moore, M.R., Thompson, G.G., and Goldberg, A. (1982) Clin. Sei. 62: 183-191. Margolis, F. (1971) Arch. Bioehem. Biophys. 145- 373-381. Matilla, A., and Molland, E.A. (1974) J. Clin. Path. 27: 698-709. Payne, A.P. (1977) J. Endoer. 73: 191-192. Payne, A.P. (1979) Anita. Behav. 27: 897-904. Payne, A.P, McGadey,J., and Johnston, H.S. (1985) J. Anat. 1 4 0 : 25-36. Payne, A.P, McGadey, J., Moore, M.R, and Thompson, G.G. (1977) J. Endoer. 75z 73-82. Payne, A.P., and Moore, M.R. (1987) Boll. dell' Ist. Dermatol. S. Gallicano 13: 21-28. Pevet, P. (1985) In The Pineal Gland. (B. Mess, C.S. Ruzsas, L. Tima and P. Pevet, eds.) Elsevier (Amsterdam) 163-186. Reiter, R.J., Richardson, B.A., Matthews, S.A., Lane, S.J., and Ferguson, B.N. (1983) Life Sci. 32: 1229-1236. Sakai, T. (1981) Arch. histol. Jap. 44: 299-333. Spike, R.C., Payne, A.P., and Moore, M.R. (1988)J. Anat. 160: 157-160. Sun, C.Y., and Nadakavukaren, M.J. (1980) Cell and Tissue Res. 207: 511-517. Thiessen, D.D., Claney, A., and Goodwin, M. (1976) J. Chem. Ecol. 2: 231-238. Thiessen, D.D., and Kittrell, M.W. (1980) Physiol. Behav. 24 417-424. Thompson, G.G. Hordevatzi, X., Moore, M.R, MeGadey, J, and Payne, A.P. (1984) Int. J. Bioehem. 16: 849-852.
SECRETORY PRODUCT OF THE RAT HARDERIAN GLAND M. Vokurka, J. H u g o and V. J a n o u s e k Charles University Medical Faculty, Department of Pathdogieai Physiology U. nemecnice 5, 128 53 Prague 2, Czechoslovakia.
Rodent Harderian gland (HG) is used as a model of porphyrin biosynthesis due to its production of large amounts of free porphyrins. A remarkable feature of the porphyrin metaboliam in the HG is the exocrine nature of the gland. Massive secretion of porphyrins containing secretory product (SP) can be induced in rat HG by intraperitoneal administration of para-sympathami'netic drugs, such as ehrcmodacryorrhoea. We studied quantity and concentration of porphyrins in the SP after intraperitoneal administration of a single dose of carbachol to the rat. SP was collected from the inner canthus of both eyes. 1he quantity of porphyrim secreted from the pair of glands was 58.1 + 7.8 ug/54% of the unstimulated pair of glands in concentration 503.5 + 89.4 u g / g . A part of the pooled SP was spun in the mierohaematoerit centrifuge. Three main layers could be seen with even more sub-layers discernible. The sediment layer (2% of the total volume) contained 94.8% of the total porphyrin concentration (23.1 rag/g), the
150
A.P. Payne
aqueous layer (92% volume) contained 4% of the total porphyrin concentration (16 ug/g) and the upper (lipid) layer (6% volume) contained 1% of the total porphyrin concentration (80 ug/g). Occasionally, a thin colourless sub-layer on the top of the lipid layer was visible; this layer seems to be porphyrin--free. PHOTOPROTECTION AS A POSSIBLE FUNCTION OF THE RODENT HARDERIAN GLAND PORPHYRINS. FIRST DESCRIPTION OF PORPHYRINS IN THE LACRIMAL GLANDS J. Hugo, M. Vokurka, J. Krijt and V. J a n o u s e k Depariment of Pathological Physiology, Charles University, Prague 2. Czechoslovakia The Harderian gland (HG) produces large amounts of free porphyrins in several rodent species adapted for living in the dark environment. The gland secretes its deep red secretory product (SP) into the conjunctival sac. The functions of the HG and its SP are not yet known. Recently, we found in rat HG, increased porphyrin secretion in response to light. The absorption spectrwn of the fresh SP obtained after administration of carbachol to rat did not show any Soret peak in the region 380-700 nm, maximum being between 465-475 nm with absorbance O.8-1.2/O.1 ram. B a s e d on these findings, we have proposed a hypothesis on poss~le photoprotective role of the HG porphyrins for the rodent eye. The lacrimal glands, similarly to the HG, secrete their product into the conjunetival sac. Although the HG is considered to be the only tissue with physiological occurrence of high amounts of free porphyrins, we have found considerable concentrations of porphyrins both in the extra- and intra-orbital lacrimal glands in young and old rats. In the old males, the concentrations ranged fram 30-120 ug/g wet tissue, thus being much higher than those in both young males and old females. The porphyrins were assayed also in the male rat salivary glands. The concentrations were higher in old than in young males, but usually not exceeding 2 ug/g wet tissue. TISSUE PORPHYRINS IN ATHYMIC NUDE (nu/nu) MICE V. J a n o u s e k , J. S a n i t r a k , J. Krijt and M. Holub