MICROSCOPY RESEARCH AND TECHNIQUE 37:572–582 (1997) Postnatal Development of the Harderian Gland in the Rabbit: Light and Electron Microscopic Observations KAZUHIKO SHIRAMA,1* SATOSHI OZAWA,2 YOUSUKE SEYAMA,2 MASANORI KOBAYASHI,1 SHOHETSU SAWAMURA, 1 AND JINZO YAMADA1 1Department of Anatomy, Tokyo Medical College Shinjuku-ku, Shinjuku, Tokyo 160 Japan of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo 113, Japan 2Department KEY WORDS Harderian gland; development; morphology; rabbit ABSTRACT We have investigated the development of the Harderian glands of Japanese white rabbits from birth to 4 months of age. Although two types of secretory cells comprise the glandular epithelium of the pink and white lobes in fully developed glands, the time of neonatal appearance is different between the two. Cells consisting of the pink lobe first appear on the third day of life, while cells of the white appear around seventh day of life. The ultrastructure of the Harderian glands from 1-week-old rabbits resembles that of adult animals. The gland can be divided into three parts on the basis of their epithelial cell composition at the electron microscopic level. The respective parts are composed of: (1) one type of cells with large vacuoles (pink lobe), (2) one type of cells with small vacuoles (white lobe), and (3) two types of cells with large and small vacuoles (pink-white mixed portion). The relative number of plasma cells per 1 mm2 is low in both pink and white lobes during early postnatal life. However, in adult animals, the white lobe has a larger number of plasma cells than the pink lobe. These results suggest the possibility that the white lobe participates in the immune system more than does the pink. Microsc. Res. Tech. 37:572–582, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION The Harderian gland is a large orbital gland present in most terrestrial vertebrates which possess a nictitating membrane. Although it plays a role in the lubrication of the cornea, there is evidence suggesting involvement in other functions such as pheromone production (Payne, 1977), thermoregulation (Thiessen and Kittrel, 1980), indole production (Menéndez-Pláez, 1990; Reiter, 1989) and growth factor synthesis (Yokoyama et al., 1989). At this time, however, its function(s) is not yet clear. To date, several morphological studies on the Harderian gland have been reported in many species, according to which it differs in structure. In rodents such as mouse, rat and hamster, the glands consist of two secretory cell types (type A and B or type I and II), the usual difference being a significant disparity in the size of lipid vacuoles. The cell with large vacuoles is termed type A in mouse (Shirama and Hokano, 1991; Strum and Shear, 1982; Watanabe, 1980) and in rat (Brownscheidle and Niewenhuis, 1978), or type II in hamster (Bucana and Nadakavukaren, 1973; Hoffman, 1971; Woolley and Woreley, 1954), while the cell with small vacuoles is termed type B or type I. In the rabbit, two distinct lobes (pink and white) can be identified; both produce lipoidal secretions although their epithelial cells have different morphocal characteristics. The vacuoles in the cells of the pink lobe are large, while those of the white lobe are small (Björkman et al., 1960; Jost et al., 1974; Kühnel, 1971, 1992; Winterhager and Kühnel, 1983). Even though the gland is composed of two cell types in both species, the localization of these cells are respectively different. Two r 1997 WILEY-LISS, INC. cell types are completely separated in the rabbit Harderian gland but are intermingled throughout the gland in rodents. However, some authors have found that besides these lobes, a pink-white mixed area can be distinguished around the boundary between the two lobes on the basis of the epithelial cell composition (Björkman et al., 1960; Kühnel, 1971, 1992; Shirama et al., 1996; Winterhager and Kühnel, 1983). The two cell types in this portion are intermingled in an alveolus like that which occurs in rodent Harderian glands. It has been demonstrated that mast cells were present in the Harderian glands of the Syrian hamster (Menéndez-Peláez et al., 1992; Payne et al., 1982) and the mouse (Shirama et al., 1988b). The cells showed marked sex differences in these counts. In addition to mast cells, plasma cells also occurred in the interstitium of the rabbit Harderian