SlideShare a Scribd company logo
1 of 9
Download to read offline
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
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
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
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
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,
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-
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
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.

References

Berridge, M.J., Oschman, J.L.: Transporting epithelia. New York: Academic Press 1972
Berrill, N.J.: The origin of vertebrates. Oxford: Clarendon Press 1955
Biava, C.: The identification and structural forms of human particulate glycogen. Lab. Invest. 12,
   1179 1197(1963)
Bloom, W., Fawcett, D.W.: A textbook of histology. Philadelphia: W.B. Saunders Co. 1968
Brown, A.C., Davies, A.B.: The fate of thorium dioxide introduced into the body cavity of Ciona
   intestinalis. J. Invert. Path. 18, 276-279 (1971)
Burighel, P., Milanesi, C.: Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri.
   Vacuolated and zymogen cells. Z. Zellforsch. 145, 541-555 (1973)
Burighel, P., Milanesi, C.: Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri.
   Mucous, endocrine and plicated cells. Cell Tiss. Res. 158, 481-496 (1975)
Cardell, R.R., Larner, J., Babcock, M.B.: Correlation between structure and glycogen content of
   livers from rats on a controlled feeding schedule. Anat. Rec. 177, 23-38 (1973)
Chatterjee, B., Ghose, K.C.: Seasonal variation in stored glycogen and lipid in the digestive gland and
   genital organs of two freshwater prosobranchs. Proc. Mal. Soc. Lond. 40, 407-412 (1974)
Coimbra, A.: Evaluation of the glycogenolytic effect of alpha amylase using radioautography and
   electron microscopy. J. Histochem. Cytochem. 14, 898-905 (1967)
Coimbra, A., Leblond, C.P.: Sites of glycogen synthesis in rat liver cells as shown by EM radioauto-
   graphy after administration of glucose-H3. J. Cell Biol. 30, 151-176 (1966)
Degail, L., Levi, C.: Etude au microscope 61ectronique de la glande digestive des Pyuridae (Ascidies).
   Cah. Biol. Mar. 5, 411-422 (1964)
Doezema, P., Phillips Jr., J.H.: Glycogen storage and synthesis in the gut of the purple sea urchin,
   Strongylocentrotus purpuratus. Comp. Biochem. Phys. 34, 691-697 (1970)
Glycogen in Ascidian Pyloric Gland                                                                     55

Eakin, R.M., Kuda, A.: Glycogen in lens of tunicate tadpole (Chordata: Ascidiacea). J. exp. Zool.
    180, 26%270 (1972)
Ermak, T.H.: Cell proliferation in the digestive tract of Styela clara (Urochordata: Ascidiacea) as
    revealed by autoradiography with tritiated thymidine. J. exp. Zool. 194, 449-466 (1975)
Fouque, G.: Contribution /l l'6tude de la glande pylorique des ascidiac6s. Ann. Inst. Oceanogr.
    (Paris) 28, 189-317 (1953)
Gaill, F.: Etude histologique de la glande pylorique de Synoicum argus (Polyclinidae, Tuniciers).
    Arch. Zool. Exp. Gen. 114, 97-110 (1973)
Gaill, F.: Aspect ultrastructural de la glande pylorique et de l'intestin post6rieur de Sidnyum argus
    (Polyclinidae, Tuniciers). Cah. Biol. Mar. 15, 337 341 (1974a)
Gaill, F.: Observations ultrastructurales de l'epithelium r6nal de Molgula occulta (Ascidies, Tuniciers).
    C.R. Acad. Sci. (Paris) 279, 351-353 (1974b)
Goodbody, I.: The physiology of ascidians. Adv. Mar. Biol. 12, 1-150 (1974)
Leeson, C.R., Leeson, T.S.: Staining methods for sections of epon-embedded tissues for light micro-
    scopy. Canad. J. Zool. 48, 189-191 (1970)
Mackie, G.O., Paul, D.H., Singla, C.M., Sleigh, M.A., Williams, D.E.: Branchial innervation and
   ciliary control in the ascidian Corella. Proc. roy. Soc. B 187, 1-35 (1974)
Millar, R.H.: Concretions in the pyloric gland of Ciona intestinalis. Nature (Lond.) 164, 717-718
    (1949)
Millar, R.H.: Ciona, L.M.B.C. Memoirs. Liverpool: Colman 1953
Overton, J.: The fine structure of blood cells in the ascidian Perophora viridis. J. Morph. 119, 305-326
   (1966)
Stetten, D. Jr., Stetten, M.R.: Glycogen metabolism. Physiol. Rev. 40, 505-537 (1960)
Tandler, B. : Subsurface cisterns in mouse hepatocytes. Anat. Rec. 179, 273-284 (1974)
Vonk, H.J.: Digestion and metabolism. In: Physiology of crustacea (T.H. Waterman, ed.), Vol. 1,
   p. 291. New York: Academic Press 1960


