Postnatal development of the harderian gland in the Syrian golden hamster Mesocricetus auratusA light and electron microscopic study.код для вставкиСкачать
THE ANATOMICAL RECORD 233597-616 (1992) Postnatal Development of the Harderian Gland in the Syrian Golden Hamster (Mesocricetus auratus): A Light and Electron Microscopic Study JOSB M. L6PEZ, JORGE TOLIVIA, AND MANUEL ALVAREZ-URfA Bepartamento de Morfologia y Biologia Celular, Facultades de Biologia y Medicina, Universidad de Oviedo, Ouiedo, Spain ABSTRACT The main objective of the present investigation was to study the morphological and chronological aspects of the development of the Harderian gland in the Syrian golden hamster. Tissues were obtained from male and female hamsters at days 1,3,5,7,10,12,15,17,20,27,37,46,and 90 after birth and processed for light and transmission electron microscopy. The present observations indicate that a well-defined temporal sequence in microscopic and ultrastructural modification is recognizable in the development of the hamster Harderian gland. Four stages of development were proposed. Between days 1-5 (first stage), the gland shows characteristics of an immature structure. The glandular cells contain many free ribosomes, few and small organelles, and large irregular-shape nuclei. Between days 7-17 (second stage), there is a marked increase of organelles involved in synthesis and secretion. The gland begins the secretion of lipids and porphyrins, but no morphological differences between male and female glands are observed. Between days 20-36 (third stage), the morphological differences between the two sexes appear and progressively develop. In 45-day-old hamsters, the Harderian gland possesses the structural characteristics of adult glands, and further developmental changes are essentially quantitative in nature (fourth stage). At all stages of development, the population of secretory cells has a uniform appearance. The morphological results are discussed as well as the possible relationship of this temporal sequence with hormonal changes. o 1992 Wiley-Liss, Inc. The Harderian gland (HG) is a large, compound, tubulo-alveolar gland located in the orbital cavity of most terrestrial vertebrates possessing nictitating membranes (Olcese and Wesche, 1989). In mammals, it is especially prominent in rodents, where it is usually larger than the orbital globe itself. However, the HG is not present in primates, terrestrial carnivores, or chiropterans (Sakai, 1981). In rodents, this gland is one of the most active sites of both porphyrin and lipid biosynthesis known (Davidheiser and Figge, 1955; Margolis, 1971; Jost et al., 1974). Porphyrin amounts are visible as brown pigment accretions within the alveolar lumens (Kennedy, 1970; Johnston et al., 1985). In addition to lipids and porphyrins, it is believed to synthesize methoxyindoles, mainly melatonin (Pang et al., 1976; Bubenik et al., 1976a,b). The fact that the HG could synthesize three such different compounds is remarkable, taking into account that in some rodents (e.g., female hamsters), the gland has only one secretory cell type and thus, presumably, the three products are synthesized by the same cell. In spite of its relatively large size and the presence of potentially important chemical compounds, the functional role of this gland remains unknown. It may lubricate the eye (Davis, 1929; Cohn, 19551, transmit photic information to the pineal gland in neonatal rats (Wetterberg et al., 1970a,c), modulate reproductive 0 1992 WILEY-LISS. INC function (Hoffman, 1971), produce pheromones (Thiessen et al., 1976; Payne, 19771,or play a role in temperature regulation (Thiessen et al., 1977). Due to the presence of melatonin, the HG may also be involved in the functioning of the pineal-hypothalamic-pituitarygonadal axis (Hoffman et al., 1985). Nevertheless, despite the observation of a circadian rhythm in the activity of HG melatonin-synthesizing enzymes (Pevet et al., 1980; Balemans et al., 1983; Menendez-Pelaez et al., 1987a, 1988, 19891, the existence of endocrine secretion has not been demonstrated. The HG of the adult Syrian golden hamster is particularly interesting as it exhibits a marked sexual dichotomy in morphology and secretory activity. Glands of males contain no observable pigment accretions, low porphyrin concentration, and two cell types: type I, containing numerous small vacuoles, and type 11, containing large vacuoles. In female glands, only cells containing small vacuoles are present (type I), and the lumens Received August 19, 1991; accepted December 24, 1991. Address reprint requests to Dr. Jose M. L6pez, Dept. Morfologia y Biologia Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo 33006, Spain. 598 J.M. LOPEZ ET AL. Fig. 1. Frontal section of the whole head of a newborn female hamster showing the arrangement of the Harderian gland (arrows). Note the relatively large size of the gland. x 20. Fig. 3. Semithin section of the Harderian gland of a newborn male hamster showing light and dark cells. Note the dark cell in mitosis (arrow). x 590. Fig. 2.Parafin section of the Harderian gland of a newborn male hamster. Note that the secretory tubules are widely separated by connective tissue and have narrow lumina. x 160. contain concretions of a pigmented material chemically identified as porphyrin (Christensen and Dam, 1953; Woolley and Worley, 1954; Hoffman, 1971; Payne et al., 1975). Ultrastructurally, Bucana and Nadakavukaren (1972a) reported the presence of clusters of cylindrical tubules in secretory cells of only the males, whereas numerous membrane formations in the secretory cells of only the females were observed. It has been well documented that this sexual dimorphism is influenced by gonadal hormones (Woolley and Worley, 1954; HARDERIANGLANDDEVELOPMENT 599 Fig. 4. Low power electron micrograph of the Harderian gland of a many polysomes, Golgi complexes, and mitochondria. Note the exisnewborn female hamster. Note the presence of electron-dense and tence of intercellular junctions between the cells (arrows). x 6,350. electron-lucent cells. The cells are loosely apposed showing many intercellular spaces (arrowheads). Note the existence of two luminal Fig. 6. Electron micrograph of an epithelial cell from a newborn spaces (asterisks) and a dark cell undergoing mitosis (arrow). x 3,310. female hamster gland. Note the existence of a continuous basal lamina (arrows). The intercellular spaces contain small podia-like projections (arrowheads). x 10,400. Fig. 5. Electron micrograph of a n epithelial cell from a newborn male hamster gland. The cytoplasm contains a few cisternae of RER, 600 J.M. LOPEZ ET AL. Fig. 7. Light and dark cells in a newborn male hamster gland. Note the presence of many polysomes and abundant mitochondria in both cells. No evident differences are observed between them in the organelle composition. X 14,950. Fig. 8.Region of cytoplasm from a newborn female hamster glandular cell containing Golgi and a large number of vesicles. x 19,650. Fig. 9. Membrane-bound structure resembling a n autophagic or heterophagic vacuole in close proximity with the nucleus in a glandular cell from a newborn female hamster. Note the existence of an extensive Golgi complex (arrows). x 8,880. Fig. 10. Membrane-bound structure resembling that of Figure 9 in a dark cell from a newborn male hamster. Note the existence of irregular clumps