Deparlment of Pathological Physiology, Onarles University, Prague 2 and Institute for Clinical and Experimental Medicine, Prague 2. Czech osl ovaki a Athymic hairless (nu/nu) mice are an ideal model for immunological studies. A number of changes of biochemical parameters as well as elevated thermogenic activity have also been described. In this regard porphyrin metabolism is of interest as porphyrins are indispensable components of substances necessary for energy metaboli~n. For the e~periments, female and male nu/nu mice derived from the BALB/c strain were used for tissue porphyrin determination - nu/+ animals served as controls. The animals were divided into two groups according to age - a) animals younger than 60 days and b) animals older than 100 days. Total porphyrins in the liver, kidneys and Harderian glands were determined spectrofluorimetrically. Individual porphyrin esters were separated by HPLC on a silica column after isolation and preconcentration on reverse phase C18 cartridges. No differences in total porphyrin levels or porphyrin patterns between nu/nu and nu/+ were observed in the liver, kidneys and Harderian glands. In all groups a higher porphyrin content in young animals' liver in comparison to older ones was observed - the difference was significant in female nu/nu mice (0.67+ O.1 ug/g w.w. in 60 days old animals as compared to O.31 + 0.07 ug/g w.w. in 100 days old animals) as well as in nu/+ mice (0.57 + O.21 ug/g w.w. in 60 days old animals as compared to 0.24 + 0.08 ug/g w.w. in 100 days old animals). In all analyzed tissues a prevalence of proteporphyrin was found. After the administration of 2.5% griseofulvin in the diet, given for 72 hours to young female nu/nu mice, the porphyrin~ content in the liver and
The Harderian Gland
151
kidneys was increased about 100 and 10 times, respectively (240 ug/g w.w. in the liver and 6.5 ug/g w.w. in the kidneys). In the liver of griseofulvin-treated animals an increase of protoporphyrin content (from 77.4% to 93.3%) and in the kidneys (from 68.3% to 69.5%) was observed, whereas the content of uroporphyrin was decreased (liver:.from 13.7% to 0.4%; kidneys: from 16.7% to 8.3%). There was practically no change in the content or spectram of porphyrins in Harderian glands, the porphyrin content being about 10 to 40 ug/g w.w. in all groups studied, more than 95% of which was protoporphyrin. The results indicate that there was a difference between the young and old animals in porphyrin content in the liver. No other differences in porphyrin content or porphyrin pattern in nu/nu and nu/+ mice were observed. Both in nu/nu and nu/+ mice, experimental protoporphyria can be induced by administering griseofulvin for 72 hours. H A K D E R I A N E ~ M Y AND PORPHYRIN SYNTHESIS Rosemary C. Spike, A.P. P a y n e and M.R. Moore University Departments of Anatomy and Medicine
Western Infirmary, Glasgow. The rodent Harderian gland is a large orbital structure, usually larger than the eye itself. Of unknown function, in many species, it synthesises and stores porphyrins. In the female golden hamster Harderian gland, the porphyrin content is several hundred times higher than that of the liver or kidney, while the male hamster Harderian gland has a low porphyrin content and enzyme activity. Surprisingly, it is not known what effect the gland has on porphyrin synthesis elsewhere in the body. To examine this, ALA-synthase activity and porphyrin content were measured in the liver and kidney of intact male and female hamsters and in males and females which had been Harderianectemised for 5 months. Blood porphyrin levels were also measured. Harderianectcmy had no effect on porphyrin content or A L A S activity in the kidney. Unexpectedly, there was a marked and significant decrease in liver porphyrin content in both males and females following Harderianectemy. ALA-synthase activity was also significantly decreased in the liver of Harderianectemised males, but this did not reach significance in the male. Both sexes showed a trend towards elevated blood porphyrin levels. The data suggest some link between porphyrin biosynthesis in the liver and the Harderian gland. [Supported by S E R C Grant No. GR/B 83049~ PORPHYRIN SYNTHESIS IN THE HARDERIAN GLAND AND OTHER TISSUES OF THE GOLDEN HAMSTER DURING PREGNANCY AND LACTATION 1LC. Spike, S.L. Stewart, IL Murray, F.M. Kelly, J.A. Maharaj, A.P. P a y n e and M.R. Moore University Departments of Anatomy and Medicine Western Infirmary, Glasgow, Scotland.