Accepted August 6, 1976

More Related Content

What's hot

The golgi apparatus
The golgi apparatusThe golgi apparatus
The golgi apparatusDilip Pandya
 
Presentation on Golgi complex, Ribosome and Plastid
Presentation on Golgi complex, Ribosome and PlastidPresentation on Golgi complex, Ribosome and Plastid
Presentation on Golgi complex, Ribosome and PlastidDr. Kaushik Kumar Panigrahi
 
Rough endoplasmic reticulam (rer) ( introduction structure & function)
Rough endoplasmic reticulam (rer) ( introduction structure & function)Rough endoplasmic reticulam (rer) ( introduction structure & function)
Rough endoplasmic reticulam (rer) ( introduction structure & function)Dryogeshcsv
 
Protein Sorting and Transport Through Golgi complex
Protein Sorting and Transport Through Golgi complexProtein Sorting and Transport Through Golgi complex
Protein Sorting and Transport Through Golgi complexKaushal Sharma
 
Endomembrane system presentation
Endomembrane system presentationEndomembrane system presentation
Endomembrane system presentationAindrila Majumdar
 
ENDOPLASMIC RETICULUM
ENDOPLASMIC RETICULUMENDOPLASMIC RETICULUM
ENDOPLASMIC RETICULUMkaushikakumar
 
11.11(dr. husun bano) mitochondria lysosomes &peroxisomes
11.11(dr. husun bano) mitochondria lysosomes &peroxisomes11.11(dr. husun bano) mitochondria lysosomes &peroxisomes
11.11(dr. husun bano) mitochondria lysosomes &peroxisomesFati Naqvi
 
Unit 3-cell cell interactions
Unit 3-cell cell interactionsUnit 3-cell cell interactions
Unit 3-cell cell interactionsKomal Kp
 
Unit 2-plasma membrane and membrane transport
Unit 2-plasma membrane and membrane transportUnit 2-plasma membrane and membrane transport
Unit 2-plasma membrane and membrane transportKomal Kp
 
cell organelles- Endoplasmic reticulum and Golgi bodies
cell organelles- Endoplasmic reticulum and Golgi bodiescell organelles- Endoplasmic reticulum and Golgi bodies
cell organelles- Endoplasmic reticulum and Golgi bodiesDivya S
 
Struktur sel dan fungsinya OSN
Struktur sel dan fungsinya OSNStruktur sel dan fungsinya OSN
Struktur sel dan fungsinya OSNDeis Sappa
 

What's hot (19)

Lysosomes mbb
Lysosomes mbbLysosomes mbb
Lysosomes mbb
 
The golgi apparatus
The golgi apparatusThe golgi apparatus
The golgi apparatus
 
Presentation on Golgi complex, Ribosome and Plastid
Presentation on Golgi complex, Ribosome and PlastidPresentation on Golgi complex, Ribosome and Plastid
Presentation on Golgi complex, Ribosome and Plastid
 
