1. Cell Tiss. Res. 176,47 55 (1977) Cell and Tissue
Research
9 by Springer-Verlag 1977
Glycogen Deposits in the Pyloric Gland
of the Ascidian Styela clara (Urochordata) *
Thomas H. Ermak**
Department of Zoology, Universityof California, Berkeley,California, USA
Summary. The pyloric gland of Styela clava contains large glycogen deposits
that are digested by treatment with alpha amylase and depleted by 15 days
starvation. The deposits are surrounded by cytoplasmic regions containing
smooth endoplasmic reticulum and mitochondria. The cells also have rough
endoplasmic reticulum, Golgi cisterns, lysosomes, microvilli, cilia, and lateral
infoldings of the plasma membrane. The fine structure of the pyloric cells
and the position of tubules between the absorptive epithelium and general
circulation suggest that the gland functions as the vertebrate liver in carbo-
hydrate metabolism. The pyloric ceils of Styela do not appear to be excretory
in a 'renal' sense, since there is no infolding of the basal plasmalemma and
mitochondria are usually associated only with the glycogen deposits. However,
a hepatic-like excretory role is consistent with current findings. In light of
the phylogenic affinities of vertebrates and ascidians, it is possible that the
pyloric gland is homologous to the liver.
Key words: Glycogen - Pyloric gland - Ascidian - Ultrastructure.
Introduction
The ascidian pyloric gland is an enigmatic organ to which have been ascribed
excretory, osmoregulatory, and digestive functions (Fouque, 1953; Millar, 1953;
Gaill, 1973; G o o d b o d y , 1974). In Styela clara, an advanced solitary ascidian,
the distribution of pyloric tubules beneath the renewing intestinal epithelium
suggests that these structures are involved in nutrient assimilation (Ermak, 1975).
In a colonial ascidian, Sidnyum argus, small glycogen deposits were recently
observed in the pyloric cells (Gaill, 1974a). The present electron microscopic
Send offprint requests to : Dr. Thomas H. Ermak, Department of Physiology,Universityof California
Medical Center, San Francisco, California 94143, USA
* Supported by USPHS grant GM 10292 to Dr. Richard M. Eakin
** I am grateful to Dr. Eakin for his support and valuable criticism of the manuscript

2. 48 Th.H. Ermak
investigation was undertaken to examine the putative glycogen deposits in Styela
clava and to re-evaluate the function of the pyloric gland. The identity of glycogen
was elucidated by enzymatic digestion and the periodic acid Schiff (PAS) reaction.
The cellular structure ofa pyloric tubule was also studied under starved conditions.
Materials and Methods
Specimens of S t y e l a clava were collected from Mission Bay, San Diego, and the Berkeley Marina,
Berkeley, California. Parts of the intestinal wall immediately posterior to the stomach were fixed in
3 ~ glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) with 0.7 M sucrose for 1.5 h at room temperature.
Some specimens were then incubated at 35~ in 0.5 ~o alpha amylase (type III A, Sigma Chemical Co.;
activity 50-100 units/mg) in 0.1 M phosphate buffer for 1 to 3 h. Prior to incubation, intestinal samples
were rinsed in 0.1 M phosphate buffer to remove the sucrose in the glutaraldehyde fixative. Controls
were incubated in heat-treated amylase in 0.I M phosphate buffer. Several animals were starved for
15 days before fixation.
All specimens were postfixed in 1~o ice cold osmium tetroxide in 0.1 M phosphate buffer (pH 7.3)
with 0.7 M sucrose for 1 h and embedded in Epon. Thick sections (1 ~tm) were stained with PAS
(Leeson and Leeson, 1970) or toluidine blue. Silver-gold sections were stained with uranyl acetate
and lead citrate and examined with an RCA-3G electron microscope operating at 100 kv.
Results
The pyloric gland of Styela clava is composed of numerous diffuse tubules which
lie in the gut wall. The tubules are most concentrated in the intestine, the region
of most intense nutrient absorption, and collect into a canal that leads into the
stomach at its junction with the intestine.
The intestinal wall (Fig. 1) consists of 3 layers: (1) an inner epithelium con-
taining absorptive and secretory cells; (2) a middle layer of connective tissue,
blood channels, blood cells, and pyloric tubules; and (3) an outer atrial epithelium,
the lining of the body cavity. The pyloric tubules lie directly below the gut epi-
thelium; none are located next to the atrial epithelium. Light microscopy of
thick sections stained by the PAS technique shows large regions of intense
reaction in the pyloric cells (Fig. 1). The atrial epithelium and some blood cells
also show small regions of positive reaction, attributable possibly to glycogen
or PAS positive mucous granules.
Electron microscopy of the pyloric cells (Fig. 2) reveals large regions containing
single (400 A) and aggregated particles of glycogen contained within a matrix of
light, granular material. The aggregates are not arranged in typical alpha rosettes,
and the single units appear compound (Fig. 3). The glycogen deposit, which
excludes most cytoplasm and cytoplasmic organelles, is always surrounded by a
layer of cytoplasm containing smooth endoplasmic reticulum and never borders
Fig. 1. Light micrograph of intestinal wall, PAS staining, ae atrial epithelium; ct connective tissue;
ie intestinal epithelium; pt pyloric tubules, x 250
Fig. 2. Electron micrograph of part of pyloric tubule showing large glycogen deposit (g). er endo-
plasmic reticulum; M interdigitation; m mitochondrion; m y microvilli; tj tight junction, x 24,000