of electron-dense material. x 11,OOOX. Fig. 11. Two membrane-bound structures containing disorganized organelles within a glandular cell from a newborn male hamster. x 15,720. Hoffman, 1971; Payne e t al., 1977; Lin and Nadaka- However, knowledge on the process of differentiation of vukaren, 1979; Sun and Nadakavukaren, 1980; Mc- this gland remains poorly documented. Master and Hoffman, 1984). The present study describes the structural and ultraThe development of the HG in the hamster is a n structural features of the HG during different periods interesting system for studying the factors affect- of Syrian hamster postnatal development in order to: ing regulation of the dimorphism. Furthermore, the (1)characterize the cell modifications associated with gland may act as a n extraretinal photoreceptor in neo- the differentiation of the gland, especially those related natal rats (Wetterberg et al., 1970a1, but no morpho- to both the beginning of the secretory activity and the logical evidence to support this statement has been re- establishment of sexual dimorphism, and (2) establish ported. the precise chronology of the process and discuss posFew reports could be found on the development of the sible parallels with the developmental profiles of difHG in the hamster (Bucana and Nadakavukaren, ferent hormones reported in the literature. 1972c, 1973) or in other mammals (Muller, 1969a,b; The information provided in this study on the normal Wetterberg et al., 1970b). In a previous work, we re- development of the HG in the hamster may serve as a ported the existence of some unusual organelles includ- baseline for further experimental studies on the effect ing lamellar and nucleolus-like bodies in the hamster of different factors (photoperiod, hormonal treatment, HG during the perinatal period (L6pez et al., 1990). etc) on the development of this gland. 601 HARDERIAN GLAND DEVELOPMENT TABLE 1. Area of HG cell profiles (expressed in pm2) at different ages' Age (days) 1 3 5 7 10 12 15 17 20 27 37 46 90 Female 52.39 t 3.92 48.93 i 2.84 55.61 2 3.81 77.18 i 4.92 92.56 f 3.22 130.62 t 5.36 166.72 + 7.69 203.82 f 6.62 248.02 t 8.93 340.36 t 7.62 392.64 i 10.39 417.09 f 11.44 425.46 f 13.16 Male Type I cells Type I1 cells 49.63 f 2.83 50.31 i 4.02 58.44 f 3.67 74.63 f 4.37 99.31 2 2.85 122.41 i 4.89 152.31 + 2.82 192.40 i 7.61 233.50 f 9.06 328.12 f 8.82 316.14 i 9.87 422.09 Ifr 9.22 410.32 f 7.61 448.92 9.63 467.90 t 7.80 452.92 t 8.86 489.36 f 10.02 * lEach point represents the mean of 150 cells (50 per animal) f SEM. TABLE 3. Absolute area of the intracellular lipid vacuole profiles (ex ressed in pm2) at different ages of €fG development' Age (days) 1 3 5 7 10 12 15 17 20 27 37 46 90 Female 0 0 0 0.12 f 0.09 8.80 f 1.12 9.97 f 0.63 16.61 f 1.11 31.41 t 1.22 47.30 f 1.67 88.76 f 1.65 102.49 t 2.83 111.09 f 3.95 126.07 f 2.91 Male Type I cells Type I1 cells 0 0 0 0.21 2 0.11 7.91 f 0.97 12.31 + 0.77 19.23 2 1.07 28.63 f 0.76 52.61 2 1.92 143.39 f 1.93 98.32 f 1.03 112.63 t 1.97 188.61 + 3.07 121.66 t 2.78 212.63 t 3.01 261.91 t 3.69 143.86 t 1.77 'Each point represents the mean of 150 cells (50 per animal) +- SEM. TABLE 2. Percentage of the total cell area occupied by the nuclear profile at different ages of HG development' Age (days) 1 3 5 7 10 12 15 17 20 27 37 46 90 Male Female 52.74 t 3.32 65.03 + 4.81 60.98 2 3.86 41.23 t 6.32 32.54 t 3.16 21.90 2 2.17 20.77 t 2.33 16.72 t 3.61 14.44 t 1.77 9.74 t 1.63 9.27 t 1.12 9.92 i 0.97 9.49 t 1.12 TvDe 1 cells 61.47 t 2.91 57.12 t 2.99 55.83 t 4.01 44.90 t 3.94 34.86 + 4.01 24.64 f 3.23 21.25 t 2.79 15.02 i 2.22 13.04 t 2.08 10.80 t 1.54 8.44 t 1.08 8.70 f 1.13 8.02 2 0.45 TvDe 11 cells Animals were sacrificed on days 1(a few hours after birth), 3, 5, 7, 10, 12, 15, 17, 20, 27, 37, 46, and 90. We used six animals per age (three males and three females) except on days 1 , 7 , and 12, where a n additional animal was used to obtain sections of the whole head. Cycling females were sacrificed always on the day of estrus, when a maximal concentration of porphyrins has been reported (Payne et al., 1979). Histological Procedures 10.09 t 1.79 7.92 2 1.03 7.58 f 0.88 6.63 t 0.93 'Each point represents the mean of 150 cells (50 per animal) f SEM. MATERIALS AND METHODS Animals Adult male and female Syrian golden hamsters (Mesocricetus auratus) were maintained on a 14-hr light: 10-hr dark regimen (light on at 0600). Animals were confined individually in plastic cages in a controlled environment (24 i 1 "C) and provided with food and water ad libitum. Under these conditions, female hamsters had a 4-day estrous cycle. Female hamsters (95110 g) were checked daily (between 09.00 and 10.00 hr) for the characteristic postovulatory discharge. These animals were therefore expected to come into estrus 3 days later. Females in estrus were housed overnight with a male, copulatory behavior of the male (mounting) and female (lordosis) was ascertained, and the pair was separated the following morning. This day was designated as the first day postcoitum (1dpc). The day of delivery was 16 dpc in most animals, but it varied from 15 to 18 dpc. In the present study we used only those animals born on 16 dpc (- 80%). The day of birth was designated as day 1. To determine the time of first ovulation, copulatory behaviour and a postovulatory discharge were checked every day beginning at 27 days of age. Hamsters younger than 10 days of age were sacrified by cervical dislocation; the glands were immediately removed and fixed by immersion for 5 h r at 4°C in 4% glutaraldehyde and 1 mM calcium chloride in 0.5 M cacodylate buffer (pH 7.4). Animals older than 10 days were anaesthetized with ether and perfused via the left ventricle with physiological saline followed by the fixative solution mentioned above. The glands were dissected out of the eye socket after 15 min of perfusion at room temperature and placed in the same fixative for 5 h r at 4°C. The HGs from the right side of each animal were processed for light microscopy. They were rinsed in tap water, dehydrated in a graded ethanol series (u to 80%), embedded in glycol methacrylate (Technovit 71001, and serially sectioned at 3 pm. The sections were stained with thionin (Tolivia and Tolivia, 1988a). To study the spatial relationship between the HG and its surrounding tissues in perinatal stages, serial frontal paraffin sections of the whole head from hamsters 1, 7, and 12 days old were obtained, and stained with a trichrom method (Tolivia and Tolivia, 1988b). For electron microscopy, the left glands were cut into 1-mm3 pieces, rinsed in buffer, postfixed for 2 h r in a solution of 1%osmium tetroxide and 2% potassium ferrocyanide, dehydrated with a graded series of acetone, cleared in propylene oxide, and embedded in Durkupan-ACM (Fluka). Ultrathin sections were cut on a LKB ultramicrotome using glass knives, stained with uranyl acetate and lead citrate, and viewed with a Zeiss EM 109 electron microscope. Some semithin sections 1 pm thick were stained with Ziehl's fuchsin (Tolivia and Tolivia, 1985). E 602 J.M. LOPEZ ET AL. Morphornetry For light microscopic quantification, one methacrylate section was selected from each animal, taking into account the criterion that its profile area was maximal. These sections were examined in a Nikon microscope equipped with a drawing tube. The outlines of sections were drawn a t a magnification of