The Harderian gland of the female golden hamster is a major site for the synthesis and storage of porphyrins. Both synthesis and gland structure alter with hormone manipulations, such as ovariectomy or androgen administration. The present experiment was undertaken to discover what changes might occur in females experiencing a first pregnancy (PRIMS) and whether such changes also occur in females experiencing a third or later one (MULTS), when conditions such as age and season are controlled. For females experiencing a first pregnancy (PRIMS), porphyrin levels and ALA-synthase activity in the Harderian gland rose and fell again during lactation. There was no obvious trend in multiparous females (MULTS). PRIMS and MULTS differed in porphyrin content during mid and late pregnancy and in A L A S activity just after delivery. A L A S activity was also significantly higher in PRIMS than MULTS during late pregnancy in liver and kidney; faecal porphyrin content increased markedly in PRIMS during lactation. Porphyrins are normally stored in the hrnina of the Harderian gland tubules
152
A.P. Payne
as solid deposits. Duyring a first pregnancy there is an increase in abnormaUy--loeated stores. These include i) large interstitial deposits surrounded by foreign body giant oells and ii) small interstitial deposits in free maerepheges. Mast cell r~mbers also increase. These histdegical differences are maintained in non-pregnant MULTS. These data suggest that pregnancy and lactation cause rises in porphyrin synthesis in pr~nigravid females, but that the consequences are not so marked in multiparous ones. [Supported by SERC Grant No. GR/B 83049 and The Scottish Home and Health Vacation Grants].
0098-2997/90 $0.00 + .50 (~) 1989 Pergamon Press plc.
THE HARDERIAN GLAND: ITS BIOLOGY AND ROLE IN PORPHYRIN SYNTHESIS Anthony P. Payne University Department of Anatomy, Glasgow University, G12 8QQ, U.K.
The Harderian gland has been known for nearly 300 years, yet its functions remain largely speculative. In one group, the rodents, it is a major site for the synthesis and storage of porphyrins. In the most useful laboratory species i) porphyrin content and enzyme activities are much higher than in other tissues; ii) gland structure is inherently linked to synthetic capacity and iii) the gland may exhibit extraordinary lability of both structure and porphyrin synthesis in response to a variety of manipulations. It deserves to be far more widely used in porphyrin research than it is at present. 1) ~-IE NATURAL HISTOI~Y OF THE HARDERIAN GLAND The Harderian gland (first described in deer by Harder in 1694) occurs in most terrestrial vertebrates, that is the anuran amphibia, reptiles, birds and mammals. The known exceptions are mammalian carnivores (which possess a-different gland system), bats and the higher primates. It also occurs in species which are believed to be secondarily aquatic, such as crocodiles and cetaceans. Early reports suggested that the gland occurs in man as a transient embryological feature which is normally lost but may persist in some instances (Fleiseher, 1907), though this requires modern confirmation. The gland is located within the bony orbit and is frequently larger than the eye itself. Its chief characteristic (distinguishing it from other orbital glands) is that its duet opens onto the surface of the nictitating membrane but its glandular part (which is massive) is outside the nictitating membrane. Rather, the glandular portion surrounds much of the eyeball and optic nerve on its nasal or medial side; in species such as the rat, it even interdigitates with other orbital contents such as the extraocular muscles (Grafflint 1942). The secretions produeed by the gland vary greatly and may be serous (as in reptiles, where it may function as an accessory salivary gland), mucous (as in birds and ar~hibia) or lipid (as in most man~nals); for a review, see Sakai (1981). Histologically, the secretory part of the rodent gland consists of tubules formed from a single layer of epithelial cells surrounded by a network of myoepithelial cells. The epithelial cells are usually colt~nnar with basal nuclei, apical microvilli and large numbers of lipid vacuoles. There are many speeialisations and differences between species. In rodents, there is no morphologically identifiable duct system within the gland itself and the epithelial cell types (which vary from 1-3 depending on the species) seem uniformly distributed. The simple description of the rodent gland as compound 'tubular' may, therefore, be more useful than the widespread assertion that the gland is compound 'tubulo-alveolar' in strue~re.