Rough endoplasmic reticulam (rer) ( introduction structure & function)
Rough endoplasmic reticulam (rer) ( introduction structure & function)Rough endoplasmic reticulam (rer) ( introduction structure & function)
Rough endoplasmic reticulam (rer) ( introduction structure & function)
 
Peroxisome
PeroxisomePeroxisome
Peroxisome
 
Protein Sorting and Transport Through Golgi complex
Protein Sorting and Transport Through Golgi complexProtein Sorting and Transport Through Golgi complex
Protein Sorting and Transport Through Golgi complex
 
Endomembrane system presentation
Endomembrane system presentationEndomembrane system presentation
Endomembrane system presentation
 
ENDOPLASMIC RETICULUM
ENDOPLASMIC RETICULUMENDOPLASMIC RETICULUM
ENDOPLASMIC RETICULUM
 
Golgi appratus
Golgi appratusGolgi appratus
Golgi appratus
 
11.11(dr. husun bano) mitochondria lysosomes &peroxisomes
11.11(dr. husun bano) mitochondria lysosomes &peroxisomes11.11(dr. husun bano) mitochondria lysosomes &peroxisomes
11.11(dr. husun bano) mitochondria lysosomes &peroxisomes
 
Bio
BioBio
Bio
 
Golgi bodies
Golgi bodiesGolgi bodies
Golgi bodies
 
Golgi complex
Golgi complexGolgi complex
Golgi complex
 
Unit 3-cell cell interactions
Unit 3-cell cell interactionsUnit 3-cell cell interactions
Unit 3-cell cell interactions
 
Unit 2-plasma membrane and membrane transport
Unit 2-plasma membrane and membrane transportUnit 2-plasma membrane and membrane transport
Unit 2-plasma membrane and membrane transport
 
Golgi apparatus
Golgi apparatusGolgi apparatus
Golgi apparatus
 
HUMAN TISSUE ANATOMY & PHYSIOLOGY
HUMAN TISSUE ANATOMY & PHYSIOLOGYHUMAN TISSUE ANATOMY & PHYSIOLOGY
HUMAN TISSUE ANATOMY & PHYSIOLOGY
 
cell organelles- Endoplasmic reticulum and Golgi bodies
cell organelles- Endoplasmic reticulum and Golgi bodiescell organelles- Endoplasmic reticulum and Golgi bodies
cell organelles- Endoplasmic reticulum and Golgi bodies
 
Struktur sel dan fungsinya OSN
Struktur sel dan fungsinya OSNStruktur sel dan fungsinya OSN
Struktur sel dan fungsinya OSN
 

Similar to Ermak styela clava glycogen deposits 1977

Age Related Histomorphological and Transmission Electron Microscopic Studies ...
Age Related Histomorphological and Transmission Electron Microscopic Studies ...Age Related Histomorphological and Transmission Electron Microscopic Studies ...
Age Related Histomorphological and Transmission Electron Microscopic Studies ...iosrjce
 
Types of Epithelia
Types of EpitheliaTypes of Epithelia
Types of EpitheliaEneutron
 
Parietal cells in health & diseases
Parietal cells in health & diseasesParietal cells in health & diseases
Parietal cells in health & diseasesChirantan MD
 
Ermak styela clava cell proliferation j exp zool 1975
Ermak styela clava cell proliferation j exp zool 1975Ermak styela clava cell proliferation j exp zool 1975
Ermak styela clava cell proliferation j exp zool 1975Thomas Ermak
 
Histology & Function of The Pancreas
Histology & Function of The Pancreas Histology & Function of The Pancreas
Histology & Function of The Pancreas FatmaGhoneim3
 
Food theorymay2013.withfigsnopage1
Food theorymay2013.withfigsnopage1Food theorymay2013.withfigsnopage1
Food theorymay2013.withfigsnopage1Andy Sutton
 
Cell Physiology Spring 2016 Name Page 1 This test is .docx
Cell Physiology Spring 2016 Name Page 1 This test is .docxCell Physiology Spring 2016 Name Page 1 This test is .docx
Cell Physiology Spring 2016 Name Page 1 This test is .docxtidwellveronique
 