3. Glycogen in Ascidian Pyloric Gland 49
Fig. 3. Cytoplasmic matrix containing smooth endoplasmic reticulum (ser) in parallel with lateral
plasma membranes (pm). g glycogen. • 48,000

4. 50 Th.H. Ermak
Fig. 4. Light micrograph of pyloric tubule treated with alpha amylase for 1 h, toluidine blue staining.
bc blood cells; l lumen; s space, x 640
Fig. 5. Electron micrograph ofpyloric cell treated with alpha amylase for 1 h. c connective tissue fibers;
er endoplasmic reticulum; ly lysosome; m mitochondrion; n nucleus; s space, x 24,000
directly upon the plasma membrane. When the glycogen lies close to a lateral
plasma membrane, the intervening cytoplasmic matrix may be as narrow as
0.1 to 0.2 gm, and the tubules of endoplasmic reticulum are arranged in parallel
(Fig. 3). Mitochondria occur throughout the cytoplasm and frequently border
directly on a glycogen deposit (Fig. 2). The cell also contains Golgi cisterns and
lysosomes.
Although no infoldings of the basal plasmalemma are observed, the pyloric

5. Glycogenin Ascidian Pyloric Gland 51
Fig. 6. Pyloric ceils from ascidian starved 15 days. g a Golgi apparatus; M interdigitation; m mito-
chondrion; m v microvilli;c! cilia; tj tight junction, x 35,000
cells exhibit interdigitations of lateral plasma membranes (Figs. 2, 6). A tight
junction (Mackie et al., 1974) occurs at the apical cell border (Figs. 1, 6); several
gap-like junctions are seen between the tight junction and the basal border. The
cell apex bears numerous microvilli 2 gm in length and at least one long cilium.
Cilia in the tubules are sometimes so numerous that they fill the lumen.
Treatment with alpha amylase eliminates the PAS-reaction leaving large
unstained regions in the pyloric tubules but not in most other tissues. These
spaces are particularly evident in toluidine blue stained 1 gm thick sections
(Fig. 4). In thin sections, amylase removes the glycogen particles leaving large
spaces (Fig. 5). A 1 h incubation period produces little alteration in cytoplasm,
cellular membranes, and connective tissue fibers. Longer incubation, however,

