Histochemie 10, 33--43 (1967)

The Uhrastructural Localization of Acid Phosphatase, Alkaline Phosphatase and Adenosine Triphosphatase

in Induced Goitres ofXenopus laevis Daudin Tadpoles*

R. COLEMAN, P. J . EV~NN~TT, and J . M. DODD

Department of Zoology, The University of Leeds, Leeds 2, England

Received February 10, 1967

Summary. Tadpoles of Xenopus laevis were maintained in solutions of potassium per- chlorate, potassium thiocyanate, thiourea and propyl-thiouracil {0.005 % w/v). Metamorphosis was inhibited and within a short time large well-vascularized goitres developed. The locali- zation of certain phosphatases at the ultrastructural level in these goitres is described and the possible roles played by these enzymes is discussed. Acid phosphatase was localized in follicle epithelial cells in dense droplets thought to be lysosomes or cytolysomes. Alkaline phospha- tase and a Mg++-activated ATP-ase were found on the outer surfaces of the membranes of follicular cells and on the walls of capillary blood vessels.

I t is now well es tabl i shed t h a t the t h y r o i d glands of a m p h i b i a n l a rvae are h igh ly ac t ive t h roughou t me tamorphos i s and a t the m e t a m o r p h i c c l imax t h e y are u n d o u b t e d l y amongs t the most ac t ive thy ro ids encounte red in the An ima l K ingdom. I n view of th is t h e y would a p p e a r to be pa r t i cu l a r ly su i tab le for s tudies on the local iza t ion of enzymes a t the level of u l t r a s t ruc tu re . So far such s tudies appea r to have been res t r i c ted to mammals .

I n the present work the a l r eady high level of a c t i v i t y of the t hy ro id cells of Xenopus l a rvae has been enhanced b y treatment with a variety o] goitrogens. GASCHE (1946) and GASCItE and DI~UEY (1946) showed t h a t a n u m b e r of chemicals in solut ion in wa te r conta in ing la rvae of Xenopus resul ted in m e t a m o r p h i c stasis and in gross cellular h y p e r t r o p h y and hyperp la s i a of the t h y r o i d glands. W e have ex t ended this work to include o the r goi trogens and have found t h a t t he ensuing goitres can account for up to 12% of the t o t a l body weight of the t r e a t e d larvae . This p r o b a b l y represents a g rea te r re la t ive degree of t hy ro id s t imula t ion t h a n has been p roduced in a n y o ther an imal and is fu r the r evidence of the except ion- a l ly high levels of a c t i v i t y shown b y the t h y r o i d glands of tadpoles .

The in t roduc t ion of new techniques for localizing enzymes in cells w i thou t des t roy ing a n y of the cell a rchi tec ture , and the more recent combina t ion of these wi th e lect ron microscopy, has enabled work to s t a r t on corre la t ing s t ruc ture and funct ion a t the level of u l t r a s t rue tu re . A number of such s tudies has a l r e a dy been r epor t ed on m a m m a l i a n t hy ro id t issue (see Discussion), b u t this is the ]irst work to appear on the ultrastructural enzymology o/the amphibian thyroid gland. I n i t , the cellular local iza t ion of acid phospha tase , a lkal ine phospha tase and aden- osine t r i phospha ta se (ATP-ase) is descr ibed in induced Xenopus goitres and the

* This work was carried out during the tenure by one of us (R.C.) of a Medical Research Council Scholarship and forms part of a programme of research in amphibian thyroid physi- ology supported by a grant from the Medical Research Council, which we gratefully acknow- ledge.