X40. The outlines of all the intraluminal porphyrin accretions in each section were drawn at a magnification of X200. Both the section and porphyrin accretions area were measured using a n automatic picture analyzing system (Kontron Bildanalyse IMCO 10). The area of intraluminal porphyrin accretions was expressed in pm2 of porphyrins per mm2 of tissue section. For ultrastructural quantification, 50 secretory cell profiles of each cell type were randomly selected from each animal. In all cell profiles examined, the nucleus and a portion of both basal and apical membrane were visible. They were photographed and enlarged to a final magnification of X4,OOO. The following parameters were measured in each cell profile: (1)area of the total cell profile, (2) area of the nuclear profile, and (3) area of the intracytoplasmic lipid vacuole profiles. These areas were planimetrically assessed on photographs. In addition, the percentage of secretory cells containing clusters of cylindrical tubules, the distinctive structure of adult male cells described by Bucana and Nadakavukaren (1972a1, was calculated. A mean and its standard error (SEM) were calculated for each parameter on each group of hamsters. RESULTS The Harderian gland (HG) varied appreciably during postnatal life in both histological organization and ultrastructural characteristics of cellular constituents. On the basis of the observations obtained, we have proposed four developmental stages in the maturation process of the hamster HG: a first stage that precedes the appearance of the cytoplasmic lipid vacuoles (days 15); a second stage in which the synthesis and secretion of both lipids and porphyrins begins (days 7-17); a third stage, when the morphological differences between the two sexes appear and develop (days 20-36); and the fourth stage, during which the gland shows the adult aspect, characterized by a n intense secretory activity and a n evident sexual dimorphism (hamsters older than 45 days). Stage 1: Days 1-5 In newborn hamsters, the HG was well defined (Fig. 1). It was cup-shaped and occupied a considerable part of the orbit, in direct contact with the posterior and nasal aspects of the eyeball (Figs. 1, 2). At birth, the gland showed the same localization and relative size to other ocular structures as in adults. Histologically, the gland was composed of branched secretory tubules with narrow lumina, widely separated from each other by loose interacinar connective tissue (Fig. 2). The wall of these tubules was composed of cells destined to differentiate into secretory cells. The cell shape varied from cuboidal to low columnar and the epithelium appeared multilayered. In semithin epoxy sections stained with Ziehl’s fuchsin (Tolivia and Tolivia, 19851, the cells of the tubules could be divided in two types, referred to a s light and dark (Fig. 3). Both the dark and the light cells were similar in height and shape. Mitotic figures were observed in both types. By electron microscopy, the gland showed characteristics of a n immature structure. The epithelial cells were loosely apposed to adjacent cells (Fig. 4).Some luminal epithelial cells were moderately polarized. They showed junctional complexes with zonula occludens on the apical luminal side, and many intercellular spaces where small processes were seen with some small podia-like projections (Figs. 4,5). The intercellular matrix showed low electron-density. The luminal surface presented scattered short microvilli. In the basal portion, it was very difficult to distinguish between prospective secretory and prospective myoepithelial cells (Fig. 4). Prospective secretory cells had large irregular-shape nuclei. Euchromatin was finely dispersed throughout the nucleoplasm, and a few chromatin clumps were associated with the nuclear envelope. Nucleoli were large, irregular, and frequently eccentrically placed. At this period, most of the cells had small size and were characterized by a high nuc1ear:cytoplasmic ratio (Tables 1,2). The cytoplasm contained abundant polysomes, some mitochondria, and a moderately developed Golgi apparatus, but other cellular organelles as the endoplasmic reticulum were very scarce (Figs. 5-8). Polysomes consisted of 5-20 ribosomes arranged in rosette or linear formations. They were evenly scattered throughout the cell cytoplasm, being very evident in the organelle-free areas (Figs. 5,6). Mitochondria were rod-shaped and presented moderately electron-dense matrices and loosely lamellar cristae (Figs. 5-7). The Golgi apparatus consisted of stacks of flattened saccules ending in distentions associated with small vesicular profiles (Fig. 8). The stacks were multiple and not confined to the supranuclear region. The very few cisterns of the endoplasmic reticulum were dilated by amorphous material of moderate electron density and presented attached ribosomes. These cisterns frequently displayed a n intimate spatial relationship to mitochondria (Figs. 6, 7). At this age, a common feature of the secretory epithelial cells was the existence of large spherical membrane-bound structures closely associated with the nucleus (Figs. 9-11). Such structures, which could be visualized in semithin sections, showed a heterogeneous appearance, resembling autophagic or heterophagic vacuoles. Most contained irregular clumps of a n amorphous electron-dense material and some disorganized organelles in a matrix of diffuse finely granular or flocculent electron-lucent material. No difference was seen between male and female glands in the morphology of these structures. The light-dark difference observed at the light microscopic level among the glandular cells also appeared at the ultrastructural level. Based upon the cytoplasm electron density, two kinds of cells were identified: electron-dense and electron-lucent cells (Figs. 4,7). In spite of the difference in electron density, no difference was seen between the two cell types in the organelle composition (Fig. 7). Gradual but not dramatic changes in gland morphology were observed during days 1-5 of the neonatal period. As a result of continued cell proliferation, the ep- 603 HARDERIAN GLAND DEVELOPMENT Fig. 12. Semithin section of the gland of a 5-day-old female hamster. The interstitial connective tissue is scarce. Note that myoepithelial cells stain more intense than luminal epithelial cells (arrows). Severa1 cells are in mitosis (arrowheads). X 350. Fig. 13. Low power electron micrograph of a secretory tubule from a 5-day-old male hamster. Note the existence of small microvilli projecting into the lumen. Three myoepithelial cells are observed a t the base of the tubule (arrows). x 3,720. ithelial tubules branched and increased to a large extent throughout the connective tissue matrix. At growing tips or new branching points, cell proliferations created multilayered thickenings. However, epithelial cells gradually became aligned in two layers and the basal ones differentiated into myoepithelial cells. In 5-day-old hamsters, the arrangement of the secretory tubules was significantly more condensed than in newborn hamsters (Fig. 12). At this period, a low percentage of the tubules showed small lumina. In semithin sections, the myoepithelial cells could be easily identified. They showed oral or elongated nuclei that lay parallel to the basement membrane and stained more intensely than the epithelial cells (Fig. 12). The