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A cytochemical and electron microscopic study of the thumb pad in Rana pipiens
Experimental
280
Cell Research
32, 280-288
A CYTOCHEMICAL AND ELECTRON MICROSCOPIC OF THE THUMB PAD IN RAW4 PIPIENS’ P. F. PARAKKAL Department
of
and
Dermatology, Boston University oj’ Biology, Brown University, Received
R.
STUDY
A. ELLIS
Medical Center, Boston, Mass. Providence, R. I., U.S.A.
December
(1963)
and Department
3, 1962
THE thumb pads are a secondary male sexual characteristic of Rana pipiens. The swelling of the thumb pads in the male is due to the growth of the mucous glands in the dermis and to the thickening of the epidermis. The thumb pad undergoes yearly cyclic changes. In the fall before the breeding season, the mucous glands begin to enlarge and the epidermis becomes thickened and folded. These changes reach their culmination during the breeding season when the glands attain their maximum size and the epidermis achieves maximum thickness. In late spring and summer the glands become smaller and the epidermis becomes thinner resulting in an overall reduction in size of the thumb pads. It has been demonstrated that the thumb pad cycle is under the control of the pituitary-gonadal axis [l, 4, 7, 161. Since the fine structure and chemical properties of the cellular constituents of the thumb pad of the male frog have not yet been studied in detail we carried out electron microscopic and histochemical studies to broaden our knowledge on this subject. MATERIALS
AND
METHODS
Thumb pads were excised from pithed frogs and fixed in Bouin’s and Helly’s fluids and embedded in paraffin. The sections were stained with Hematoxylin and eosin, PAS for glycogen and other mucopolysaccharides and Toluidine Blue buffered to pH 4.5 for basophilia. Fresh frozen sections were used for the demonstration of succinic dehydrogenase, cytochrome oxidase and monoamine oxidase by the methods of Farber and Louviere [3], Burstone [2] and Glenner et al. [5] respectively. Alkaline phosphatase [6], acid phosphatase [15], non-specific esterases [12], and cholinesterase [9] were studied in 20-50 ,U frozen sections of tissues after fixation for 4 hr in chilled 10 per cent formalin. For electron microscopy the thumb pads were excised and fixed in ice cold 2 per cent osmium tetroxide in veronal-acetate buffer at pH 7.4-7.6 [13]. Small pieces of 1 This work AM-K3-H938, Experimental
was supported GhI-08380-02 Cell Research
32
by grants and RG2125
from (Cll).
The
National
Institutes
of Health
No. A5779,
Thumb
pad of Rana
pipiens
epidermis and glands were isolated from these pads under the dissecting microscope and then placed in fresh buffered osmium and fixed for 2 hr. The tissue was rinsed in distilled water, dehydrated through an ethanol series and embedded either in methacrylate or Epoxy resins [8]. Sections 250-600 A thick were cut with glass knives on a Porter Blum microtome and after staining with lead acetate [17], they were examined with an RCA EMU 3D electron microscope equipped with a 40 p platinum objective aperture. Electron micrographs were taken at initial magnification of 4000 to 8000 and enlarged photographically as desired. RESULTS
Epidermis.-The thumb pad epidermis of the male Rana pipiens is highly papillated (Fig. 2) during the breeding season. The epidermal cell boundaries and the numerous “intercellular bridges” are well demonstrated with toluidine blue. The cytoplasm contains small basophilic granules and a PASpositive, saliva-resistant material. The outer two-thirds of the epidermis reacts for alkaline phosphatase, acid phosphatase and alpha naphthol esterase and is moderately reactive for monoamine oxidase. The formazan granules produced by the succinic dehydrogenase and cytochrome oxidase techniques are uniformly distributed throughout the entire epidermis. When the epidermal cells are examined under the electron microscope the ubiquitous distribution of mitochondria is consistent with the pattern revealed histochemically. Numerous ribosomes are randomly dispersed in the cytoplasm. Rough-surfaced endoplasmic reticulum is almost non-existent in these cells, and organized Golgi zones are not apparent. Cytoplasmic tilaments and mucous granules are scattered throughout the entire cytoplasm. The filaments in many instances appear to terminate at the sites of desmosomes. The desmosomes are numerous and seem to be regularly spaced along the cell surface. Glands.