Carbohydrates 3.pdf
Carbohydrates 3.pdfCarbohydrates 3.pdf
Carbohydrates 3.pdfyinehov
 
Yeast “contraceptives” also novel drugs
Yeast “contraceptives” also novel drugsYeast “contraceptives” also novel drugs
Yeast “contraceptives” also novel drugsInternet Medical Society
 
Structure of cell membrane and Transport
Structure of cell membrane and TransportStructure of cell membrane and Transport
Structure of cell membrane and TransportMohit Adhikary
 
CELL BIOLOGY
CELL BIOLOGYCELL BIOLOGY
CELL BIOLOGYYESANNA
 
Salivary gland by Dr Deepak Kumar
Salivary gland by Dr Deepak KumarSalivary gland by Dr Deepak Kumar
Salivary gland by Dr Deepak KumarDr Deepak Kumar
 
Cell ultrastructure and cell organelles
Cell ultrastructure and cell organellesCell ultrastructure and cell organelles
Cell ultrastructure and cell organellesDev Hingra
 

Similar to Ermak styela clava glycogen deposits 1977 (20)

Age Related Histomorphological and Transmission Electron Microscopic Studies ...
Age Related Histomorphological and Transmission Electron Microscopic Studies ...Age Related Histomorphological and Transmission Electron Microscopic Studies ...
Age Related Histomorphological and Transmission Electron Microscopic Studies ...
 
Types of Epithelia
Types of EpitheliaTypes of Epithelia
Types of Epithelia
 
Parietal cells in health & diseases
Parietal cells in health & diseasesParietal cells in health & diseases
Parietal cells in health & diseases
 
Ermak styela clava cell proliferation j exp zool 1975
Ermak styela clava cell proliferation j exp zool 1975Ermak styela clava cell proliferation j exp zool 1975
Ermak styela clava cell proliferation j exp zool 1975
 
Histology & Function of The Pancreas
Histology & Function of The Pancreas Histology & Function of The Pancreas
Histology & Function of The Pancreas
 
Histology of the pancreas, gall bladder and appendix by Zachariah Richard
Histology of the pancreas, gall bladder and appendix by Zachariah RichardHistology of the pancreas, gall bladder and appendix by Zachariah Richard
Histology of the pancreas, gall bladder and appendix by Zachariah Richard
 
Food theorymay2013.withfigsnopage1
Food theorymay2013.withfigsnopage1Food theorymay2013.withfigsnopage1
Food theorymay2013.withfigsnopage1
 
Cell Physiology Spring 2016 Name Page 1 This test is .docx
Cell Physiology Spring 2016 Name Page 1 This test is .docxCell Physiology Spring 2016 Name Page 1 This test is .docx
Cell Physiology Spring 2016 Name Page 1 This test is .docx
 
Carbohydrates 3.pdf
Carbohydrates 3.pdfCarbohydrates 3.pdf
Carbohydrates 3.pdf
 
Bioartificial pancreas
Bioartificial pancreasBioartificial pancreas
Bioartificial pancreas
 
Yeast “contraceptives” also novel drugs
Yeast “contraceptives” also novel drugsYeast “contraceptives” also novel drugs
Yeast “contraceptives” also novel drugs
 
Structure of cell membrane and Transport
Structure of cell membrane and TransportStructure of cell membrane and Transport
Structure of cell membrane and Transport
 
Biomaterials-2
Biomaterials-2Biomaterials-2
Biomaterials-2
 
Cell bio
Cell bioCell bio
Cell bio
 
Git class-1
Git class-1Git class-1
Git class-1
 
Introductiondata
IntroductiondataIntroductiondata
Introductiondata
 
CELL BIOLOGY
CELL BIOLOGYCELL BIOLOGY
CELL BIOLOGY
 
Salivary gland by Dr Deepak Kumar
Salivary gland by Dr Deepak KumarSalivary gland by Dr Deepak Kumar
Salivary gland by Dr Deepak Kumar
 