ultrastructural features of the epithelial cells in 3- and 5-day-old hamsters were basically similar to those described in newborn hamsters. There was a gradual increase in the amount of cytoplasm (Table 1) but their ultrastructural features did not change significantly. In 5-day-old hamsters, the luminal epithelial cells had large moderately irregular nuclei with nucleoli, pale staining euchromatin, and few heterochromatin clumps. Surrounding the nucleus, there was a small amount of cytoplasm that contained an abundance of polysomes, some Golgi complexes, and mitochondria. The mitochondria were increased slightly in number. Luminal epithelial cells became polarized. Polarity was evident in both the characteristics of the plasma membrane and the pattern of intracellular organization. Myoepithelial cells were easily identified at this period. They were located between the luminal epithelial cells and the basement membrane, forming a discontinuous layer around the acini (Fig. 13). Their nuclei were oval or elongated and, occasionally, showed identations. The cytoplasmic organelles were scarce. Stage 2: Days 7-1 7 At postnatal day 7, intracytoplasmatic lipid vacuoles were first observed. They appeared in a small percentage of epithelial cells in both male and female glands (Fig. 14). The appearance of these secretory vacuoles could be considered as morphological evidence of the onset of gland secretory activity. From 7 days of age, the percentage of cells containing lipid vacuoles rapidly increased and after 10 days of age, most of the secretory cells showed lipid vacuoles. Concomitant with the appearance and increase of the lipid vacuoles, there was an enlargement in cell volume and a marked reorganization of the cellular ultrastructure (Tables 1-3). On day 7 the variability in cell morphology was marked, ranging from secretory cells containing several intracytoplasmic lipid vacuoles and a considerable development of the morphological secretory apparatus 604 J.M. LdPEZ ET AL. Fig. 14. Semithin section of the gland from a 7-day-old male hamster. Note the existence of several cells containing lipid vacuoles (arrows). x570. membrane-bound cluster of cylindrical tubules a t lower left (arrows) and cylindrical tubules associated with one of the lipid vacuoles (arrowheads). x 20,610. Fig. 15, Electron micrograph of secretory cells from a 7-day-old female hamster. Note the existence of small lipid vacuoles (arrows). x 5,325. Fig. 17. Region of cytoplasm showing a prominent Golgi complex adjacent to the nucleus from a 7-day-old male hamster. x 24,050. Fig. 16. Detail of a secretory cell from a 7-day-old female hamster showing a small network of typical anastomosing tubules of SER, two cisterns of RER and three lipid vacuoles. Note the existence of a Fig. 18. Electron micrograph of a tubular acinus from a 10-day-old male hamster. Lipid vacuoles are relatively large and moderately abundant. Note the existence of a lipid vacuole in the process of secretion (arrow). x 3,350. to small cells whose fine structural cytoplasmic char- cells showed lipid vacuoles. Cells gradually increased in size, whereas the lipid vacuoles became more numeracteristics resembled those of the earlier stage. Cells containing lipid vacuoles showed changes. ous and arranged in the apical portion of the cell (TaThey had spherical nuclei and a more extensive cyto- bles 1-3; Fig. 18). At this time, the first example of plasm, which contained moderate numbers of free p l y - exocytosis at the luminal surfaces of secretory cells was somes, well-developed Golgi complexes, abundant observed (Fig. 18). The onset of exocytotic activity mitochondria, and a more prominent endoplasmic re- coincided with the observation of the first solid intraluticulum (Figs. 15-17). The endoplasmic reticulum con- minal accretions of pigmented material (possibly porsisted of a small network of smooth tubules and a few phyrins) (Table 4). These small accretions were homogprofiles of rough endoplasmic reticulum (RER). The enous in appearance and quite pale in color. They were scarce lipid vacuoles were preferentially located close only observed in one of the six hamsters studied at this to the nucleus. At this time, no lipid vacuoles were age (a female). By 12 days after birth, the HG had developed conobserved in the process of secreting from the cell into the lumen. The observation of lipid vacuoles coincided siderably and had a more adult pattern. The gland was with the appearance of clusters of cylindrical tubules surrounded by a thin connective tissue capsule and (see Table 5). These tubules, whose diameter was ap- septa from this capsule divided it into unequal-size lobproximately 40 nm, were first observed in close associ- ules. Most of the gland volume was occupied by branchation with lipid vacuoles (Fig. 16).Juxtanuclear struc- ing secretory tubules separated from each other by tures resembling autophagic or heterophagic vacuoles small amounts of connective tissue (Fig. 19). The secretory tubules were formed by a single layer of epithelial were scarce in cells containing lipid vacuoles. At 10 days after birth, 90% of the luminal epithelial cells surrounded by myoepithelial cells surrounding HARDERIANGLANDDEVELOPMENT TABLE 4. Area of intraluminal porphyrin accretion profiies (expressed in pm2 of porphyrins per mm2 of tissue section) at different ages of HG development' 1 3 5 7 10 12 15 17 20 27 37 46 90 Female 0 0 0 0 47.56 +111.52 180.09 270.01 2 903.65 2,192.25 4.260.70 8i203.84 10,511.06 2 Male 0 0 0 0 0 47.56 * 26.39 * 9.58 82.51 2 11.66 197.09 2 6.41 213.19 ?c 15.04 888.92 2 64.88 169.92 2 11.75 37.63 t 19.85 0 0 62.00 * 78.76 * 147.71 * 500.53 * 264.68 626.28 'Each point represents the mean of 3 sections (1 per animal) ? SEM. TABLE 5. Percentage of cells containing clusters of cylindrical tubules at different ages of HG development' Age (days) 1 3 5 7 10 12 15 17 20 27 37 46 90 Male Female 0 0 0 2.00 2 2.67 2 4.00 2 3.33 2 4.00 2 2.00 2 1.33 2 0 0 0 1.15 0.67 1.15 0.67 1.15 1.15 0.67 Type I cells 0 0 0 1.33 2 0.67 4.00 2 1.15 3.33 2 1.33 4.66 t 2.69 5.33 2 0.67 8.00 1.15 16.00 2.31 32.66 3.71 48.66 2 4.67 71.33 +. 4.37 * * * Type I1 cells 1.33 2 0.67 2.67 2 0.67 3.33 2 0.67 2.67 t 1.33 'Each point represents the mean of 150 cells (50 per animal) ? SEM. central lumina of varying width. They contained only one type of glandular cell characterized by the presence of numerous small lipid vacuoles (Fig. 20). The gland contained no histologically specialized ducts within itself, but there was a single extraglandular duct. Some of the secretory tubules began near the hilus and constituted a trunk with a wide lumen that divided into numerous branches (Fig. 19). Small irregular accumulations of pigmented secretion were found in a small number of lumina in glands from both sexes (Table 4). At the electron microscope level, secretory cells were characterized by marked changes in cytoplasm size and structure. Morphometric analysis indicated that the nuclear volume fraction substantially decreased (Table 2). This fact was due to the enlargement of the cytoplasm (Table 1). At this time, the organelles of the cell showed a pronounced development. The Golgi apparatus occupied a n extensive zone around and above the upper pole of the nucleus (Fig. 21). I t consisted of parallel sets of closely packed elongated saccules with associated vesicles. The width