3 Histochemie, Bd. 10

34 R. COLEMAN, P. J. EVENNETT and J . M . DODD:

poss ib l e ro les of t h e s e e n z y m e s is d i s c u s s e d 1. T h e s i tes of l o c a l i z a t i o n of t h e s e

e n z y m e s a r e i d e n t i c a l t o t h o s e f o u n d in u n t r e a t e d t a d p o l e s a t a c o r r e s p o n d i n g

s t a g e of d e v e l o p m e n t t h o u g h , of course , t h e a b s o l u t e a m o u n t of e n z y m e r e a c t i o n

p r o d u c t l oca l i zed i n t h e go i t r c s is m u c h g r e a t e r . T h u s t h e i n c r e a s e in cell size,

m e t a b o l i c a c t i v i t y a n d e n z y m e a c t i v i t y m a k e s t h e s e g o i t r o u s g l a n d s e spec ia l ly

s u i t a b l e fo r c y t o c h e m i c a l s t u d i e s of e n z y m e loca l i za t ion .

M a t e r i a l a n d M e t h o d s

Material Fertilized eggs of Xenopus laevis were obtained by injecting adult toads of both sexes

with chorionic gonadotropin. The resulting tadpoles were reared a t 22~ in a constant tem- perature room and fed on liver powder. Stage of development was determined by reference to the Normal Table for Xenopus (NIEuW~:OOP and FABleR, 1956).

To obtain a continuing supply of goitrous thyroid tissue, 60 tadpoles at stage 55/56 were immersed in each of the following goitrogens: potassium thiocyanate, potassium perchlorate, thiourea and propyl-thiouracil, a t a concentration of 0.005% w/v in tap water. A similar series from a second ba tch of tadpoles was set up at stage 58. The tadpoles were kept in the goitrogens in glass jars of 3 litre capacity, each containing 30 animals. Solutions were changed twice weekly. The development of the goitres was examined over a period of 20 months.

Methods

a) P r e p a r a t i o n of S p e c i m e n s

The thyroid tissue was fixed by immersion in cold buffered glutaraldehyde fixative. The goitre was immediately cut or teased into small (c. 1 mm a) pieces in the fixative and following f ixation and washing the pieces of goitre were incubated in the relevant incubation medium. Tissues were post-fixed in veronal acetate-buffered 1% osmium tetroxide (pH 7.4), dehy- dra ted in graded ethanols, t reated with propylene oxide and embedded in Shell Epikote Resin (epon 812) which was polymerized at 60~ for 24 hours. Thin sections (silver) were cut with glass knives on a Cambridge (Huxley) ul tramicrotome and mounted on uncoated or carbon-coated copper grids. Sections were stained with 5% aqueous uranyl acetate for up to 8 hours and examined in an AEI EM 6 B electron microscope with a 25 ~z or 50 ~ objective aperture a t 60 or 80 kv. Micrographs were taken at original magnifications varying from 2,000 to 40,000.

Some pieces of tissue were briefly immersed in 1% ammonium sulphide after incubation, embedded as described above and sectioned a t 0.5--2.0 [z. These sections were stained with Azur I I in borax (JEoN, 1965) and examined in the light microscope. In sulphide-treated blocks, deposits of lead sulphide reveal sites of enzyme action.

Sections at 7.5 ~ were cut from wax-embedded whole goitres and stained with haema- toxylin and eosin, or Periodic acid-Schiff (PAS) to observe gross morphological changes in the gland.

b) T e c h n i q u e s for E n z y m e L o c a l i z a t i o n

Lead-salt replacement techniques were used for enzyme localization (GOMORI, 1952). Electron-dense lead ions are captured at the site of enzyme hydrolysis and are seen in the electron microscope as clumps of lead phosphate, which can be converted to black lead sulphide deposits for light microscope study. Fixat ion and all preparative techniques up to ethanol dehydrat ion were performed at 4~ expect for the incubation in the substrate media.

For the sake of brevi ty the enzymes are referred to directly as, e.g., "ATP-ase" ra ther than the more precise "ATP-ase act ivi ty". I t is also recognized t ha t each of the phosphatases described is likely to consist of several enzymes.

Phosphatases in Tadpole Goitres 35

I. Acid Phosphatase (based on MILLER, 1962; ERICSSON and T~uMP, 1965).

1. Fix in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 (2 hrs). 2. Wash in 0.1 M cacodylate buffer containing 11.5% sucrose (15 mins). 3. Rinse in 0.05 M acetate buffer containing 7.5% sucrose (1 min). 4. Incuba te in Gomori medium pH 5 at 37~ (20 rains). 0.6 g lead ni t ra te is dissolved

in 500 ml of 0.05 M acetate buffer. To this solution is added 50 ml freshly prepared 3 % sodium fi-glycerophosphate. The turbid solution is kept at 37~ overnight and filtered before use.