145
146
A.P. Payne
2) FUNCTIONS OF THE HARDERIAN GLAND It is widely assumed that the original or primary function of the gland is to provide a lubricating secretion for the front of the eye. W h e r e the duct opens onto the posterior surface of the nictitating membrane the value is clear, though there are many cases where it opens onto the front. However, there are many alternative functions which have been proposed. These include:a) That the gland is a site of immune response for the conjunctival sac. In many species, particularly birds, the gland contains immunocompetent cells such as B-lymphoeytes (Burns, 1975) which respond to both topic and systemic application of bovine serum albumen (Burns, 1976). In rabbits and guinea pigs, much of the lipid secretion is in the form of alkyldiacylglycerols,which may be bactericidal (Jest, 1974). b) That the gland may be a source of thermoregulatory lip ids. In species such as the gerbil, it has been suggested that up to 40% of pelage lipids (necessary for thermal insulation and waterproofing) may be derived from the Harderian gland and distributed to the fur by grooming patterns which commence at the head and progress backwards. When gerbils are immersed for short periods in ice cold saline, core temperature only drops slowly. In Harderianectemised gerbils temperature drop is rapid, as it is in gerbils which have been shampooed to remove pelage lipids (Thiessen and Kittrell, 1980). c) That the gland is a source of pheromones. In mammals, pheromones are often produced caudally, e.g. urine, faeces and the secretions of preputial glands, vagina and anal glands. However,it is likely that facial cues, emanating from salivary or orbital glands, are also important. In gerbils, Harderian secretions are released and investigated during social interactions (Thiessen et al, 1976); m a l e hamsters actively investigate smears of whole female Harderian gland and, when these are placed on the face of an individual male, the aggressive responses of others towards it are reduced (Payne, 1977; 1979). d) That the gland may be part of a retinal-pineal axis. The Harderian gland shares a con~non biochemistry with the retina and pineal in the production of melatonin and other 5-methoxyindoles and in exhibiting diurnal and seasonal rhythms (Pevet, 1985; Menendez-Pelaez et al, 1987). In the hamster, there is far more melatonin in the Harderian gland than in the pineal (Reiter et al, 1983) and Harderianectomy decreases (or shifts) the rise in pineal melatonin which occurs during the dark period (Panke et ai, 1979). e) That the gland may act as a photoprotector. Considerable depletion of Harderian gland porphyrin content occurs when rats are subjected to sudden strong light (Hugo et al, 1987). When the secretory product from the conjunctival sac is spun, a sedimentary fraction is rich in porphyrin (Hugo et al these proceedings). This may absorb and hence protect the eye from excessive ultraviolet light. 3) THE HARDERIAN GLAND IN PORPHYRIN RESEARCH Although the functions of the Harderian gland remain obscure, its importance in the present context is that, in rodents, it is a major site for the synthesis and storage of porphyrins. Again, their function within this particular tissue is unknown. Porphyrins have been found in all rodent species so far essayed although the amount varies considerably (see Table 1). The major form is usually proteporphyrin. In hamsters, this represents up to 95% of gland porphyrins, w i t h minor traces of copro- and uroporphyrins, as well as penta-, hexa- and heptacarboxylic forms. In rats, Hardereporphyrin was initially thought to form up to 30% of total porphyrins (Kennedy, 1970), but recent studies have found only traces (J. Hugo, Personal Communication). In many species, the porphyrin content of the Harderian gland is considerably higher than
The Harderian Gland
147
in tissues such as the liver or kidney. In t h e female hamster (see below) this d i f f e r e n c e is of the order of a hundred fold. N o r is i t c l e a r t h a t the Harderian gland is controlled in a s~nilar way to other tissues. Thus, Margolis (1971) found that DDC and AIA would raise liver porphyrins in mice but not H a r d e r i a n porphyrins. Janousek and co-workers (see t h e s e Proceedings) found that griseofulvin, which elevates liver porphyrin t o t h e point of i n t r a c e l l u l a r c r y s t a l formation (Matilla and Molland, 1974) had no e f f e c t on t h e Herderian g l a n d . Nevertheless, t h e r e may be links between porphyrin