of each saccule was between 15 and 30 nm and they varied in length. The cavity of the sac- 605 cules was dilated a t each end to form vesicles that usually had a pale content (see Fig. 23). Mitochondria were numerous, being plentiful throughout the cytoplasm (Fig. 21). The details of their fine structure did not vary from those of earlier stages. They were oval in profile and presented lamellar cristae, which usually extended the full width of the mitochondrion. The mitochondria1 matrix was finely granular and varied from a moderate to a high electron density. At this period, the most remarkable feature of the secretory cells was the extensive development of the endoplasmic reticulum. The cells showed a complex system of vesicles and tubules of smooth endoplasmic reticulum (SER), which constituted a dense network around mitochondria and lipid vacuoles (Fig. 22). Sometimes, the SER tubules seemed to be morphologically related to stacks of the Golgi complex (Fig. 23). By contrast with the SER, the RER was poorly developed, with only occasional stacked profiles. In addition to SER and RER, the cells showed a third specialization of the endoplasmic membrane system consisting of stacks of parallel cisterns (Figs. 21, 24). These structures, identified as lamellar bodies (LBs), have been described in an earlier work (Lopez et al., 1990). Briefly, they were formed by two different types of cisterns: One was often continuous with a surrounding RER cistern and presented a n intraluminal electronlucent material. The second type showed closed marginal dilatations and contained a n homogeneous electron-dense material. These two types of cisterns were alternately arranged in the stacks. Irregular cytoplasmic electron-dense bodies, identified as nucleolus-like bodies (NLBs) or nematosomes, frequently appeared in the secretory cells at this period (Figs. 21, 25). Like the LBs, the NLBs have been described in a previous work (Lopez et al., 1990). The cells contained abundant lipid vacuoles located chiefly in the apical half of the cytoplasm. The vacuoles consisted of a lipid droplet limited by a 2-4-nm-thick electrondense line, surrounded by a triple-layer membrane. The unit membrane was separated from the dense line by a gap of variable size whose electron density resembled that of the cytoplasm (Fig. 26). The content of the lipid droplets appeared electron-lucent, probably due to the action of fat solvents used during embedding. Images of lipid vacuoles in the process of secretion from the cell were frequent (Figs. 20,271. In this process, the lipid vacuole membrane fused with the apical plasmalemma and then broke down, opening the vacuole to the luminal space. The membrane of the vacuole was translocated into the plasma membrane (Figs. 27, 28). Frequently, the intravacuolar space sandwiched between the lipid droplet and the unit membrane contained clusters of cylindrical tubules. In some cases, vacuoles containing tubular clusters were observed in close apposition to the apical plasmalemma (Fig. 29). This localization could be interpreted as the first stage of a n exocytotic process. However, we have never observed such tubules in the luminal space. Occasionally, a cilium was observed extending from the secretory cells at this period (Fig. 30). Epithelial cell proliferation continued through this period. Frequent mitotic figures were observed. Cells in mitosis had a comparable organelle composition to those in interphase, including lipid vacuoles, which 606 J.M. LOPEZ ET AL. Fig. 19. Methacrylate section of the gland from a 12-day-old female hamster. Note the existence of a straight secretory tubules with wide lumen from which some smaller tubules arise (arrows). The secretory tubules are separated from each other by small amounts of connective tissue. ~ 6 0 . Fig. 20. Low power electron micrograph of a secretory tubule from a 12-day-old male hamster showing presence of only one secretory cell type. Note the existence of lipid droplets in the lumen (arrows). Myoepithelial cells lie between secretory cell and basal lamina. x 1,620. Fig. 21. High magnification of a portion of the secretory tubule shown in Figure 21. The secretory cells are characterized by an abundance of SER, mitochondria, and lipid vacuoles. The Golgi complex is well developed and located adjacent to the nucleus (arrows). Note the existence of several lamellar bodies (arrowheads) and an electrondense body (asterisk). Two pale myoepithelial cell processes are observed between the secretory cells and the basal lamina (stars). x 4,860. Fig. 22. High magnification of a glandular cell from a 12-day-old female hamster. The SER constitutes a dense network, which occupies wide areas in the cytoplasm. x 22,670. dense line. Note that the unit membrane surrounding the droplet is separated from the dense line by a gap whose electron density is similar to that of the cytoplasm. x 45,400. Fig. 23. Portion of eytoplasm from a 12-day-old female hamster gland showing a Golgi complex located between a lipid vacuole and membranes of SER. x 23,360. Fig. 27. Vacuole in the process of secretion from the luminal surface of a glandular cell from a 12-day-old male hamster. x 20,500. Fig. 28. High magnification of Figure 27. x 67,000. Fig. 24. Lamellar body in the cytoplasm of a glandular cell from a 12-day-old female hamster. Note the existence of two types of cisterns forming the structure. x 33,500. Fig. 25. Electron-dense body (NLB) in the cytoplasm of a glandular cell from a 12-day-old male hamster. x 29,500. Fig. 26. Lipid vacuole in the cytoplasm of a glandular cell from a 12-day-old male hamster. The lipid droplet is limited by an electron- Fig. 29. Lipid vacuole containing cylindrical tubules in the space sandwiched between the unit membrane and the lipid droplet in a 12-day-old male hamster gland. Note the close proximity between the membrane of the vacuole and the plasmalemma. x 48,000. Fig. 30. Electron micrograph of a secretory cell containing a cilium from a 12-day-old female hamster gland. x 31,780. 608 J.M. LOPEZ ET AL. Fig. 31.Secretory cell showing late telophase from a 12-day-old male hamster. Note that the daughter cells in mitosis have a comparable composition of organelles to their neighbors in interphase. x 5,200. Fig. 31.Portion of a myoepithelial cell from a 12-day-old male hamster gland. Note the filaments near the nucleus (arrows). X 35,100. ences were observed in the amount of intraluminal accretions between the sexes. However, the frequency of cells containing clusters of cylindrical tubules was four times greater in male than in female glands (Table 5). At this age, type I1 cells contained a small number of large lipid vacuoles (3-6 pm in diameter), which seemed to be formed by coalescence of small ones (Fig. 33). In addition to vacuole size, several ultrastructural features of the type I1 cells differed from those of the type I. Like type I, type I1 cells were pyramidal in shape and had round, basally located nuclei, a welldeveloped Golgi complex, and abundant mitochondria with lamellar cristae. However, these cells had quite a few ribosomes, a very low content of both lamellar bodies and clusters of cylindrical tubules and relatively abundant arrays of parallel cisterns of RER (Figs. 3336). In addition, they showed numerous smooth unbranched tubules with constant width and an electrondense content (Fig. 35). A common