6. 52 Th.H. Ermak
disrupts microvilli and washes out cytoplasmic matrix. The reaction is inhibited
by heat denaturation and sucrose in the incubation medium. Mucous granules
in atrial epithelial cells (see below) are not digested by amylase.
After 15 days starvation, glycogen deposits are lost from the pyloric epithelium
(Fig. 6). The cells appear smaller in size; presumably at least a part of this decrease
is due to the loss of glycogen. Elements of smooth and rough endoplasmic reti-
culum fill most of the cytoplasm; however, smooth endoplasmic reticulum no
longer occurs parallel to the plasma membrane (see Discussion).
Atrial epithelial cells are low columnar cells containing several membrane-
bounded mucous granules, presumably the PAS positive granules of light micro-
scopy. They also have irregular apical and basal borders, and interdigitating
baso-lateral plasma membranes. Several blood cells in the connective tissue also
contain mucous granules and glycogen.
Discussion
The results of this investigation clarify several points concerning the function of
the pyloric gland. Based upon ultrastructural evidence. Gaill (1974a) suggested
that the gland might be excretory because of infoldings of the basal plasmalemma
and the presence of microvilli and cilia. In Styela, the infoldings are actually
from the lateral plasma membrane, a condition in common with the atrial epi-
thelium and, in general, all transporting epithelia (Berridge and Oschman, 1972).
The pyloric cells in Styela differ in morphology from true ascidian renal epithelial
cells (Gaill, 1974b) and also plicated cells in the gut (Degail and Levi, 1964;
Burighel and Milanesi, 1975) which exhibit distinct basal membrane infoldings
containing numerous mitochondria. In the pyloric gland, mitochondria in the
basal part of a cell are more intimately in contact with the glycogen deposit than
with the plasmalemma.
Fouque (1953) suggested that the pyloric cells are engaged in 're-working'
substances absorbed by intestinal cells. The fine structure of the tubules supports
this contention; the pyloric gland of Styela is most likely actively engaged in
carbohydrate metabolism. Digestion of the large deposits by alpha amylase (as
demonstrated by Biava, 1963, and Coimbra, 1967) but not of other PAS-positive
sites in the intestinal wall indicates that the particles are glycogen. The deposits
in Styela are much larger than those in Sidnyum (Gaill, 1974a). Since glycogen
content typically varies with nutritional state, a difference between the two
species might merely reflect variability in storage at the time of fixation. Alter-
natively, different species of ascidians might store glycogen in the pyloric gland
to different degrees.
Among invertebrates, glycogen is usually concentrated in a specific region of
the gut, such as the crustacean hepatopancreas (Vonk, 1960), the gastropod
digestive gland (Chatterjee and Ghose, 1974), and the echinoderm intestine
(Doezema and Phillips, 1970). In Styela, the pyloric gland is the major gut region
to store glycogen. Small amounts of glycogen also occur in the ascidian stomach
(Burighel and Milanesi, 1973), intestine (Gaill, 1974 a), and digestive diverticulum
(Degail and Levi, t 964). Blood cells which contain glycogen are probably amoeb-