5. Rinse in 2% acetic acid (1 min). 6. Rinse in 0.05 M acetate buffer containing 7.5% sucrose (1 rain). 7. Post-fix and embed.

II . Alkaline Phosphatase (based on TRANZ~R, 1965).

1. Fix in 1.5 % glutaraldehyde buffered to pH 7 with Michaelis buffer (1.5 hours). 2. Incuba te in Gomori-type medium at 37~ (20 rains): 3% Sodium fl-glycerophosphate

0.1 ml; 3% Veronal 6 ml; 0.1 M lead n i t ra te 1 ml; 0.1 M Trisodium citrate 1.1 mh Adjust pH to about 9.

3. Rinse in buffer (1 min). 4. Rinse in 2% acetic acid (1 rain). 5. Rinse in buffer (1 rain). 6. Post-fix and embed.

III . Adenosine Triphosphatase (based on WAeHSTEI~ and MEISEL, 1957; FARQVEAR and

PALADE, 1966). 1. Fix in 1.5% glutaraldehyde in 0.067 M sodium cacodylate buffer pH 7.2 (2 hours). 2. Wash in 0.1 M sodium cacodylate buffer containing 7 To sucrose (2 changes of 30 mins). 3. Incuba te at 37~ (50 mins): 125 mg per cent ATP (disodium salt) 20 ml; 0.2 M TRIS-

HC1 buffer pI-I 7.2 20 ml; 2% lead ni t rate 3 ml; 0.1 M magnesium sulphate 5 ml; Distilled water 2 ml. Adjust pH to 7.2 with 0.5 M NaOH before incubation.

4. Rinse in veronal-acetate buffer containing 7 % sucrose (pH 7.4). 5. Post-fix and embed. Identical media were used for some pieces of tissue except t h a t 0.1 mM ouabain (Stro-

phanth in-G) or 5 • 10 s M BAL (2,3,dimercapto-l-propanol) were added. I n a fur ther medium the ATP was replaced by sodium fl-glycerophosphate. Control media were prepared by omit t ing the substrate from the incubat ion medium.

Results The first noticeable result of treatment of tadpoles with goitrogen is the

retardation or cessation of metamorphosis. They may complete one or two further stages of metamorphosis but subsequently no further development occurs except in a small proportion of animals, which slowly change into young toads, but even in these the thyroid glands are goitrous. Over a period of months the neotenic tadpoles grow to a large size (up to 10 em in length) and the devel- opment of the goitre, seen as a well-vascularized outgrowth from the lower jaw, is apparent (Figs. 1 a, b). No differences can be detected either in the external appearance or in the histology of the goitres induced by the various different goitrogens, although these goitrogens are believed to affect different cellular mechanisms.

In the follicular cells of the normal tadpole thyroid there is a central nucleus, the infra-nuclear (basal) region being occupied by ergastoplasm. The supra- nuclear (apical) region contains dense droplets, less dense colloid droplets and Golgi apparatus. At the apical edge of the cell are varying numbers of micro- villi which project into the lumen of the follicle which is usually large and filled

3*

Fig. 1 a - - c (for legend see p. 37)

R. COLEMAN, P. J. EVENNISTT and J. M. DODD: Phosphatases in Tadpole Goitres 37

with colloid. Pronounced intercellular spaces, which expand into lacunae at intervals, are often found between contiguous follicular cells and the cell mem- brane bordering the lacunae is usually microvillous.