feature of all types of secretory cells at this period was the presence of autophagic vacuole-like structures. In female gland cells, membrane-bound cytoplasmic vacuoles of large size containing lipid droplets, membranous vesicles, cylindrical tubules, and myelin bodies were observed (Figs. 37, 38). Both types of male gland cells presented concentric whorls of membranes containing organelles such as mitochondria and Stage 3: Days 20-37 multivesicular bodies (Fig. 39). These membranous By the 20th day of age, the first secretory cells con- structures showed a certain resemblance to disorgataining large lipid vacuoles were observed in glands nized lamellar bodies. The presence of autophagic vacfrom male hamsters (Fig. 33). This type of cell, termed uole-like structures suggests a turnover of organelles type 11, was not found in the female HG. This was the during this period. first morphological difference between the two sexes. In hamsters aged 27 days, considerable morphologiAt this age, both male and female glands showed cal differences between male and female glands were intraluminal deposits of pigment (Table 4). No differ- observed. Both sexes possessed intraluminal accretions were normally seen in both of the daughter cells resulting from a single mitosis (Fig. 31). At this age, myoepithelial cells showed some ultrastructural characteristics of mature cells. They contained a small number of cytoplasmic filaments resembling those of smooth muscle (Fig. 32). In addition to filaments, their cytoplasm contained free ribosomes, mitochondria, rough endoplasmic reticulum, and lysosome-like vesicles. Myoepithelial cells had numerous cytoplasmic extensions and an enlarged central portion containing the nucleus. They usually were electronlucent in comparison to the adjacent secretory cells (Fig. 21). At this period, the features of both the secretory and myoepithelial cells characterized them as being at an advanced stage of differentiation. The degree of HG development showed little variation in 15- and 17-day-oldhamsters. In secretory cells, there was a gradual increase in cytoplasmic volume, in the amount of cytoplasmic lipid vacuoles and intraluminal pigment accretions, and in the extent of various organelles (Tables 1,3,4).However, the fine structural characteristics of the gland at these ages were almost identical to those reported in 12-day-old hamsters. In particular, no morphological differences between male and female glands were seen. HARDERIANGLANDDEVELOPMENT 609 Fig. 33. Electron micrograph of a cell containing few lipid vacuoles of large size (type I1 cell) from a 20-day-old male hamster gland. Small lipid vacuoles seem to fuse with large ones (arrows). x 5,860. Fig. 35. High magnification of the cytoplasm of a type I1 cell. Note the existence of two Golgi complexes (arrows) and several smooth electron-dense tubules (arrowheads). 20,500 X . Fig. 34. Electron micrograph of the gland from a 20-day-old male hamster showing portions of the cytoplasm of type I (bottom) and I1 (upper side) cells. x 12,580. Fig. 36. Portion of cytoplasm of a type I1 cell from a 20-day-old male hamster gland containing many polysomes. x 28,890. of pigment, but the amount of pigment in females was much higher than in males (Table 4).The pigment content in 27-day-old male glands decreased as compared with that of the 20-day-old male hamster. In contrast, it showed a marked increase in female glands. Furthermore, type I1 cells appeared in more than 90% of the male secretory tubules, whereas they were virtually absent in females. Cells containing clusters of cylindri- 610 J.M. LOPEZ ET AL. Fig. 37. Electron micrograph of a glandular cell from a 20-day-old female hamster. Note the existence of a large membrane-bound vacuole containing lipid droplets and cell debris (arrows). x 6,140. Fig. 38. High magnification of the vacuole shown in Figure 36. Note the existence of cylindrical tubules inside the vacuole (arrows). x 16,900. Fig. 39. Electron micrograph of a n autophagic vacuole within a cell from a 20-day-old male hamster. Note the existence of a mitochondrion and another inclusion inside the structure. x 41,220. Fig. 40. Portion of cytoplasm of a type I cell from a 27-day-old male hamster showing clusters of cylindrical tubules and lamellar bodies in the same cell. X 27,480. Fig. 41. Light micrograph from a 27-day-old male hamster showing a type I1 cell undergoing mitosis (arrow). x 800. Stage 4: Animals Older Than 45 Days cal tubules were more frequent in male than in female glands (Table 5). At this period, cylindrical tubules and lamellar bodies frequently occurred in the same glanIn 45-day-old hamsters, the HG possessed the strucdular cell (Fig. 40). It was also remarkable to observe tural characteristics of adult glands. At this age, the mitotic figures among the type 11cells (Fig. 41). gland displayed marked morphological differences beThe glandular maturation continued in 37-day-old tween the sexes at both structural and ultrastructural hamsters, as evidenced by the gradual increase of mor- levels. At the light microscopic level (Figs. 42, 431, the phological sex differences. Intraluminal accretions of HG of males had no observable intraluminal pigment pigment were found to rise in female glands; they were granules and their secretory tubules were composed of very scarce in males (Table 4). In contrast, female approximately equal numbers of both type I and type I1 glands did not contain tubular clusters, but these struc- cells. Female HG presented secretory tubules mainly tures were present in a high percentage of the male composed of cells containing minute lipid droplets, and type I cells (Table 5). many of these secretory tubules showed large intralu- HARDERIAN GLAND DEVELOPMENT Fig. 42. Semithin section of the gland from a 45-day-old female hamster. The gland shows secretory tubules lined by type I cells. Two of the tubular alveoli contain solid intraluminal accretions of pigmented material, possibly porphyrins (arrows). x 280. Fig. 43. Semithin section of the gland from a 45-day-old male hamster. Type I and I1 cells are found lining the lumina. Type I1 cells are characterized by the presence of large lipid vacuoles. x 1,300. 611 Fig. 45. A Golgi complex in a secretory cell from a 45-day-old female hamster. Note the proximity of the Golgi complex to a lipid vacuole. x 17,860. Fig. 46. The basal region of a secretory cell from a 45-day-old male hamster. The cytoplasm contains many mitochondria showing a conventional morphology. Note the infolding of the cell basal plasma membrane (arrows). X 17,860. Fig. 44. The apical region of a secretory cell from a 45-day-old female hamster. The cytoplasm is packed with lipid vacuoles, and some of them are in the process of undergoing exocytosis (arrows). Note the existence of pigment accumulation in the alveolar lumen (asterisk). x 3,070. Fig. 47. The apical region of a secretory cell from a 45-day-old male hamster. Note the presence of numerous clusters of cylindrical tubules. x 24,050. minal accretions of reddish-brown pigment. Ultrastructurally, type I cells from male glands presented abundant clusters of cylindrical tubules. Such structures were not found in female glands (Table 5). Moreover, the percentage of cells containing lamellar bodies was very low in male cells and high in female cells. All secretory cell types showed cytological features