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Postnatal development of the harderian gland in the Syrian golden hamster Mesocricetus auratusA light and electron microscopic study.

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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
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Breuker, H. 1982 Seasonal spermatogenesis in the mute swan
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Brownscheidle, C.M., and R.J. Niewenhuis 1978 Ultrastructure of the
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HARDERIANGLANDDEVELOPMENT
ization of N-acetylated indolealkylamines in CNS and the Harderian gland using immunohistology. Brain. Res., 118:417-427.
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