7. Glycogenin Ascidian PyloricGland 53
ocytes. In Perophora viridis, granular amoebocytes, which are presumed to be
nutritive storage cells, contain masses of 200-300 A particles of glycogen (Overton,
1966). In an ascidian tadpole ocellus, the lens is a large glycogen deposit composed
of beta granules (300-400 A in diameter) and surrounded by numerous mito-
chondria (Eakin and Kuda, 1972).
Refringent granules (Fouque, 1953) and solid concretions (Millar, 1949; 1953)
have also been described in the pyloric gland. In light of the refractive qualities
of glycogen, such as that in an ascidian tadpole lens (Eakin and Kuda, 1972),
it is possible that the granules are actually glycogen. Those concretions illustrated
by Millar (1953, Fig. 4, p. 33) in the pyloric gland of Ciona intestinalis correspond
in size and shape to the glycogen deposits in Styela (compare to Fig. 1, this
investigation). It is also possible that the vacuoles described in a pyloric cell
(Millar, 1953) represent spaces where glycogen was lost in the preparation of
the sections, since glycogen is frequently removed during routine histological
procedures (Bloom and Fawcett, 1968, p. 591). The disposition of vacuoles in
Ciona is like the distribution of spaces in Styela created by the digestion of glycogen
by alpha amylase.
Glycogen in a pyloric cell disappears with starvation. As in a mammalian
hepatocyte (Cardell et al., 1973), beta and alpha particles of glycogen are associated
with regions of smooth endoplasmic reticulum and mitochondria. Rough endo-
plasmic reticulum, however, is not so well developed as that in the mammalian
hepatocyte. In autoradiograms of liver cells exposed to tritiated glucose, label is
localized over both glycogen particles and smooth endoplasmic reticulum
(Coimbra and Leblond, 1966). Glucose is apparently added to growing glycogen
particles, since peripheral D-glucosyl residues are renewed more rapidly than
those in the core of a glycogen molecule (see Stetten and Stetten, 1960). The
parallel arrangement of smooth endoplasmic reticulum between a glycogen
deposit and a lateral plasma membrane is similar to subsurface endoplasmic
reticula in mouse hepatocytes (Tandler, 1974). However, since this pattern
disappears with the loss of glycogen during starvation, the tubules of smooth
endoplasmic reticulum are probably not subsurface cisterns but only aligned in
parallel because of the small amount of cytoplasm surrounding them.
The fine structural similarities between ascidian pyloric cells and vertebrate
hepatic cells as well as the distribution of pyloric tubules beneath the intestinal
epithelium suggest that the pyloric gland might play a wider role in nutrient
assimilation. In Styela, the entire intestinal lining is a renewing epithelium
(Ermak, 1975). A groove of relatively undifferentiated cells runs along one side
of the intestine. These germinal cells replace the absorptive and secretory cells
on the side walls in about a month. Beneath the germinal cells, the pyloric tubules
are few in number. Under the absorptive cells, however, the tubules are exceedingly
numerous. All food products passing from the gut lumen to the blood channels
pass through this meshwork of tubules. Like the vertebrate liver, which is inter-
posed between the digestive tract and general circulation, the pyloric gland might
act as a processing station transforming nutrients absorbed by the gut lining and
passing them to blood cells which then distribute them to the rest of the body.
Absorbed products of digestion, whether metabolized or transformed by the
pyloric gland, would quickly be distributed throughout the body by way of

8. 54 Th.H. Ermak
o p e n b l o o d channels (lined only by connective tissue), since the posterior end
o f the heart leads directly to the gut. In ascidians, the heart is unusual in that it
periodically reverses beat direction, one time p u m p i n g blood towards the pharynx,
the next time towards the viscera.
Like the vertebrate liver, the pyloric gland develops as a diverticulum o f the
gut. Sections o f closely packed tubules in Styela are reminescent o f sections o f
liver cords. A l t h o u g h the pyloric cells are involved in c a r b o h y d r a t e metabolism,
lipid metabolism, which is also a function o f the vertebrate liver, is p r o b a b l y
handled directly by the gut epithelium. Lipid droplets occur in great n u m b e r in
the s t o m a c h lining (Burighel and Milanesi, 1973) but usually not in pyloric cells.
In Styela, lipid reserves in absorptive cells o f the s t o m a c h and intestine fluctuate
seasonally and are also depleted by starvation (Ermak, unpublished results).
A l t h o u g h the pyloric gland does not have the ultrastructural features o f a
renal o r g a n (see above), it p r o b a b l y has an hepatic-like excretory function.
T h o r i u m dioxide which has been injected into the b o d y cavity o f Ciona intestinalis
is p h a g o c y t o s e d by blood cells and eliminated by the intestine via the pyloric
gland (Brown and Davies, 1971). In Styela plieata, the pyloric gland takes up
vital dyes f r o m the blood (Fouque, 1953). Cilia in a pyloric tubule would, then,
aid in transporting any secreted substances d o w n the pyloric duct to the intestine
(see G o o d b o d y , 1974). Thus the ultrastructural, positional, and functional
characteristics o f the pyloric gland are strikingly similar to those o f the vertebrate
liver. Indeed, the likelihood that vertebrates evolved f r o m primitive ascidian
stocks (Berrill, 1955) suggests that the pyloric gland is h o m o l o g o u s to the liver.
If this is the case, the pyloric gland might be expected to possess other hepatic
functions.
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Accepted August 6, 1976