-During the breeding season there are numerous mucous glands under the thickened epidermis of the thumb pad of the male frog (Fig. 2). These glands are much larger than the mucous glands found elsewhere in the skin (Fig. 1) and much larger than the mucous glands observed at similar sites in the skin of female frogs. They consist of columnar cells with basally placed nuclei. The glandular epithelium reveals many PAS-positive and saliva-resistant granules. It reacts strongly for alkaline phosphatase, acid phosphatase and alpha-naphthol esterase. Acid phosphatase and alpha naphthol esterase are uniformly distributed in the whole gland, while alkaline phosphatase is concentrated at the apices of the cells (Fig. 3). The glands are moderately reactive for monoamine oxidase and show no reaction for both acetyl and butyrylcholinesterase. 19 -
621816
Experimental
Cell Research
32
P. F. Parakkal
and R. A. Ellis
In contrast to the uniform distribution in the glands of enzymes mentioned above, marked cell differences were recorded in succinic dehydrogenase and cytochrome oxidase activity. A few cells randomly scattered in the glands reacted intensely when tested for succinic dehydrogenase and cytochrome oxidase while the remaining epithelium was non-reactive. The reactive cells are shown in Fig. 4 and 5. The presence of succinic dehydrogenase and cytochrome oxidase in certain cells of the gland and their absence from the others serves as a chemical mark that facilitates the distinction of two cell types within the glands. Electron microscopic studies confirmed that the glands consist of two different types of epithelial cells. The most numerous are the secretory cells. These have a basally placed pleomorphic nucleus that is elongated in the direction of the long axis of the cell and a deeply folded nuclear envelope. The cytoplasm contains a few mitochondria, a well-developed rough-surfaced endoplasmic reticulum, many vesicles and membrane bound mucous granules (Fig. 7). A well-developed Golgi zone was not noted. The basal portion of the cells is filled mainly with rough-surfaced endoplasmic reticulum, while the apical part contains many membrane bound mucous granules. The endoplasmic reticulum seemed to be involved in the formation of the mucous granules. Dilatations of the endoplasmic reticulum and the appearance of some amorphous granular material within these cisterns are the first stage in the formation of the mucous granules. The small and large vesicles containing an amorphous granular substance and many RNP particles at the outer rim seem to correspond to advanced stages of mucous granule formation. In the center of the fully formed granules, condensations of mucus can be seen as variously shaped structures (Fig. 8). The terminal granules are uniform in size and attain a maximum diameter of about 1.5 p.
Fig. thin
I.-The skin of female epidermis. x 50.
Fig. Z.-The and highly
thumb papillated
Fig. 3.-Mucous apical region.
frog,
stained
with
pad of male frog during epidermis. x 50.
glands x 100.
of the thumb
H-E.
Note
the breeding
pad reveal
intense
small
mucous
season.
Note
reaction
glands
and non-papillated
the enlarged
for alkaline
mucous
phosphatase
Fig. 4.-Arrows point to non-secretory cells of the frog’s thumb pad gland. They react for succinic dehydrogenase during the breeding season. The secretory cells are non-reactive. Fig.
5.-Same
as Fig.
Fig. 6.-Mucous gland during the non-breeding Experimenfal
4 at higher
magnification.
of the thumb pad showing season. x 450.