Plant cell Overiew
Plant cell Overiew Plant cell Overiew
Plant cell Overiew
 
Cell ultrastructure and cell organelles
Cell ultrastructure and cell organellesCell ultrastructure and cell organelles
Cell ultrastructure and cell organelles
 

More from Thomas Ermak

Ermak styela clava hematogenic tissues 1976
Ermak styela clava hematogenic tissues 1976Ermak styela clava hematogenic tissues 1976
Ermak styela clava hematogenic tissues 1976Thomas Ermak
 
Ermak styela clava renewal blood cells experientia 1975
Ermak styela clava renewal blood cells experientia 1975Ermak styela clava renewal blood cells experientia 1975
Ermak styela clava renewal blood cells experientia 1975Thomas Ermak
 
Ermak styela clava kinetics stomach j exp zool 1976
Ermak styela clava kinetics stomach j exp zool 1976Ermak styela clava kinetics stomach j exp zool 1976
Ermak styela clava kinetics stomach j exp zool 1976Thomas Ermak
 
Ermak styela clava renewal gonads tiss cell 1976
Ermak styela clava renewal gonads tiss cell 1976Ermak styela clava renewal gonads tiss cell 1976
Ermak styela clava renewal gonads tiss cell 1976Thomas Ermak
 
Ermak renewing cells of ascidians 1982
Ermak renewing cells of ascidians 1982Ermak renewing cells of ascidians 1982
Ermak renewing cells of ascidians 1982Thomas Ermak
 
Ermak comparative cell proliferation j exp zool 1981
Ermak comparative cell proliferation j exp zool 1981Ermak comparative cell proliferation j exp zool 1981
Ermak comparative cell proliferation j exp zool 1981Thomas Ermak
 

More from Thomas Ermak (6)

Ermak styela clava hematogenic tissues 1976
Ermak styela clava hematogenic tissues 1976Ermak styela clava hematogenic tissues 1976
Ermak styela clava hematogenic tissues 1976
 
Ermak styela clava renewal blood cells experientia 1975
Ermak styela clava renewal blood cells experientia 1975Ermak styela clava renewal blood cells experientia 1975
Ermak styela clava renewal blood cells experientia 1975
 
Ermak styela clava kinetics stomach j exp zool 1976
Ermak styela clava kinetics stomach j exp zool 1976Ermak styela clava kinetics stomach j exp zool 1976
Ermak styela clava kinetics stomach j exp zool 1976
 
Ermak styela clava renewal gonads tiss cell 1976
Ermak styela clava renewal gonads tiss cell 1976Ermak styela clava renewal gonads tiss cell 1976
Ermak styela clava renewal gonads tiss cell 1976
 
Ermak renewing cells of ascidians 1982
Ermak renewing cells of ascidians 1982Ermak renewing cells of ascidians 1982
Ermak renewing cells of ascidians 1982
 
Ermak comparative cell proliferation j exp zool 1981
Ermak comparative cell proliferation j exp zool 1981Ermak comparative cell proliferation j exp zool 1981
Ermak comparative cell proliferation j exp zool 1981
 