gland (Shirama et al., 1996). In the present work, the development of the rabbit Harderian gland was studied, especially its epithelial cell types and its plasma cell counts. MATERIALS AND METHODS Japanese white rabbits (Oryctolagu cuniculus) were obtained from a commercial colony (Sankyo Labo Service Corporation, Tokyo, Japan). They were housed in stainless steel cages with food and water available ad Contract grant sponsor: Ministry of Education, Science and Culture; Contract grant number: 05670029. *Correspondence to: Dr. K. Shirama, Department of Anatomy, Tokyo Medical College, Shinjuku-ku, Shinjuku, Tokyo 160, Japan. Received 30 May 1996; accepted 27 January 1997. RABBIT HARDERIAN GLAND Fig. 1. Photomicrograph of the Harderian gland from a 0-day-old rabbit, showing some alveoli. Toluidine blue (epon section) 3240. Fig. 2. Photomicrograph of the Harderian gland from a 3-day-old rabbit, showing some alveoli. The vacuoles which seem to be those found in the pink lobe first appear in the cytoplasm. Toluidine blue (epon section) 3240. 573 Fig. 3. Photomicrograph of the Harderian gland from a 1-week-old rabbit, showing the white lobe. Note the glandular epithelial cells with large vacuoles. Toluidine blue (epon section) 3240. Fig. 4. Photomicrograph of the Harderian gland from a 1-week-old rabbit, showing a white lobe. Note the glandular epithelial cells with small vacuoles. Toluidine blue (epon section) 3240. 574 K. SHIRAMA ET AL. Fig. 5. Electron micrograph of the Harderian gland from a 0-dayold rabbit showing the glandular cells and myoepithelial cells. Various stages of mitoses are frequent at this stage of development (arrow). A few short microvilli are seen in the lumen (L). 32,700; bar: 5 µm. Fig. 6. Electron micrograph of the Harderian gland from a 0-dayold rabbit, showing the basal portion of the glandular epithelial cells (G). Note the myoepithelial cell (ME) which is ovoid (but not laminar) in shape with no contractile fibrils. Large arrow, nerve fibers; small arrows, desmosomes; arrowheads, basal lamina. 39,400, bar: 2 µm. 575 RABBIT HARDERIAN GLAND Fig. 7. Electron micrograph of the Harderian gland from a 3-dayold rabbit, showing the glandular cells. The vacuoles which seem to be those found in the pink lobe first appeared in the cytoplasm. Mitosis (arrow) which shows telophase is found within the glandular cell libitum, and were kept in a light- and temperaturecontrolled room (12 hour light-12 hour dark; 24 6 2°C). To examine the development of the Harderian glands, male and female rabbits were anesthetized with Nembutal and sacrificed by intracardial injection of Nembutal at 0, 3, 7, 14, 28, and 120 days of age (comprising four rabbits each). At autopsy, the Harderian glands were removed and fixed as quickly as possible after death. Portions of the Harderian glands which were collected for electron microscopic evaluation were fixed by immersion in 3.0% buffered glutaraldehyde (0.1 M cacodylate buffer, pH 7.3) for 2 hours at room temperature. The specimens were transferred to 0.1 M cacodylate buffer for 2 hours and then postfixed for 3 hours in 1.0% osmic acid in 0.1 M cacodylate buffer. After further washing in buffer, the tissues were dehydrated in graded ethanol concentrations and embedded in Quetol 812. Semithin sections were cut and stained with toluidine blue and used to study the number and morphological features of Harderian gland plasma cells. Four sections were taken from the tissue block of each animal. For each section, the number of plasma cells per mm2 was recorded at a final magnification of 31,000 in a Olympus Labophot. The groups were compared by a one-way analysis for variance (F test). Ultrathin sections (50–70 nm) of selected regions of the epon-embedded materials were then cut with a diamond knife, mounted on uncoated grids and stained with uranylacetate and lead citrate before being examined with a H-7000 (Hitachi) transmission electron microscope. mass. Glandular cells contain large numbers of mitochondria and well-developed rough endoplasmic reticulum within their cytoplasm. 