The first noticeable histological change in the thyroids following administra- tion of goitrogen is a drastic reduction in the amount of PAS-stainable colloid in the lumina of follicles ; thisis apparent within a few days. The follicular epithelial cells assume a columnar appearance and reach a height of up to 80 [z. This hyper- t rophy is mainly due to the great increase in the amount of infra-nuclear endo- plasmic reticulum. Cellular hypertrophy is accompanied by hyperplasia and increased vascularization of the gland. In many cases after prolonged administra- tion of goitrogen the hypertrophy and hyperplasia is so great as to cause the follicular lumina to be greatly diminished in size or completely occluded and many extensive compact cellular regions may develop in the gland. Many more mitotic figures are seen in the goitre than is usual in the untreated tadpole gland. In addition, there is a great increase in the number of Azur II-staining droplets found in the supra-nuclear zone of the epithelial cells (Fig. 1 c). Goitrogens also appear to cause an increase in the numbers of "parafollicular" cells which are of two types, "light" cells and others filled with many densely staining droplets. In many of the goitres examined colloid cells (UHL]SNHUTH, 1924) were found in the epithelium, and these were filled with PAS-positive material resembling col- loid. "Parafollicular" cells and colloid cells will not be considered further in this paper as they are the subjects of continuing investigations.

At the ultrastructural level acid phosphatase reaction product (lead phos- phate) appeared in the form of relatively large clumps of electron-dense deposits formed by the aggregation of fine particles. Acid phosphatase was detected in many of the membrane-limited electron-dense droplets in the supra-nuclear zone of follicular epithelial cells (Fig. 2a). There appeared from micrographs to be up to six or seven sites of clumping in each of these droplets. Acid phosphatase reaction product was also detected in dense droplets containing heterogeneous membranous structures (Fig. 2b). Acid phosphatase is known as a typical lyso- somal enzyme (DE Duv]~, 1963; DE Duvs~ and WATTXAUK, 1966) and presumably the dense bodies described here, which correspond to the densely-staining Azur II-positive droplets of light microscope studies, are lysosomes and cytolysomes. Occasionally some lead deposits were detected in nuclei, but this localization was not thought to indicate sites of acid phosphatase activity (see DEANE, 1963).

Non-specific alkaline phosphatase was localized as deposits of lead phosphate in capillary walls and in the region of sinusoids. Alkaline phosphatase was also prominently localized on the outer cell surface of epithelial ceils especially in the intercellular spaces (Fig. 3a). Reaction product was also found on the microvilli that project from the lateral walls of the follicular cells into the prominent inter- cellular lacunae (Fig. 3b).

Fig. l a--c. Tadpole of X. laevis after 9 months immersion in 0.005% Propyl-thiouracil. Note large well-vascularized goitre, a side view; b front view; c Potassium thiocyanate- induced goitre. Epon embedded, cut at 1 tz, stained in Azur II. Acid phosphatase seen in

densely stained droplets (arrowed)

38 R. COLEMAN, P. J. EVEN:NETT and J. M. DODD:

Fig. 2a and b. Potassium thiocyanate-induced goitre, a Dense droplet, which may be a lysosome, showing localized regions of acid phosphatase activity, b Cytolysome showing

regions rich in acid phosphatase

The sites of localization of ATP-ase were examined only in the th iocyanatc and perchlorate goitres and were found to be identical to those seen for alkaline phosphatase (Fig. 3c). 5 • l0 -3 M BAL added to the incubat ion medium did not inhib i t the degree of localization of ATP-ase. 0,1 mM ouabain added to the incuba t ion medium had little, if any, noticeable effect and did not apparen t ly inhib i t the degree of localization of ATP-ase. When the A T P in the incuba t ion med ium was replaced by sodium fl-glycerophosphate and incubat ion was carried out at pI~ 7.2 far fewer react ion sites were seen.

Phosphatases in Tadpole Goitres 39

Fig. 3. a, b Potassium thiocyanate-induced goitre showing localization of alkaline phos- phatase on intercellular membranes, c Potassium perchlorate-induced goitre showing

localization of ATP-ase

40 R. COLEMAN, P. J. EVENNETT and J. M. ])ODD :

No enzyme reaction product was detected in any of the control incubation media, which lacked the substrate.