Ceil Research
32
glands at the intensely x 450.
x 900. uniform
reactivity
for succinic
dehydrogenase
Thumb
pad of Rana
283
pipiens
Experimental
Cell Research
32
284
P. F. Parakkal
and R. A. Ellis
The second cell type is less numerous. Its nucleus is more regularly shaped than that of the secretory cells. The cytoplasm as a whole is clear in comparison to that of the surrounding secretory cells. No rough-surfaced endoplasmic reticulum or mucous granules were observed in their cytoplasm, but mitochondria are abundant and there is a well-developed Golgi region (Fig. 9). The glands become smaller during the non-breeding season and their columnar secretory cells are replaced by cuboidal or squamous cells. At this time two cell types can no longer be distinguished histochemically since all the cells in the glands are reactive for both succinic dehydrogenase and cytochrome oxidase (Fig. 6). DISCUSSION
The results of this study show that the mucous glands of the thumb pad of the male frog increase enormously in size during the breeding season and consist of two morphologically different cell types. The bulk of the gland is made up of secretory cells. This cell type is similar to the goblet cell of the intestine [ 111 and trachea [141 since it possesses an irregularly shaped or indented nucleus. Distinct differences however between these and other mucous secreting cells can be noticed when the cytoplasmic constituents are examined critically. In the goblet cells of the intestinal mucosa, the apical cytoplasm is occupied by coalesced mucous droplets [ll]. The individual mucous granules are thought to be elaborated through the Golgi membranes and when formed, are surrounded by unit membranes that disappear when goblets are produced [ 11, 141. In the secretory cells of the gland of the thumb pad the mucous granules are never found to coalesce and each granule is limited by a unit membrane. A well-developed Golgi zone is not discernible in the secretory cell, although they possess a well-organized rough-surfaced endoplasmic reticulum. The secretory granules are formed in the dilatations of the endoplasmic reticulum and not within the Golgi apparatus. Thus, the formation of secretory granules in the mucous glands of the thumb pad seems to follow a different pattern from that in the goblet cells and resembles more closely the pattern of the formation of zymogen granules seen in the pancreatic acinar cells [lo].
Fig. 7.-Electron micrograph of secretory cells of the mucous gland of the thumb pad. The cells contain a well-developed rough-surfaced endoplasmic reticulum (ER) and many mucous granules in various stages of formation (arrows). Fully formed mucous granules reveal condensation of material in the central portion (MU). x 28,800. Experimental
Cell Research
32
Thumb
pad of Rana
pipiens
Experimental
Cell
Research
32
P. F. Parakkal
and R. A. Ellis
The second cell type in the gland of the thumb pad appears to be nonsecretory. These cells are present only in small numbers during the breeding season. They are devoid of mucous granules but contain a large number of mitochondria. The exact role of this cell type is not known, but it is probable that they represent a stem cell that may be responsible for the yearly renewal of the cell population in the gland. The fact that the mucous glands are devoid of secretory cells during the non-breeding season but are tilled with nonsecretory cells that are positive for succinic dehydrogenase and cytochrome oxidase seems to favor this hypothesis, but additional information is needed for final confirmation. SUMMARY
The epidermis and the mucous gland of the thumb pad of male Rana were studied by histochemical and electron microscopic methods. During the breeding season the epidermis of the thumb thickens and becomes highly papillated while the mucous glands beneath increase enormously in size. The outer two-thirds of the epidermis reacts positively for alkaline phosphatase, acid phosphatase, alpha naphthol esterase, monoamine oxidase, succinic dehydrogenase and cytochrome oxidase. The mucous glands were found to consist of two different cell types. Most numerous are the typical secretory cells that give positive histochemical tests for alkaline phosphatase, acid phosphatase, alpha naphthol esterase but are unreactive for succinic dehydrogenase and cytochrome oxidase. Electron microscopic studies of these cells reveal typical secretory granules closely associated with the endoplasmic reticulum. The second cell type is nonsecretory that gives a strong positive reaction for succinic dehydrogenase and cytochrome oxidase and contains a rich complement of mitochondria. pipiens
We wish to thank Dr. A. G. Matoltsy making helpful suggestions.
for critically
reading
the manuscript
and
REFERENCES 1. 2. 3. 4.
ARON, M., Arch. Biol. 36, 3 (1926). BURSTONE, M. S., J. Histochem. Cytochem. 8, 63 (1960). FARBER, E. and LOUVIERE, C. D., J. Histochem. Cytochem. GLASS, F. M. and RUGH, R., J. Morphol. 74, 409 (1944).