32,700; bar: 5 µm. RESULTS In light microscopic examinations of toluidine bluestained epoxy sections, the Harderian gland at the day of birth appeared as groups of alveoli. Each group was composed of 4–10 alveoli which varied in shape and were often still nonluminated (Fig. 1). On the third day, the gland developed and a large number of alveoli (8–15) formed, some of which were luminated (Fig. 2). On the seventh day of neonatal development, two portions could be discerned by naked eye, and characterized on the basis of their color as the pink and white lobes. The pink lobe was about twice the size of the white lobe, reflecting the adult gland. Conspicuous changes in the morphology of the Harderian gland became evident: there was a pronounced increase in the diameters of alveoli. The two cell types comprising the acinus had completed their characteristic feature by this time (Figs. 3, 4). The 2-week postnatal Harderian gland appeared to be almost fully developed; subsequent changes occurred mainly in the interstitium. Plasma cells were first noted in both lobes at around the second week after birth. After 4 months, the white lobe contained a larger number of plasma cells than did the pink lobe (Figs. 14, 15). In electron microscopic observations, one type of glandular epithelium as well as myoepithelial cells were observed in the Harderian gland of 0-day-old rabbits (Figs. 5, 6). The epithelial cells were small and contained small numbers of vacuoles within their highdensity cytoplasm, apparently due to greater concentra- 576 K. SHIRAMA ET AL. Fig. 8. Electron micrograph of the Harderian gland from a 7-day-old rabbit, showing the white lobe. Note the shape and size of the vacuoles. Cell mass formed by mitoses at alveolar walls is forming a new appositional and continuing alveoli in collaboration with actively generated fibroblasts beneath the alveoli (arrows). 33,400; bar: 5 µm. tion of ribonucleo-protein per unit area. The vacuoles had a maximum diameter of about 1 µm and their shape was spherical or ovoid. The cytoplasm contained abundant round mitochondria, measuring up to 1 µm in diameter. Various stages of mitoses in the glandular epithelial and myoepithelial cells were frequent at this stage of development. Myoepithelial cells occurred in loose contact between glandular epithelial cells with basal lamina (Fig. 6). On the third day after birth, the vacuoles further increased in diameter, measuring about 3 µm while maintaining their earlier shape. On the basis of vacuole size, these cells appeared to be the cells comprising only the pink lobe, none being seen in the white lobe (Fig. 7). From this stage the number of alveoli actively increased. The process of alveolar formation progressed as follows: (1) a part of an epithelial bud with basal lamina was constricted by partial reproduction of connective tissue cell elements, and/or proliferation and growth of an epithelial bud toward connective tissue were constricted by myoepithelial cell contraction; (2) cells comprising the alveolus increased in number (mitosis); and (3) a lumen was newly formed within the cell mass and became a continuation of the original one (Fig. 8). The two cell types characterized by the large or small vacuoles within the cytoplasm were first noted on the seventh postnatal day. As each type of vacuole was still developing, they were smaller than that of adults (large vacuoles: 4.5 µm; small vacuoles: 1.4 µm). Each size was about 3 µm or 1 µm in diameter, respectively. The glandular epithelium of both the pink and white lobes was characterized by a large number of secretory vacuoles occupying most of the cytoplasm. The mitochondria were numerous, small and either spherical or elongated. The Golgi complexes were prominent at the juxtanuclear region. Numerous profiles of rough endoplasmic reticulum were scattered within the cytoplasm. By 2 weeks after birth, the vacuoles in both cell types had developed further with diameters of 4.0 µm (pink lobe) and 1.2 µm (white lobe). At the same time, the development of the other cell organelles in both types of cells was that of 7-day-old animals. At the fourth week, RABBIT HARDERIAN GLAND 577 Fig. 9. Electron micrograph of the Harderian gland from a 4-month-old rabbit showing the pink (P) and white (W) lobes. 