synthesis in the Harderian gland and in other tissues. For example, Harderianectomy resulted in unexpected decreases in porphyrin content and ALA-synthase activity in the liver but not in the kidney and increases in blood porphyrin levels (Spike et al - these Proceedings). TABLE 1 LEVELS OF PORPHYRIN IN ~-IE HARDERIAN GLANDS OF SEVERAL SPECIES OF RODENT (nrnol/g tissue) Male
Plains mouse (Pseudomys aus~alis)
Female
237 ~
83
1932 ±
772
Mongolian gerbil (Meriones unguiculatus)
89 ±
35
213 ±
69
Woodmouse (Apodemus sylvaticus)
61 ±
22
125 +
64
Golden hamster (Mesoericetus auratus)
42 ±
9
3300 +_ 302
The Harderian gland as a model for porph~rin biosynthesis The Harderian gland has been p o r t r a y e d as a l a r g e s t r u c t u r e whose functions are poorly understood; porphyrin synthesis~ which seems t o occur only in rodents, may b e c o n t r o l l e d d i f f e r e n t l y from other porphyrin-forming tissues. What then is the relevance of this gland t o porphyrin research? Firstly, the Harderian gland is of importance b e c a u s e of the amount of porphyrin i t produces. Initially thought t o b e purely a s t o r a g e o r g a n for bloed-borne porphyrins produced elsewhere, it is now known t h a t t h e gland is an active s i t e of synthesis. In the female golden hamster, for example, both the porphyrin c o n t e n t and the activity of ALA-synthase a r e g r e a t l y in excess of levels found in tissues such as liver or kidney ( Table 2). Secondly, because the gland exhibits considerable lability of s t r u c t u r e and synthesis, p a r t i c u l a r l y in response t o c h a n g e s in hormone levels and reproductive s t a t u s . Nowhere is this seen more c l e a r l y than in the gland of the golden hamster. Sex d i f f e r e n c e s in porphyrin c o n t e n t a r e commonly found in r o d e n t Harderian g l a n d s (see Table 1) but none a r e so marked as in the hamster. Again, t h e r e a r e major sex differences in t h e activity of the enzymes ALA-synthase, PBC_,-~leaminase, URO-d ac arb oxylase and COPRO-oxidase (see Thompson et al, 1984). Furthermore, t h e r e a r e morphological sex d i f f e r e n c e s in: i) epithelial ceil types; Type I iwhich has small lipid vacuoles) is the only cell type in the female gland, while the male also possesses Type II cells which have very l a r g e lipid vacuoles. I t is likely t h e s e a r e d i f f e r e n t forms of the same c e l l .
148
A. P. Payne ii) UItrastructural features such as the polytubular complexes (prabably derived from SER) occur only in male cells. iii) interstitial mast ceils are some 40 times more numerous in female glands than in
male ones, TABLE 2 THE PORPHYRIN ~ T E N T AND ALA SYNTHASE ACrlVrIY OF LIVER, KIDNEY AND HARDERIAN GLAND IN THE FEMALE GOLDEN HAMSTER. These data are representative means taken from Spike et al these Proceedings.
P orphyrin content
(nmol/g)
ALA-S Activity (nmol ALA/b/g protein)
Liver
25
•
2
180
+
29
Kidney
27
=~
5
380
+
75
539
1800
Harderian gland
3400 +
_+ 511
Castration changes all male characteristics to the female pattern, including porphyrin content, enzyme activities and all morphological indice~ androgen replacement restores them (Payne et al, 1977; Sun and Nadakavukaren, 1980). As such, it has been argued that androgens constitute a 'eoarse tuning' mechanism by which porphyrin synthesis is allowed or inhibited (Payne and Moore, 1987). In each kind of menipulation, it is noteworthy that changes in porphyrin synthesis are invariably aceompanied by changes in gland structure. The relationship of the ovary to porphyrin synthesis in the female gland is more complex (Spike et al~ 1986). Ovariectomy lowers ALA-synthase activity and appears to be accompanied by degenerative changes in the gland, including attenuation of the epithelial cells, invasion of the tubules by neutrephils, the appearance of large porphyrin accretions in the interstitial tissue (presumably where tubule walls have disintegrated) and the appearance of maerophages with crystalline porphyrin deposits. These events have been interpreted as an escalating series of degenerative changes (Payne et al, 1985). Agairb therefore, changes in porphyrin synthesis are accompanied by changes in gland structure. The role of the ovary in controlling porphyrin synthesis is clearly less dr~natic than that of testicular androgens and has been interpreted as a 'fine tuning' mechanism (Payne and Moore, 1987). Nevertheless, there are other sorts of data which support the notion of ovarian control:i) Changes over pregnancy; and lactation Levels of ALA-synthase in the Harderian gland rise during a first pregnancy in the hamster and reach a peak just after birth. Similar changes also occur in ALA-synthase levels in liver and kidney which reach a peak just before birth. These changes do not occur during a third or subsequent pregnancy (Spike et al - these Proceedings).