Fig. S.-Electron cell. x 15,600.
micrograph
Fig. B.-Electron thumb pad. Note
micrograph shows a portion of a non-secretory cell in the mncons gland of the clear cytoplasm, many mitochondria (Mi), and Golgi zone (C). x 10,500.
Experimental
Cell
Research
shows
32
terminal
mucous
granules
4, 347 (1956).
located
at the apex
of a secretory
Thumb
pad of Rana
pipiens
287
Experimental
Cell Research
32
P. F. Parakkal and R. A. Ellis 5. GLENNER, G. G., BURTNER, H. J. and BROWN, G. W., JR., J. Histochem. Cyfochem. 5, 591 (1957). 6. GOMORI, G., in Microscopic Histochemistry. Principles and Practice, p. 184. The University of Chicago Press, Chicago, 1951. 7. KERMIT, C., And. Record 48, 241 (1948). 8. LUFT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 9. MONTAGNA, W. and ELLIS, R. A., J. Inuest. Derm. 29, 151 (1957). 10. PALADE, G. E. and SIEKEVITZ, P., J. Biophys. Biochem. Cytol. 2, 671 (1956). 11. PALAY, S. L., in Frontiers of Cytology, p. 305. Yale University Press, New Haven, 1958. 12. PEARSE, A. G. E., in Histochemistry, Theoretical and Applied, p. 462. Little, Brown and co., 1954. 13. PEASE, D., in Histological Techniques for Electron Microscopy, p. 36. Academic Press, New York, 1960. 14. RHODIN, J. and DALHAMN, T., 2. Zellforsch. Mikroskop. Anat. 44. 345 (1956). 15. RUTENBURG, A. M. and SEUG~AN, A.‘M., J. Histochem. Cytochem: 3, 55‘(1955). 16. SLUITER, J. W., VAN OORDT, G. J. and MIGHORST, C. A., fluart. J. Microscop Sci. 91. 131 _ (1950). 17. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958).
Experimental
Cell Research
32
280
Cell Research
32, 280-288
A CYTOCHEMICAL AND ELECTRON MICROSCOPIC OF THE THUMB PAD IN RAW4 PIPIENS’ P. F. PARAKKAL Department
of
and
Dermatology, Boston University oj’ Biology, Brown University, Received
R.
STUDY
A. ELLIS
Medical Center, Boston, Mass. Providence, R. I., U.S.A.
December
(1963)
and Department
3, 1962
THE thumb pads are a secondary male sexual characteristic of Rana pipiens. The swelling of the thumb pads in the male is due to the growth of the mucous glands in the dermis and to the thickening of the epidermis. The thumb pad undergoes yearly cyclic changes. In the fall before the breeding season, the mucous glands begin to enlarge and the epidermis becomes thickened and folded. These changes reach their culmination during the breeding season when the glands attain their maximum size and the epidermis achieves maximum thickness. In late spring and summer the glands become smaller and the epidermis becomes thinner resulting in an overall reduction in size of the thumb pads. It has been demonstrated that the thumb pad cycle is under the control of the pituitary-gonadal axis [l, 4, 7, 161. Since the fine structure and chemical properties of the cellular constituents of the thumb pad of the male frog have not yet been studied in detail we carried out electron microscopic and histochemical studies to broaden our knowledge on this subject. MATERIALS
AND
METHODS
Thumb pads were excised from pithed frogs and fixed in Bouin’s and Helly’s fluids and embedded in paraffin. The sections were stained with Hematoxylin and eosin, PAS for glycogen and other mucopolysaccharides and Toluidine Blue buffered to pH 4.5 for basophilia. Fresh frozen sections were used for the demonstration of succinic dehydrogenase, cytochrome oxidase and monoamine oxidase by the methods of Farber and Louviere [3], Burstone [2] and Glenner et al. [5] respectively. Alkaline phosphatase [6], acid phosphatase [15], non-specific esterases [12], and cholinesterase [9] were studied in 20-50 ,U frozen sections of tissues after fixation for 4 hr in chilled 10 per cent formalin. For electron microscopy the thumb pads were excised and fixed in ice cold 2 per cent osmium tetroxide in veronal-acetate buffer at pH 7.4-7.6 [13]. Small pieces of 1 This work AM-K3-H938, Experimental
was supported GhI-08380-02 Cell Research
32
by grants and RG2125
from (Cll).