32,300; bar: 5 µm. the sizes of both types of vacuoles reached adult levels. At this stage, the Golgi complexes and the profiles of endoplasmic reticulim were diminished in size and number, and were obscure in the cells of the pink lobe, while being prominent in the cells of the white lobe. In the pink lobe of adult rabbits, each alveolus was composed of a single layer of columnar secretory cells. Most of the vacuoles in cells lining the pink acini appeared to be completely empty with a maximum diameter of 4.5 µm. Some small vacuoles (0.5–1.0 µm) contained a heterogenous fibrilar or stringy component which was often densely stained. An electron-dense substance was frequently found around a portion of the periphery of the vacuoles, giving them a crisp boundary. In the cells of the white lobe, the vacuoles were much smaller than those in the cells of the pink lobe. These were found especially in the supranuclear region of the cytoplasm with a maximum diameter of about 1.4 µm and of irregular shape. Some small vacuoles contained varying amounts of a heterogeneous and electronopaque material within their center or along the periphery. The cytoplasm in both types of cells had numerous mitochondria that contained an electron-dense matrix. The matrix rendered them easily identifiable against the less dense ground cytoplasm. The Golgi complexes and rough endoplasmic reticulum were well developed in the cells of the white lobe, but not in those of the pink lobe (Figs. 9–11). In addition to the pink and white lobes, a pink-white mixed portion could be distinguished around the boundary between the two lobes on the basis of the epithelial cell composition by light microscopy (Fig. 12). This portion was first noted around the seventh day. The morphological feature of this portion differed from that of other areas, there being two secretory cell types. One was similar to that found in the pink lobe, while the cell type of the second was like that of the white lobe. The two cell types in this portion were intermingled in the alveolus forming the boundary between the two (Fig. 12). In both lobes, cored vesicles were noted in the apical portion of glandular cells (Fig. 13). The apical cell membrane showed only the development of small, poorly developed and scattered microvilli in both types of secretory cells through all of the developmental stages. Lateral and basal cell membranes showed some interdigitation. Cell apices were 578 K. SHIRAMA ET AL. Fig. 10. A high power electron micrograph of the glandular cells in the pink lobe. Note the cells with numerous mitochondria and large vacuoles. Some small vacuoles contain a peripheral ring of densely stained material (arrowheads). Arrow shows junctional complex elements. N, nucleus. 314,000; bar: 1 µm. Fig. 11. A high power electron micrograph of two adjacent glandu- lar cells in the white lobe. The cytoplasm shows numerous secretory vacuoles. The vacuoles have irregular shapes. The Golgi complexes are very prominent at the juxtanuclear region. Numerous profiles of RER are scattered within the cytoplasm. Compare to that in Figure 10. Arrow indicates junctional complex elements. N, nucleus. 314,000; bar: 1 µm. RABBIT HARDERIAN GLAND 579 Fig. 12. Photomicrograph of the Harderian gland from a 4-monthold rabbit showing the pink-white mixed portion. The morphological feature of this portion differed from that of other areas. The two cell types in this portion were intermingled in the acini as observed in the rat, mouse or hamster Harderian gland. 3240 (original magnification). Toluidine blue (epon section). Fig. 13. Electron micrograph showing the glandular cells of a pink-white mixed portion of the Harderian gland (4-month-old rabbit). The alveoli of this portion consist of two secretory cell types. One is similar to that found in the pink lobe (P); the cells of the other are like those of the white (W). L, lumen; ME, myoepithelial cell; large arrow; nerve fibers; small arrows, coated vesicles; arrowheads; desmosomes. 34,800; bar: 5 µm. Inset: Higher magnification of an area presenting ‘‘coated vesicles.’’ joined by typical tight junctional complexes, and desmosomes were found scattered between adjacent cell membranes helping to maintain the contiguous epithelial lining of each secretory unit. Each microvillus of the secretory cells in both lobes had about 20 actin filaments which appeared to form a most highly organized cytoskeletal apparatus. Microvilli from the cells of the white lobe, however, differed from that of the pink lobe 580 K. SHIRAMA ET AL. Fig. 14. Electron micrograph of the Harderian gland from a 4-month-old rabbit showing the white lobe. Note some plasma cells (P) with expanded RER. ME, myoepithelial cell. 33,600; bar: 10 µm. in the amount of surface