2) Changes with age The female hamster ceases to be fertile somewhere between 9 and 15 months of age. Harderian gland porphyrin and ALA-synthase are significantly reduced in females at 18 and 24 months compared with ones at 6 months. Furthermore, the degenerative changes in gland morphology found after ovariectomy also occur in post-reproductive senescence (Spike et al, 1988). Conclusions- The Harderian gland may prove a particularly useful model of porphyrin biosynthesis. Its porphyrin content and enzyme activity are very high compared with some other tissues. Also, it has particular attributes in some species of laboratory rodent (conspicuous sex differences and marked hormonal co0trol) which may make it a
The Harderian Gland
149
suitable model for human porphyrias such as acute intermittent porphyria which are ecarnoner after puberty than before, which occur more frequently in women and which are aggravated by hormone disturbances such as the menstrual cycle, pregnancy or the contraceptive pill (Brodie et al, 1977; MeCOll et al, 1982). References Brodie, M.J., Moore, M.R., Thompson, G.G., Goldberg, A., and Low, R.A.L. (1977). Obstet. Gynaeeol. 84- 726-731.
Br. &
Burns, R.B. (1975) Canad. J. Zool. 53- 1258-1269 Burns, R.B. (1976) Clin. exp. Immunol. 26- 371-374. Fleiseher, D. (1907) Anat. Anz. 30: 465-470. Grafflin, A.C. (1942) Am. J. Anat. 71-* 43-64. Hugo, J., Krijt, J., Vokurka, M, and Janousak, V. (1987) Gen. Physiol. Biophys. 8: 401-40. Jost, U. (1974) Hoppe Seyler's Z. fur Physiol. Chem. 355: 422-426 Kennedy, G.Y. (1970) Comp. Bioeham. Physiol. 36- 21-36. McCoU, K.E.L., Wallace, A.M., Moore, M.R., Thompson, G.G., and Goldberg, A. (1982) Clin. Sei. 62: 183-191. Margolis, F. (1971) Arch. Bioehem. Biophys. 145- 373-381. Matilla, A., and Molland, E.A. (1974) J. Clin. Path. 27: 698-709. Payne, A.P. (1977) J. Endoer. 73: 191-192. Payne, A.P. (1979) Anita. Behav. 27: 897-904. Payne, A.P, McGadey,J., and Johnston, H.S. (1985) J. Anat. 1 4 0 : 25-36. Payne, A.P, McGadey, J., Moore, M.R, and Thompson, G.G. (1977) J. Endoer. 75z 73-82. Payne, A.P., and Moore, M.R. (1987) Boll. dell' Ist. Dermatol. S. Gallicano 13: 21-28. Pevet, P. (1985) In The Pineal Gland. (B. Mess, C.S. Ruzsas, L. Tima and P. Pevet, eds.) Elsevier (Amsterdam) 163-186. Reiter, R.J., Richardson, B.A., Matthews, S.A., Lane, S.J., and Ferguson, B.N. (1983) Life Sci. 32: 1229-1236. Sakai, T. (1981) Arch. histol. Jap. 44: 299-333. Spike, R.C., Payne, A.P., and Moore, M.R. (1988)J. Anat. 160: 157-160. Sun, C.Y., and Nadakavukaren, M.J. (1980) Cell and Tissue Res. 207: 511-517. Thiessen, D.D., Claney, A., and Goodwin, M. (1976) J. Chem. Ecol. 2: 231-238. Thiessen, D.D., and Kittrell, M.W. (1980) Physiol. Behav. 24 417-424. Thompson, G.G. Hordevatzi, X., Moore, M.R, MeGadey, J, and Payne, A.P. (1984) Int. J. Bioehem. 16: 849-852.
SECRETORY PRODUCT OF THE RAT HARDERIAN GLAND M. Vokurka, J. H u g o and V. J a n o u s e k Charles University Medical Faculty, Department of Pathdogieai Physiology U. nemecnice 5, 128 53 Prague 2, Czechoslovakia.