Ermak styela clava glycogen deposits 1977

  • 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. References Berridge, M.J., Oschman, J.L.: Transporting epithelia. New York: Academic Press 1972 Berrill, N.J.: The origin of vertebrates. Oxford: Clarendon Press 1955 Biava, C.: The identification and structural forms of human particulate glycogen. Lab. Invest. 12, 1179 1197(1963) Bloom, W., Fawcett, D.W.: A textbook of histology. Philadelphia: W.B. Saunders Co. 1968 Brown, A.C., Davies, A.B.: The fate of thorium dioxide introduced into the body cavity of Ciona intestinalis. J. Invert. Path. 18, 276-279 (1971) Burighel, P., Milanesi, C.: Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Vacuolated and zymogen cells. Z. Zellforsch. 145, 541-555 (1973) Burighel, P., Milanesi, C.: Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous, endocrine and plicated cells. Cell Tiss. Res. 158, 481-496 (1975) Cardell, R.R., Larner, J., Babcock, M.B.: Correlation between structure and glycogen content of livers from rats on a controlled feeding schedule. Anat. Rec. 177, 23-38 (1973) Chatterjee, B., Ghose, K.C.: Seasonal variation in stored glycogen and lipid in the digestive gland and genital organs of two freshwater prosobranchs. Proc. Mal. Soc. Lond. 40, 407-412 (1974) Coimbra, A.: Evaluation of the glycogenolytic effect of alpha amylase using radioautography and electron microscopy. J. Histochem. Cytochem. 14, 898-905 (1967) Coimbra, A., Leblond, C.P.: Sites of glycogen synthesis in rat liver cells as shown by EM radioauto- graphy after administration of glucose-H3. J. Cell Biol. 30, 151-176 (1966) Degail, L., Levi, C.: Etude au microscope 61ectronique de la glande digestive des Pyuridae (Ascidies). Cah. Biol. Mar. 5, 411-422 (1964) Doezema, P., Phillips Jr., J.H.: Glycogen storage and synthesis in the gut of the purple sea urchin, Strongylocentrotus purpuratus. Comp. Biochem. Phys. 34, 691-697 (1970)
  • 9. Glycogen in Ascidian Pyloric Gland 55 Eakin, R.M., Kuda, A.: Glycogen in lens of tunicate tadpole (Chordata: Ascidiacea). J. exp. Zool. 180, 26%270 (1972) Ermak, T.H.: Cell proliferation in the digestive tract of Styela clara (Urochordata: Ascidiacea) as revealed by autoradiography with tritiated thymidine. J. exp. Zool. 194, 449-466 (1975) Fouque, G.: Contribution /l l'6tude de la glande pylorique des ascidiac6s. Ann. Inst. Oceanogr. (Paris) 28, 189-317 (1953) Gaill, F.: Etude histologique de la glande pylorique de Synoicum argus (Polyclinidae, Tuniciers). Arch. Zool. Exp. Gen. 114, 97-110 (1973) Gaill, F.: Aspect ultrastructural de la glande pylorique et de l'intestin post6rieur de Sidnyum argus (Polyclinidae, Tuniciers). Cah. Biol. Mar. 15, 337 341 (1974a) Gaill, F.: Observations ultrastructurales de l'epithelium r6nal de Molgula occulta (Ascidies, Tuniciers). C.R. Acad. Sci. (Paris) 279, 351-353 (1974b) Goodbody, I.: The physiology of ascidians. Adv. Mar. Biol. 12, 1-150 (1974) Leeson, C.R., Leeson, T.S.: Staining methods for sections of epon-embedded tissues for light micro- scopy. Canad. J. Zool. 48, 189-191 (1970) Mackie, G.O., Paul, D.H., Singla, C.M., Sleigh, M.A., Williams, D.E.: Branchial innervation and ciliary control in the ascidian Corella. Proc. roy. Soc. B 187, 1-35 (1974) Millar, R.H.: Concretions in the pyloric gland of Ciona intestinalis. Nature (Lond.) 164, 717-718 (1949) Millar, R.H.: Ciona, L.M.B.C. Memoirs. Liverpool: Colman 1953 Overton, J.: The fine structure of blood cells in the ascidian Perophora viridis. J. Morph. 119, 305-326 (1966) Stetten, D. Jr., Stetten, M.R.: Glycogen metabolism. Physiol. Rev. 40, 505-537 (1960) Tandler, B. : Subsurface cisterns in mouse hepatocytes. Anat. Rec. 179, 273-284 (1974) Vonk, H.J.: Digestion and metabolism. In: Physiology of crustacea (T.H. Waterman, ed.), Vol. 1, p. 291. New York: Academic Press 1960 Accepted August 6, 1976