The
National
Institutes
of Health
No. A5779,
Thumb
pad of Rana
pipiens
epidermis and glands were isolated from these pads under the dissecting microscope and then placed in fresh buffered osmium and fixed for 2 hr. The tissue was rinsed in distilled water, dehydrated through an ethanol series and embedded either in methacrylate or Epoxy resins [8]. Sections 250-600 A thick were cut with glass knives on a Porter Blum microtome and after staining with lead acetate [17], they were examined with an RCA EMU 3D electron microscope equipped with a 40 p platinum objective aperture. Electron micrographs were taken at initial magnification of 4000 to 8000 and enlarged photographically as desired. RESULTS
Epidermis.-The thumb pad epidermis of the male Rana pipiens is highly papillated (Fig. 2) during the breeding season. The epidermal cell boundaries and the numerous “intercellular bridges” are well demonstrated with toluidine blue. The cytoplasm contains small basophilic granules and a PASpositive, saliva-resistant material. The outer two-thirds of the epidermis reacts for alkaline phosphatase, acid phosphatase and alpha naphthol esterase and is moderately reactive for monoamine oxidase. The formazan granules produced by the succinic dehydrogenase and cytochrome oxidase techniques are uniformly distributed throughout the entire epidermis. When the epidermal cells are examined under the electron microscope the ubiquitous distribution of mitochondria is consistent with the pattern revealed histochemically. Numerous ribosomes are randomly dispersed in the cytoplasm. Rough-surfaced endoplasmic reticulum is almost non-existent in these cells, and organized Golgi zones are not apparent. Cytoplasmic tilaments and mucous granules are scattered throughout the entire cytoplasm. The filaments in many instances appear to terminate at the sites of desmosomes. The desmosomes are numerous and seem to be regularly spaced along the cell surface. Glands.-During the breeding season there are numerous mucous glands under the thickened epidermis of the thumb pad of the male frog (Fig. 2). These glands are much larger than the mucous glands found elsewhere in the skin (Fig. 1) and much larger than the mucous glands observed at similar sites in the skin of female frogs. They consist of columnar cells with basally placed nuclei. The glandular epithelium reveals many PAS-positive and saliva-resistant granules. It reacts strongly for alkaline phosphatase, acid phosphatase and alpha-naphthol esterase. Acid phosphatase and alpha naphthol esterase are uniformly distributed in the whole gland, while alkaline phosphatase is concentrated at the apices of the cells (Fig. 3). The glands are moderately reactive for monoamine oxidase and show no reaction for both acetyl and butyrylcholinesterase. 19 -
621816
Experimental
Cell Research
32
P. F. Parakkal
and R. A. Ellis
In contrast to the uniform distribution in the glands of enzymes mentioned above, marked cell differences were recorded in succinic dehydrogenase and cytochrome oxidase activity. A few cells randomly scattered in the glands reacted intensely when tested for succinic dehydrogenase and cytochrome oxidase while the remaining epithelium was non-reactive. The reactive cells are shown in Fig. 4 and 5. The presence of succinic dehydrogenase and cytochrome oxidase in certain cells of the gland and their absence from the others serves as a chemical mark that facilitates the distinction of two cell types within the glands. Electron microscopic studies confirmed that the glands consist of two different types of epithelial cells. The most numerous are the secretory cells. These have a basally placed pleomorphic nucleus that is elongated in the direction of the long axis of the cell and a deeply folded nuclear envelope. The cytoplasm contains a few mitochondria, a well-developed rough-surfaced endoplasmic reticulum, many vesicles and membrane bound mucous granules (Fig. 7). A well-developed Golgi zone was not noted. The basal portion of the cells is filled mainly with rough-surfaced endoplasmic reticulum, while the apical part contains many membrane bound mucous granules. The endoplasmic reticulum seemed to be involved in the formation of the mucous granules. Dilatations of the endoplasmic reticulum and the appearance of some amorphous granular material within these cisterns are the first stage in the formation of the mucous granules. The small and large vesicles containing an amorphous granular substance and many RNP particles at the outer rim seem to correspond to advanced stages of mucous granule formation. In the center of the fully formed granules, condensations of mucus can be seen as variously shaped structures (Fig. 8). The terminal granules are uniform in size and attain a maximum diameter of about 1.5 p.