Rodent Harderian gland (HG) is used as a model of porphyrin biosynthesis due to its production of large amounts of free porphyrins. A remarkable feature of the porphyrin metaboliam in the HG is the exocrine nature of the gland. Massive secretion of porphyrins containing secretory product (SP) can be induced in rat HG by intraperitoneal administration of para-sympathami'netic drugs, such as ehrcmodacryorrhoea. We studied quantity and concentration of porphyrins in the SP after intraperitoneal administration of a single dose of carbachol to the rat. SP was collected from the inner canthus of both eyes. 1he quantity of porphyrim secreted from the pair of glands was 58.1 + 7.8 ug/54% of the unstimulated pair of glands in concentration 503.5 + 89.4 u g / g . A part of the pooled SP was spun in the mierohaematoerit centrifuge. Three main layers could be seen with even more sub-layers discernible. The sediment layer (2% of the total volume) contained 94.8% of the total porphyrin concentration (23.1 rag/g), the
150
A.P. Payne
aqueous layer (92% volume) contained 4% of the total porphyrin concentration (16 ug/g) and the upper (lipid) layer (6% volume) contained 1% of the total porphyrin concentration (80 ug/g). Occasionally, a thin colourless sub-layer on the top of the lipid layer was visible; this layer seems to be porphyrin--free. PHOTOPROTECTION AS A POSSIBLE FUNCTION OF THE RODENT HARDERIAN GLAND PORPHYRINS. FIRST DESCRIPTION OF PORPHYRINS IN THE LACRIMAL GLANDS J. Hugo, M. Vokurka, J. Krijt and V. J a n o u s e k Depariment of Pathological Physiology, Charles University, Prague 2. Czechoslovakia The Harderian gland (HG) produces large amounts of free porphyrins in several rodent species adapted for living in the dark environment. The gland secretes its deep red secretory product (SP) into the conjunctival sac. The functions of the HG and its SP are not yet known. Recently, we found in rat HG, increased porphyrin secretion in response to light. The absorption spectrwn of the fresh SP obtained after administration of carbachol to rat did not show any Soret peak in the region 380-700 nm, maximum being between 465-475 nm with absorbance O.8-1.2/O.1 ram. B a s e d on these findings, we have proposed a hypothesis on poss~le photoprotective role of the HG porphyrins for the rodent eye. The lacrimal glands, similarly to the HG, secrete their product into the conjunetival sac. Although the HG is considered to be the only tissue with physiological occurrence of high amounts of free porphyrins, we have found considerable concentrations of porphyrins both in the extra- and intra-orbital lacrimal glands in young and old rats. In the old males, the concentrations ranged fram 30-120 ug/g wet tissue, thus being much higher than those in both young males and old females. The porphyrins were assayed also in the male rat salivary glands. The concentrations were higher in old than in young males, but usually not exceeding 2 ug/g wet tissue. TISSUE PORPHYRINS IN ATHYMIC NUDE (nu/nu) MICE V. J a n o u s e k , J. S a n i t r a k , J. Krijt and M. Holub Deparlment of Pathological Physiology, Onarles University, Prague 2 and Institute for Clinical and Experimental Medicine, Prague 2. Czech osl ovaki a Athymic hairless (nu/nu) mice are an ideal model for immunological studies. A number of changes of biochemical parameters as well as elevated thermogenic activity have also been described. In this regard porphyrin metabolism is of interest as porphyrins are indispensable components of substances necessary for energy metaboli~n. For the e~periments, female and male nu/nu mice derived from the BALB/c strain were used for tissue porphyrin determination - nu/+ animals served as controls. The animals were divided into two groups according to age - a) animals younger than 60 days and b) animals older than 100 days. Total porphyrins in the liver, kidneys and Harderian glands were determined spectrofluorimetrically. Individual porphyrin esters were separated by HPLC on a silica column after isolation and preconcentration on reverse phase C18 cartridges. No differences in total porphyrin levels or porphyrin patterns between nu/nu and nu/+ were observed in the liver, kidneys and Harderian glands. In all groups a higher porphyrin content in young animals' liver in comparison to older ones was observed - the difference was significant in female nu/nu mice (0.67+ O.1 ug/g w.w. in 60 days old animals as compared to O.31 + 0.07 ug/g w.w. in 100 days old animals) as well as in nu/+ mice (0.57 + O.21 ug/g w.w. in 60 days old animals as compared to 0.24 + 0.08 ug/g w.w. in 100 days old animals). In all analyzed tissues a prevalence of proteporphyrin was found. After the administration of 2.5% griseofulvin in the diet, given for 72 hours to young female nu/nu mice, the porphyrin~ content in the liver and