coat that consisted of mucopolysuccharide. The surface coat of the microvilli in the white lobe was thicker than that in the pink lobe, suggesting functional differences between them. Myoepithelial cells were located between the secretory epithelium and its basal lamina. The cells were ovoid during the early postnatal stage, and became a series of plates as development proceeded. The cytoplasm of myoepithelial cells had a distinct feature, the occurrence of fine fibrils which are considered to be mainly characteristic of contractile cells. The boundary between the myoepithelial cell and overlying secretory cell was usually a smooth one formed by the apposition of plasma membranes of the two cell types with rarely any intervening intercellular space, though the boundary between the two cell types had a tendency to be loosely bound during the early postnatal stage (Figs. 6, 14). DISCUSSION The postnatal development of the rabbit Harderian glands has received little attention. From our study, it seems clear that the Harderian glands from 7-day-old rabbits show basically similar morphological features to those of adult animals. The gland can be divided into two parts on the basis of their epithelial cell compositions at the electron microscopic level. Their epithelial cells differ morphologically, the vacuoles in the cells of one part being large, while those of the other part, small. A mixed part can be also noted around the boundary between the two at this time. In rodents, two types of secretory cells designated type A (type II) and type B (type I), comprise the glandular epithelium in fully developed glands (rat: Grafflin, 1942; mouse: Woodhouse and Rhodin, 1963; hamster: Christensen and Dam, 1953; gerbil: Johnston et al., 1983). The Harderian glands of the rabbit as well as many rodents manufacture porphyrins (porphyrin-lipid complex). The present authors have studied the development of the Harderian gland during immediate postnatal stages in the mouse to discover whether porphyrins are synthesized by type A or type B cells (Shirama and Hokano, 1991). We demonstrated that the time of the first appearances of the two cell types differ between them, the time in type A cells being the fifth day, while that in RABBIT HARDERIAN GLAND 581 Fig. 15. Relative number of plasma cells observed in the Harderian gland of rabbits during postnatal development. **P , 0.01 compared to the pink lobe. the type B cells, around the seventh day, corresponding to the time at which porphyrins are first detected. These results suggested that the Harderian gland porphyrins seem to be generated by type B cells which appeared around the seventh day. In the rabbit, the time of neonatal appearance was also different between the two cell types on the basis of their vacuole sizes. Cells comprising the pink lobe first appear on the third day, while cells of the white lobe appear around the seventh day. Moreover, from the fact that the white lobe contains more porphyrins than the pink, though the levels are not high (Davidheizer and Figge, 1958), it seems most likely that porphyrins are mainly generated by cells with small vacuoles (cells consisting of the white lobe) in the rabbit as found in the mouse. In the Syrian hamster, it is well documented that the Harderian glands show a strong sexual dimorphism which seems to be under androgenic control (Clabough and Norvell, 1973; Hoffman, 1971; Payne et al., 1977). The Harderian glands of the female hamster possess a single secretory cell type (type 1) which is characterized by small vacuoles, whereas the glands from male hamsters show two secretory cell types, type 1 (similar to the female type cell) and type 2 cells which are filled with large vacuoles (Clabough and Norvell, 1973; Hoffman, 1971; Payne et al., 1977). The cell with large vacuoles (type 2 cell) in the hamster is first noted at around the fourth week corresponding to the time at which hamsters reach puberty (Lopèz et al., 1992). In the present study, the composition of two cell types in the rabbit Harderian glands was the same between male and female throughout the whole experimental period. No sexual differences were observed either in the pink-white mixed portion consisting of two cell types in each alveolus as occurs in male hamsters. Although the Harderian gland porphyrins in the C3H / He strain of mice show a marked sex