typical of highly active lipid-secreting cells. They were filled with numerous lipid vacuoles that occupied a great portion of the cell volume (Table 3). The vacuoles are discharged at the cell apex by exocytosis, preserving the continuity of the cell surface while the product is released (Fig. 44). The membrane systems of the cytoplasm were highly organized. The cells had an extensively developed SER a well-developed Golgi complex (Fig. 45) and numerous mitochondria with a matrix of moderate density (Fig. 46). In a high percentage of male type I cells, numerous clusters of cylindrical tu- bules were observed (Table 5). These structures were most common in the cell apex and, in some cases, they occupied the major portions of the apical cytoplasm (Fig. 47). Following the establishment of these basic features, all further changes with maturation were quantitative in nature. Glands from 90-day-old female hamsters showed a n increase in the amount of intraluminal pigment (Table 4). A noteworthy feature was the frequent occurrence of binucleated secretory cells in female glands (Fig. 48). At this age, most of the type I male cells possessed numerous clusters of cylindrical tubules. A morphological relationship between cylindrical tubules and lipid vacuoles was frequently observed (Fig. 49). However, as in young animals, cylindrical tubules were never observed in the luminal space. Lipid secretion occurred by exocytosis in all cell types, including type I1 cells (Fig. 50). The lumen of the secre- 612 J.M. LOPEZ ET AL. Fig. 48. Low power electron micrograph showing part of tubular alveolus from a 90-day-old female hamster. Pigment accumulation is observed in the alveolar lumen. Vacuoles in the process of secretion by exocytosis are shown (arrowheads). Note the existence of two binucleated cells (asterisks). x 2,040. Inset: Higher magnification of an area of the lumen showing a secreted lipid droplet bounded by a single electron-dense line. x 22,000. Fig. 49. Cytoplasm of a type I cell from a 90-day-old male hamster showing close association of cylindrical tubules with lipid droplets. x 26,800. Fig. 50. The apical portion of a type I1 cell from a 90-day-old male hamster. Note the existence of a large vacuole in the process of exocytosis (arrows). Lipid vacuoles in the type I1 cell fuse with each other (arrowheads). x 3.720. HARDERIAN GLAND DEVELOPMENT tory tubules usually contained lipid droplets of the same size as those observed within the cells. However, these luminal droplets were not bounded by a unit membrane but only by a single electron-dense line (Fig. 48). DISCUSSION A well-defined temporal sequence in the appearance of structural components is recognizable in developing hamster HG cells. The observation that at birth the secretory cells are still immature and increase in size concomitantly with a marked reorganization in the composition of organelles after day 7 suggests that further development and maturation in this gland occurs postnatally. The electron microscopic analysis performed on the HG from animals younger than 5 days revealed that the glandular cells have morphological characteristics of undifferentiated cells, i.e., many free ribosomes, few and small organelles, and irregular-shape nuclei with mainly euchromatin. This result is in accordance with the earlier observation of Bucana and Nadakavukaren (1972c, 1973). A striking feature that characterizes the neonatal HG is the frequent observation of large membranebounded structures located in the juxtanuclear cytoplasm of the epithelial cells. Similar structures were described in the HG of hamsters younger than 21 days by Bucana and Nadakavukaren (1973). These authors reported sexual differences in the morphological characteristics of these complexes and, taking into account their constant juxtanuclear localization, suggested that they could result from a nuclear-cytoplasmic interaction. The results in this study partially differ from those of these authors in that the structures observed were always membrane-bound and had the same appearance in both sexes. They seem to contain nuclear fragments and disorganized organelles, having similar ultrastructural features to those identified as autophagic or heterophagic vacuoles in different embryonic or proliferating tissues such as the subfornical organ (Dellmann and Stahl, 19841,antral epithelium of the stomach (Lee and Leblond, 1985), baboon blastocyst (Enders et al., 1990). Nevertheless, by day 1 a considerable percentage of the epithelial cells shows autophagic or heterophagic vacuoles. Since these structures are not seen in myoepithelial or interstitial cells and since they disappear in later stages of development, they are unlikely to be a random occurrence. Their presence may indicate a high turnover activity during this period. Degeneration indeed is a consistent feature in the developing tissues. Light and dark cells have been described frequently in many tissues, including adult HG (Woodhouse and Rhodin, 1963; Tsutsumi et al., 1966; Bucana and Nadakavukaren, 1973). However, there still is controversy over the interpretation of this phenomenon. Whereas some authors interpret their appearance as an artefact caused by the amount of trauma involved in processing the tissue (Chemes et al., 1977; Sinowartz and Amselgrober, 1986), others propose that light and dark cells may reflect different states of metabolic or cell activity (Solari and Fritz, 1978; Osman, 1978; Breuker, 1982). Tsutsumi et al. (1966) suggested that 613 the dark cells of the rat HG may have more secretory activity than the light cells. This suggestion was supported by the observations of Bucana and Nadakavukaren (1972a),who described variations in the number of ribosomes, mitochondria, and vacuoles between light and dark cells in the HG of adult hamsters. However, these same authors did not describe light and dark cells at neonatal stages (Bucana and Nadakavukaren, 1973). In the present study we have not observed variations in the number or structure of cell organelles between dark and light cells at neonatal stages. Furthermore, mitotic figures were observed in both cell types. These observations argue against the possibility that light or dark staining could be a reflection of functional activities. However, the assumption of an artifact due purely to tissue processing is unlikely due to the fact that light and dark cells are closely apposed. The first ultrastructural study on hamster HG reported the existence of a sexual dimorphism a t the ultrastructural level (Bucana and Nadakavukaren, 1972a). Clusters of cylindrical tubules were found only in male glands, whereas female showed abundant membranous structures arranged in concentric lamellae. Subsequently, it was reported that the presence of tubular structures in male or female glands occurred concomitantly with the maintenance of elevated levels of androgens (Lin and Nadakavukaren, 1979; Sun and Nadakavukaren, 1980; Spike et al., 1985). In these experimental studies, the appearance of cylindrical tubules coincided with the disappearance of pigment granules. Bucana and Nadakavukaren (1972a) proposed that cylindrical tubules could be packets of pure enzymes necessary for the breakdown of the pigment molecules. Alternatively, Jones and Hoffman (1976) suggested that these structures might prevent porphyrin biosynthesis by inhibiting the condensation