Discussion

Azur ILposi t ive droplets seen in the light microscope studies of the goitrcs correspond to the dense droplets seen in electron micrographs. These are believed, from recent cytochemical studies on the mammalian thyroid, to play a role in the proteolytic release of thyroid hormones from PAS-positive "colloid" droplets containing thyroglobulin and bound iodinated proteins in thyroid acinar cells (NOVlKOFF and VORBRODT, 1963; SELJELID, 1965; SOBEL, 1964; WETZ]~L et al., 1963, 1965; WOLLMA]~ and SrlCER, 1963; WOLLMAN et al., 1964). I t has been demonstrated tha t the dense droplets contain acid hydrolases (acid phosphatase; esterase) characteristic of lysosomes. Moreover, the intracellular fusion of dense droplets with I)AS-positive "colloid" droplets and the transfer of hydrolytic enzyme activity has been shown, especially following thyrotropic stimulation, at both the light and electron microscope levels. So far as the present work is concerned, goitrogens are known to inhibit thyroid hormone production and, as a consequence, the pi tui tary is released from negative feedback and hypersecre- tion of thyroid stimulating hormone (TSH) ensues. TSH is known to stimulate general metabolism and protein production in the thyroid cells (DuMoNT and ROCMANS, 1964; BRADLEY and WISSIG, 1966) and the large amounts of rough endoplasmic reticulum, which develop in the tadpole goitre, are visible evidence of a high degree of thyrotropic stimulation. The lacunae of the ergastoplasm of the goitrous thyroid cells are greatly swollen and contain material of low electron density which is presumably precursor material for numerous metabolic products of the cells. The apical dense droplets containing acid phosphatase appear to arise from the Golgi apparatus in Xenopus thyroid, especially following thyro- tropic stimulation (COLEMAN, unpublished observations, 1967). Similar results have been found in mammalian thyroid (SOBEL, 1961, 1962; SELJELID, 1965). NO hormone is produced by the goitres and little or no thyroglobulin hence the dense droplets will not be used but will continue, under thyrotropic stimulation, to accumulate at the apical end of follicular cells. Many of the dense droplets containing acid phosphatase also contain heterogeneous membranous structures. These are possibly cytolysomes and probably have an autolytic function. The acid phosphatase in these organelles probably breaks down parts of the cell which need replacing or are no longer functional.

The localization of alkaline phosphatase reported here is in accord with that found in the thyroid of many mammals (DEMI~SEu 1962; HALEYet al., 1955; KABAT and FURTH, 1941 ; LI~DSAY and JENKS, 1961 ; LINDSAY and A~ICO, 1963 ; PAKDAMAN et a l , 1961; SOBEL, 1964; ZAWlSTOWSKA and ZAWISTOWSKI, 1963). Alkaline phosphatase was found in much greater quantities in the Xenopus goitres than in untreated tadpoles at the same stage of development. This agrees with the view tha t the activity of the enzyme is related to the degree of hyper- plasia or vascularity of the gland (DEMPSEY ct al., 1949). I t has been shown that alkaline phosphatase activity paralleled the degree of thyrotropic stimulation of the gland in mammals (DEMPSEY et al., 1949 ; NEGRI and WEBER, 1953 ; SOBEL,

Phosphatases in Tadpole Goitres 41

1964). The function of this enzyme (or enzymes, as there may be more than one) is not known, though it may act as a phosphotransferase.