difference which is controlled by androgenic substances, the difference was not manifest in the morphological characteristics of the gland (Shirama et al., 1981). Thus, it remains necessary to elucidate whether or not the lipid compositions or porphyrin levels in the rabbit Harderian glands show any sexual dimorphism. Generally, a few plasma cells, lymphocytes, mast cells and macrophages occurred in the interstitial tissue of the mammalian Harderian gland (Antolı́n-González et al., 1993; Payne et al., 1982; Shirama et al., 1988ab; Tolivia et al., 1992). In contrast, in birds, a large number of plasma cells are present in the interstitial tissue of the Harderian gland (Bang and Bang, 1968). The number of plasma cells depends on both age (Dohms et al., 1981; Wight et al., 1971) and on the bursa of Fabricius (Kowalski et al., 1978; Mueller et al., 1971). There are very few plasma cells present in the Harderian gland of newly hatched chickens but they increase in number as a bird ages. Starting at 2 weeks, the plasma cells infiltrate the gland from the bursa of Fabricius (Mansikka et al., 1990) and at about 1 month after hatching, large numbers of plasma cells are found in the interstitium (Neidorf and Wolters, 1978). In the rabbit Harderian gland, plasma cells increased in number with age. However, it is not clear whether or not the plasma cells in the mammalian Harderian gland have proliferative potency or from where the cells derive, 582 K. SHIRAMA ET AL. since the bursa of Fabricius is absent in mammals. Thus, it will be necessary to demonstrate that the plasma cells would increase in number if the animals are treated with antigens. ACKNOWLEDGMENTS We thank Mr. Tohru Satoh for his excellent technical assistance. REFERENCES Antolı́n-González, I., Urı́a, H., Tolivia, D., and Menéndez-Peláez, A. (1993) The Harderian gland of the rodent Octodon degus: A structural and ultrastructural study. Tissue Cell, 25:129–139. Bang, B.G., and Bang, F.B. (1968) Localized lymphoid tissues and plasma cells in paraocular and paranasal organ system in chickens. Am. J. Pathol., 53:735–751. Björkman, N., Nicander, L., and Schantz, B. (1960) On the histology and ultrastructure of the Harderian gland in rabbits. Z. Zellforsch. Mikrosk. Anat., 52:93–104. Brownscheidle, C.M., and Niewenhuis, R.J. (1978) Ultrastructure of the Harderian gland in male albino rats. Anat. Rec., 190:735–754. Bucana, C.D., and Nadakavukaren, M.J. (1973) Ultrastructural investigation of the post-natal development of the hamster Harderian gland. II. Male and female. Z. Zellforsch. Mikrosk. Anat., 142:1–12. Christensen, F., and Dam, H. (1953) A sexual dimorphism of the Harderian gland in hamsters. Acta Physiol. Scand., 27:333–336. Clabough, J.W., and Norvell, J.E. (1973) Effects of castration, blinding, and the pineal gland on the Harderian glands of the golden hamster. Neuroendocrinology, 12:344–353. Davidheiser, R.H., and Figge, F.H.J. (1958) Comparison of porphyrinproducing enzyme activities in Harderian glands of mice and other rodents. Proc. Sci. Exp. Biol. Med., 97:775–778. Dohms, J.E., Lee, K.P., and Rosenberger, J.K. (1981) Plasma cell changes in the gland of Harder following infectious bursal disease virus infection of the chicken. Avian Pathol., 24:683–695. Grafflin, A.L. (1942) Histological observations upon the porphyrinexcreting Harderian gland of the albino rat. Am. J. Anat., 71:43–64. Hoffman, R.A. (1971) Influence of some endocrine glands, hormones and blinding on the histology and porphyrins of the Harderian glands of golden hamsters. Am. J. Anat., 132:463–478. Johnston, H.S., McGady, J., Thompson, G.G., Moore, M.R., and Payne, A.P. (1983) The Harderian gland, its secretory duct and porphyrin content in the mongolian gerbil (Meriones unguiculatus). J. Anat., 137:615–630. Jost, U., Kühnel, W., and Schimassek, H. (1974) A morphological and biochemical analysis of the Harderian gland in the rabbit. Cytobiologie, 8:440–456. Kowalski, W.J., Malkinson, M., Leslie, G.A., and Small, P.A. (1978) The secretory immunological system of the fowl. IV. The effect of chemical bursectomy on immunoglobulin concentration in tears. Immunology, 34:663–667. Kühnel, W. (1971) Struktur und Cytochemie der Harderschen Drüse von Kaninchen. Z. Zellforsch. Mikrosk. Anat., 119:384–404. Kühnel, W. (1992) Morphology of the Harderian gland in the rabbit. A short review. In: Harderian