of delta-aminolaevulinic acid to porphobilinogen. The present study shows that cylindrical tubules occur in both male and female glands between days 7 and 27 of age. Furthermore, the first tubules were located in the intravacuolar space sandwiched between the lipid droplet and the unit membrane. These results differ from those of Bucana and Nadakavukaren (1972) who did not observe cylindrical tubules in secretory cells from female hamsters a t any age. In addition, they first found cylindrical tubules located in the membrane-bounded juxtanuclear complexes of cells from 14-day-old male hamsters. Our observation that the appearance of cylindrical tubules precedes that of luminal accretions is at variance with the generally accepted negative correlation between these structures and intraluminal porphyrin accretions. In addition, we have found that the tubules are first in close relationship with lipid vacuoles. This observation supports the possibility that these structures could be secreted, an hypothesis first suggested by Bucana and Nadakavukaren (1972a). Our results show that there is a temporal coincidence between the disappearance of the cylindrical tubules (day 27) and a marked increase of porphyrin accretions in female glands. Likewise, in male glands, the disappearance of luminal accretions coincides with a increase in the tubule content of the secretory glands. This fact suggests 614 J.M. LOPEZ ET AL. that both processes could be induced or regulated by the same factor. The HG has been used increasingly a s a model of porphyrin biosynthesis (Tomio and Grinstein, 1968; Margolis, 1971; Ulrich, 1974; Jones and Hoffman, 1976; Shirama et al., 1981, 1987; Lin and Nadakavukaren, 1982; Thompson et al., 1984; Spike et al., 1988, 1990). However, in spite of these studies, the mechanism of release of porphyrin into the lumen remains controversial. Some authors have suggested that porphyrins become incorporated into the vacuoles and are released into the lumen when apical vacuoles are secreted (Brownscheidle and Niewenhuis, 1978; Watanabe, 1980; Wooding, 1980; Strum and Shear, 1982). Other investigators have reported the existence of porphyrins free in the cytoplasm in structures with a narrow profile consisting of two parallel electron-dense lines on each side of a relatively electron-lucent core. These authors have proposed that such free porphyrins could be toxic, including the degeneration of the cells, whose whole content (including porphyrins) could be discarded by holocrine secretion (Carriere, 1985). In our study we have observed neither cytoplasmic trilaminar profiles of porphyrins nor signs of cellular degeneration or holocrine secretion. Furthermore, we found a temporal coincidence between the onset of vacuole discharge and the appearance of the first luminal amounts of pigment. This coincidence suggests a possible relationship between lipid vacuoles and porphyrins. The onset of the exocytotic activity was temporally coincident with the appearance of cytoplasmic filaments in the myoepithelial cells. This observation is in accord with the suggestion that myoepithelial cells may play a role in the release of the secretory products of the HG (Chiquione, 1958; Bucana and Nadakavukaren, 1972b; Huhtala et al., 1976; Brownscheidle and Niewenhuis, 1978; Watanabe, 1980; Satoh et al., 1990). The HG may serve as a n extraretinal photoreceptor in neonatal rats (Wetterberg et al., 1970a,c). In the present study, we have not found any structure that can be recognized as a photoreceptor at any age. This coincides with the result obtained by Bucana and Nadakavukaren (1973). The only remarkable observation was the existence of some secretory cells containing a n occasional cilium in hamsters between 10 and 17 days old, a morphological feature shared with photoreceptor cells. However, we did not observe lamellar membrane profiles such as occur in the outer segments. Mitoses of glandular cells were common during postnatal development. They were frequent in early postnatal life and decreased with age. However, it is clear from the present observations that a considerable amount of mitotic activity takes place at a time when the secretory cells are already at a n advanced stage of differentiation. The HG of male hamsters possesses two distinct cell types (Types I and 11). However, it is still not known whether they are independent forms of cells that may synthetize different products or two secretory stages of the same cell (Woolley and Worley, 1954; Hoffman, 1971; Bucana and Nadakavukaren, 1973; Payne et al., 1977; McMasters and Hoffman, 1984). Some authors have suggested that type I1 cells could be degenerating cells that could be secreted in toto into the lumina (Hoffman, 1971). Our observations are not in accordance with this suggestion since type I1 cells have specific cytoplasmic characteristics, secrete lipid vacuoles by exocytosis, and are able to divide by mitosis. It is interesting to note that type I1 cells differ from type I not just in the size of the lipid vacuoles but also in the pattern of their organelles. It is known that the HG responds either directly or indirectly to a t least three groups of hormones: steroid hormones, thyroid hormones, and pineal hormones (01cese and Wesche, 1989; Hoffman et al., 1989a, 1990). The regulation of the gland would be ultimately controlled by a complex interplay of photic input, the hormones of the gonads, the pineal, and the thyroid glands (Hoffman e t al., 1989b). Vomachka and Greenwald (1979) outlined the profiles of LH, FSH, PRL, progesterone, androgens, and estradiol in the serum of developing male and female hamsters and reported that levels of these hormones were low during the first 10 days of age. From this, it appears th a t the onset of the secretory activity of the HG (days 7-10) is not temporally coincident with gonadotropin or steroid hormones changes. In the present study, first sexual differences were observed at 20 days of age. This result coincides with that of Bucana and Nadakavukaren (1973). If testosterone is responsible for maintaining the adult male type HG, these glandular changes could be correlated with a n increase of the level of testosterone in males a t this age. However, serum testosterone is a t relatively low levels from birth through 30 days in hamsters (Vomachka and Greenwald, 1979). Nevertheless, our observations show parallelism between the development of the sexual dimorphism in the HG and the sexual maturation of the hamsters. Thus first ovulation in female hamsters (day 34) temporally coincides with a marked increase in the porphyrin content. Likewise, appearance of motile epididymal sperm (day 45) (Austin, 1956) coincides with the complete maturation of the gland in both sexes. ACKNOWLEDGMENTS The authors are grateful to Dr. A.P. Payne and Dr. M.R. Moore, University of Glasgow (U.K.), for a critical review of the manuscript. A portion of this work was presented a t the First International Symposium on the Harderian Gland, Barcelona (Spain), October 1990. This work was supported by a grant from the Research Committee of the University of Oviedo (DF/90 1879). LITERATURE CITED Austin, C.R. 1956 Ovulation, fertilization and early cleavage in the hamster (Mesocricetus auratus). J . Royal Micr. SOC.,75r141-154. Balemans, M.G.M., P. Pevet, J. Van Benthem, C. 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