The Harderian Gland
151
kidneys was increased about 100 and 10 times, respectively (240 ug/g w.w. in the liver and 6.5 ug/g w.w. in the kidneys). In the liver of griseofulvin-treated animals an increase of protoporphyrin content (from 77.4% to 93.3%) and in the kidneys (from 68.3% to 69.5%) was observed, whereas the content of uroporphyrin was decreased (liver:.from 13.7% to 0.4%; kidneys: from 16.7% to 8.3%). There was practically no change in the content or spectram of porphyrins in Harderian glands, the porphyrin content being about 10 to 40 ug/g w.w. in all groups studied, more than 95% of which was protoporphyrin. The results indicate that there was a difference between the young and old animals in porphyrin content in the liver. No other differences in porphyrin content or porphyrin pattern in nu/nu and nu/+ mice were observed. Both in nu/nu and nu/+ mice, experimental protoporphyria can be induced by administering griseofulvin for 72 hours. H A K D E R I A N E ~ M Y AND PORPHYRIN SYNTHESIS Rosemary C. Spike, A.P. P a y n e and M.R. Moore University Departments of Anatomy and Medicine
Western Infirmary, Glasgow. The rodent Harderian gland is a large orbital structure, usually larger than the eye itself. Of unknown function, in many species, it synthesises and stores porphyrins. In the female golden hamster Harderian gland, the porphyrin content is several hundred times higher than that of the liver or kidney, while the male hamster Harderian gland has a low porphyrin content and enzyme activity. Surprisingly, it is not known what effect the gland has on porphyrin synthesis elsewhere in the body. To examine this, ALA-synthase activity and porphyrin content were measured in the liver and kidney of intact male and female hamsters and in males and females which had been Harderianectemised for 5 months. Blood porphyrin levels were also measured. Harderianectcmy had no effect on porphyrin content or A L A S activity in the kidney. Unexpectedly, there was a marked and significant decrease in liver porphyrin content in both males and females following Harderianectemy. ALA-synthase activity was also significantly decreased in the liver of Harderianectemised males, but this did not reach significance in the male. Both sexes showed a trend towards elevated blood porphyrin levels. The data suggest some link between porphyrin biosynthesis in the liver and the Harderian gland. [Supported by S E R C Grant No. GR/B 83049~ PORPHYRIN SYNTHESIS IN THE HARDERIAN GLAND AND OTHER TISSUES OF THE GOLDEN HAMSTER DURING PREGNANCY AND LACTATION 1LC. Spike, S.L. Stewart, IL Murray, F.M. Kelly, J.A. Maharaj, A.P. P a y n e and M.R. Moore University Departments of Anatomy and Medicine Western Infirmary, Glasgow, Scotland.
The Harderian gland of the female golden hamster is a major site for the synthesis and storage of porphyrins. Both synthesis and gland structure alter with hormone manipulations, such as ovariectomy or androgen administration. The present experiment was undertaken to discover what changes might occur in females experiencing a first pregnancy (PRIMS) and whether such changes also occur in females experiencing a third or later one (MULTS), when conditions such as age and season are controlled. For females experiencing a first pregnancy (PRIMS), porphyrin levels and ALA-synthase activity in the Harderian gland rose and fell again during lactation. There was no obvious trend in multiparous females (MULTS). PRIMS and MULTS differed in porphyrin content during mid and late pregnancy and in A L A S activity just after delivery. A L A S activity was also significantly higher in PRIMS than MULTS during late pregnancy in liver and kidney; faecal porphyrin content increased markedly in PRIMS during lactation. Porphyrins are normally stored in the hrnina of the Harderian gland tubules
152
A.P. Payne
as solid deposits. Duyring a first pregnancy there is an increase in abnormaUy--loeated stores. These include i) large interstitial deposits surrounded by foreign body giant oells and ii) small interstitial deposits in free maerepheges. Mast cell r~mbers also increase. These histdegical differences are maintained in non-pregnant MULTS. These data suggest that pregnancy and lactation cause rises in porphyrin synthesis in pr~nigravid females, but that the consequences are not so marked in multiparous ones. [Supported by SERC Grant No. GR/B 83049 and The Scottish Home and Health Vacation Grants].