Glands. S. Webb, R.A. Hoffman, M.L. Puig-Domingo, and R.J. Reiter, eds. Springer Verlag, pp. 109–125. López, J.M., Tolivia, J., and Alvarez-Urı́a, M. (1992) Postnatal development of the Harderian gland in the Cyrian golden hamster (Mesocricetus auratus): A light and electron microscopic study. Anat. Rec., 233:597–616. Mansikka, A., Jalkanen, S., Sandberg, M., Granfors, K., Lassila, O., and Toivanen, P. (1990) Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity. J. Immunol., 145:3601–3609. Menéndez-Peláez, A. (1990) Melatonin and other indoles in the rodent Harderian gland: Regulation and physiological significance. In: Advance in Pineal Research. Vol. IV. R.J. Reiter and A. Lukaszyk, eds. John Libbey, London, pp. 75–80. Menéndez-Peláez, A., Mayo, J.C., Sainz, R.M., Perez, M., Antolin, I., and Tolivia, D. (1992) Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters. Anat. Embryol., 186:91–97. Mueller, A.P., Sato, K., and Glick, B. (1971) The chicken lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody producing cells. Cell Immunol., 2:140–152. Niedorf, H.R., and Wolters, B. (1978) Development of the Harderian gland in the chicken: Light and electron microscopic observations. Invest. Cell Pathol., 1:205–215. Payne, A.P. (1977) Pheromonal effects of Harderian gland homogenates on aggressive behaviour in the hamster. J. Endocrinol., 73:191–192. Payne, A.P., McGadey, J., Moore, M.R., and Thompson, G.G. (1977) Androgenic control of the Harderian gland in the male golden hamster. J. Endocrinol., 75:73–82. Payne, A.P., McGadey, J., Johnston, H.S., Moore, M.R., and Thompson, G.G. (1982) Mast cells in the hamster Harderian gland: Sex difference, hormonal control and relationship to porphyrin. J. Anat., 135:451–461. Reiter, R.J. (1989) Melatonin: Its sources, its message and the interpretation of the message. In: Advance in Pineal Research. Vol. III. R.J. Reiter and S.F. Pang, eds. John Libbey, London, pp. 165–173. Shirama, K., Furuya, T., Takeo, Y., Shimizu, K., and Maekawa, K. (1981) Influences of some endocrine glands and of hormone replacement on the porphyrins of the Harderian glands of mice. J. Endocrinol., 91:305–311. Shirama, K., Harada, T., Kohda, M., and Hokano, M. (1988a) Fine structure of melanocytes and macrophages in the Harderian gland of the mouse. Acta Anat., 131:192–199. Shirama, K., Kohda, M., and Hokano, M. (1988b) Effects of endocrine glands and hormone replacement on the mast cell count of the Harderian gland of mice. Acta Anat., 131:327–331. Shirama, K., and Hokano, M. (1991) Electron-microscopic studies on the maturation of secretory cells in the mouse Harderian gland. Acta Anat., 140:304–312. Shirama, K., Satoh, T., Yokoyama, Y., Kano, K., Kitamura, T., and Yamada, J. (1996) Ultrastructural study on the Harderian gland of the rabbit (Oryctolagus cuniculus). Folia Morphol., 55:133–141. Strum, J.M., and Shear, C.R. (1982) Harderian glands in mice: Fluorescence, peroxidase activity and fine structure. Tissue Cell, 14:135–148. Thiessen, D.D., and Kittrell, E.M.W. (1980) The Harderian gland thermoregulation in the gerbil (Merione unguiculatus). Physiol. Behav., 24:417–424. Tolivia, D., Antolı́n, I., Menéndez-Peláez, A., and Rodorı́guez-Colunga, M.J. (1992) Lymphoid cells in the Harderian gland of the rodent, Octodon degus. Anat. Rec., 234:438–442. Watanabe, M. (1980) An autoradiographic biochemical and morphological study of the Harderian gland of mouse. J. Morphol., 163:349– 365. Wight, P.A.L., Burns, R.B., Rothwell, B., and MacKenzie, G.M. (1971) The Harderian gland of the domestic fowl. I. Histology, with reference to the genesis of plasma cells and Russell bodies. J. Anat., 110:307–315. Winterhager, E., and Kühnel, W. (1983) Membrane specializations of the cells of the Harderian gland of the rabbit with particular reference to the mechanism of exocytosis. Cell Tissue Res., 231:623– 636. Woodhouse, M.A., and Rhodin, J.A.G. (1963) The ultrastructure of the Harderian gland of the mouse with particular reference to the formation of its secretory product. J. Ultrast. Res., 9:76–98. Woolley, G.W., and Worley, J. (1954) Sex dimorphism in the Harderian gland of the hamster (Cricetus auratus). Anat. Rec., 118:416–417. Yokoyama, Y., Kano, K., Kaji, K., and Seyama, Y. (1989) Purification and characterization of a growth factor from guinea-pig Harderian gland. J. Biol. Chem., 264:17058–17063.