Fig. thin
I.-The skin of female epidermis. x 50.
Fig. Z.-The and highly
thumb papillated
Fig. 3.-Mucous apical region.
frog,
stained
with
pad of male frog during epidermis. x 50.
glands x 100.
of the thumb
H-E.
Note
the breeding
pad reveal
intense
small
mucous
season.
Note
reaction
glands
and non-papillated
the enlarged
for alkaline
mucous
phosphatase
Fig. 4.-Arrows point to non-secretory cells of the frog’s thumb pad gland. They react for succinic dehydrogenase during the breeding season. The secretory cells are non-reactive. Fig.
5.-Same
as Fig.
Fig. 6.-Mucous gland during the non-breeding Experimenfal
4 at higher
magnification.
of the thumb pad showing season. x 450.
Ceil Research
32
glands at the intensely x 450.
x 900. uniform
reactivity
for succinic
dehydrogenase
Thumb
pad of Rana
283
pipiens
Experimental
Cell Research
32
284
P. F. Parakkal
and R. A. Ellis
The second cell type is less numerous. Its nucleus is more regularly shaped than that of the secretory cells. The cytoplasm as a whole is clear in comparison to that of the surrounding secretory cells. No rough-surfaced endoplasmic reticulum or mucous granules were observed in their cytoplasm, but mitochondria are abundant and there is a well-developed Golgi region (Fig. 9). The glands become smaller during the non-breeding season and their columnar secretory cells are replaced by cuboidal or squamous cells. At this time two cell types can no longer be distinguished histochemically since all the cells in the glands are reactive for both succinic dehydrogenase and cytochrome oxidase (Fig. 6). DISCUSSION
The results of this study show that the mucous glands of the thumb pad of the male frog increase enormously in size during the breeding season and consist of two morphologically different cell types. The bulk of the gland is made up of secretory cells. This cell type is similar to the goblet cell of the intestine [ 111 and trachea [141 since it possesses an irregularly shaped or indented nucleus. Distinct differences however between these and other mucous secreting cells can be noticed when the cytoplasmic constituents are examined critically. In the goblet cells of the intestinal mucosa, the apical cytoplasm is occupied by coalesced mucous droplets [ll]. The individual mucous granules are thought to be elaborated through the Golgi membranes and when formed, are surrounded by unit membranes that disappear when goblets are produced [ 11, 141. In the secretory cells of the gland of the thumb pad the mucous granules are never found to coalesce and each granule is limited by a unit membrane. A well-developed Golgi zone is not discernible in the secretory cell, although they possess a well-organized rough-surfaced endoplasmic reticulum. The secretory granules are formed in the dilatations of the endoplasmic reticulum and not within the Golgi apparatus. Thus, the formation of secretory granules in the mucous glands of the thumb pad seems to follow a different pattern from that in the goblet cells and resembles more closely the pattern of the formation of zymogen granules seen in the pancreatic acinar cells [lo].
Fig. 7.-Electron micrograph of secretory cells of the mucous gland of the thumb pad. The cells contain a well-developed rough-surfaced endoplasmic reticulum (ER) and many mucous granules in various stages of formation (arrows). Fully formed mucous granules reveal condensation of material in the central portion (MU). x 28,800. Experimental
Cell Research
32
Thumb
pad of Rana
pipiens
Experimental
Cell
Research
32
P. F. Parakkal
and R. A. Ellis
The second cell type in the gland of the thumb pad appears to be nonsecretory. These cells are present only in small numbers during the breeding season. They are devoid of mucous granules but contain a large number of mitochondria. The exact role of this cell type is not known, but it is probable that they represent a stem cell that may be responsible for the yearly renewal of the cell population in the gland. The fact that the mucous glands are devoid of secretory cells during the non-breeding season but are tilled with nonsecretory cells that are positive for succinic dehydrogenase and cytochrome oxidase seems to favor this hypothesis, but additional information is needed for final confirmation. SUMMARY
The epidermis and the mucous gland of the thumb pad of male Rana were studied by histochemical and electron microscopic methods. During the breeding season the epidermis of the thumb thickens and becomes highly papillated while the mucous glands beneath increase enormously in size. The outer two-thirds of the epidermis reacts positively for alkaline phosphatase, acid phosphatase, alpha naphthol esterase, monoamine oxidase, succinic dehydrogenase and cytochrome oxidase. The mucous glands were found to consist of two different cell types. Most numerous are the typical secretory cells that give positive histochemical tests for alkaline phosphatase, acid phosphatase, alpha naphthol esterase but are unreactive for succinic dehydrogenase and cytochrome oxidase. Electron microscopic studies of these cells reveal typical secretory granules closely associated with the endoplasmic reticulum. The second cell type is nonsecretory that gives a strong positive reaction for succinic dehydrogenase and cytochrome oxidase and contains a rich complement of mitochondria. pipiens
We wish to thank Dr. A. G. Matoltsy making helpful suggestions.
for critically
reading
the manuscript
and
REFERENCES 1. 2. 3. 4.
ARON, M., Arch. Biol. 36, 3 (1926). BURSTONE, M. S., J. Histochem. Cytochem. 8, 63 (1960). FARBER, E. and LOUVIERE, C. D., J. Histochem. Cytochem. GLASS, F. M. and RUGH, R., J. Morphol. 74, 409 (1944).
Fig. S.-Electron cell. x 15,600.
micrograph
Fig. B.-Electron thumb pad. Note
micrograph shows a portion of a non-secretory cell in the mncons gland of the clear cytoplasm, many mitochondria (Mi), and Golgi zone (C). x 10,500.
Experimental
Cell
Research
shows
32
terminal
mucous
granules
4, 347 (1956).
located
at the apex
of a secretory
Thumb
pad of Rana
pipiens
287
Experimental
Cell Research
32
P. F. Parakkal and R. A. Ellis 5. GLENNER, G. G., BURTNER, H. J. and BROWN, G. W., JR., J. Histochem. Cyfochem. 5, 591 (1957). 6. GOMORI, G., in Microscopic Histochemistry. Principles and Practice, p. 184. The University of Chicago Press, Chicago, 1951. 7. KERMIT, C., And. Record 48, 241 (1948). 8. LUFT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 9. MONTAGNA, W. and ELLIS, R. A., J. Inuest. Derm. 29, 151 (1957). 10. PALADE, G. E. and SIEKEVITZ, P., J. Biophys. Biochem. Cytol. 2, 671 (1956). 11. PALAY, S. L., in Frontiers of Cytology, p. 305. Yale University Press, New Haven, 1958. 12. PEARSE, A. G. E., in Histochemistry, Theoretical and Applied, p. 462. Little, Brown and co., 1954. 13. PEASE, D., in Histological Techniques for Electron Microscopy, p. 36. Academic Press, New York, 1960. 14. RHODIN, J. and DALHAMN, T., 2. Zellforsch. Mikroskop. Anat. 44. 345 (1956). 15. RUTENBURG, A. M. and SEUG~AN, A.‘M., J. Histochem. Cytochem: 3, 55‘(1955). 16. SLUITER, J. W., VAN OORDT, G. J. and MIGHORST, C. A., fluart. J. Microscop Sci. 91. 131 _ (1950). 17. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958).
Experimental
Cell Research
32