I t is known that alkaline phosphatase can also dephosphorylate ATP (PADY- KULA and HERMAn, 1955; PEARS]~, 1960). The localization of ATP-ase can be demonstrated by assessing the difference between ATP-incubated and glycero- phosphate-incubated tissue. At pH 7.2 there should be little interference from alkaline phosphatase activity. The inhibitor BAL at c. 5 • 10 -a M has been shown to inhibit the activity of non-specific alkaline phosphatase, but to enhance the activity of ATP-ase (PAI)u and ttERMA~, 1955). By using this inhibitor of alkaline phosphatase activity we have shown that an actual Mg++-activated ATP-ase is present at pH 7.2 in these tadpole goitres. Biochemically a Na+, K+-dependent ouabain sensitive ATP-ase has been demonstrated in the mamma- lian thyroid and is thought to play a role in the membrane transport of iodide into thyroid cells (BRuNBERG and HALMI, 1966; WOLFF and HALMI, 1963; WOLFF, 1964; ZAKRZEWSKA-HE:NISZ, 1966). Ouabain, a cardiac glycoside, also has been shown to have some retarding influence on tadpole metamorphosis (HALONEN et al., 1952). In the Xenopus goitres investigated the addition of 0.1 mM ouabain to the incubation medium had no apparent inhibitory effect on the localization of ATP-ase. An almost identical localization to that described in the tadpole thyroid of ATP-ase has recently been shown in the amphibian epidermis (FAR- QUI~AR and PALADE, 1966). In this latter study, the authors also failed to demon- strate any inhibitory effect of 0.1 mM ouabain on the ultrastructural localization of ATP-ase. In the same study the limitations imposed on the cytochemical localization of ATP-ase and the relationship to a "transport" ATP-ase are re- viewed and discussed. An ouabain-sensitive ATP-ase has been shown to account for only about 6% of the total ATP-ase activity in the normal rat thyroid (BRuN- BERG and I-IALMI, 1966). The large amount of ouabain-resistant ATP-ase could occlude any inhibition of the ouabain-sensitive ATP-ase by the cardiac glycoside. This would account for the inability to detect ouabain-inhibition cytochemically. The ultrastructural localization of ATP-ase in Xenopus tadpole goitre is in accord with that described for the mammalian thyroid at the light microscope level (PAKDAMA~ et al., 1961; LINDSAu 1963; SOBEL, 1964).

R e f e r e n c e s

BRADLEY, A. S., and S. L. WlSSlO: The anatomy of secretion in the follicular cell of the thyroid gland. III. The acute effect in rive of thyrotropic hormone on amino-acid uptake and incorporation into protein by the mouse thyroid gland. J. Cell Biol. 80, 433-436 (1966).

BI~V~CBERO, J. A., and N. S. HAL~II: The role of ouabain-sensitive adenosine triphosphatase in the stimulating effect of thyrotropin on the iodide pump of rat thyroid. Endocrinology 29, 801--807 (1966).

DEANE, H. W.: :Nuclear location of phosphatase activity; fact or artifact? J. Histoehem. Cytoehem. ll, 443--444 (1963).

DE•PSEY, E.W.: Comparative and microscopic anatomy of the thyroid. In: The thyroid (ed. S. C. WER~ER), 2nd. ed. New York: Harper & Row 1962.

- - 1~. O. GREEP, and H. W. DEAX]~: Changes in the distribution and concentration of alka- line phosphatase in tissues of the rat after hypophysectomy or gonadectomy, and after replacement therapy. Endocrinology 44, 88--103 (1949).

DUMOXT, J. E., and P. A. ROC~IANs: In rive effects of thyrotropin on the metabolism of the thyroid gland. J. Physiol. (Lend.) 174, 2 6 ~ 5 (1964).

42 R. COLEMAN, P. J. EVENNETT and J. M. DODD:

DUVE, C. DE: The lysosome concept. In: Ciba Symposium on Lysosomes (eds. A. V. S. DE ]~EUCK and M. P. CAMERON), p. 1--31. London: J. & A. Churchill Ltd. 1963.

- - , and R. WATTIAUX: Functions of lysosomes. Ann. Rev. Physiol. 28, 4 3 5 ~ 9 2 (1966). ERICSSON, J. L. E., and B. F. TRU~IP: Observations on the application of electron microscopy

of the lead phosphate technique for the demonstration of acid phosphatase. Histochemie 4, 470--488 (1965).

FARQUHAR, M. G., and G. E. PALADE: Adenosine triphosphatase localization in amphibian epidermis. J. Cell Biol. 30, 359--379 (1966).

GASCHE, P.: Zur Frage des AngriHspunktes des Thiouraci]. Versuche an Xenopuslarven. 1. Mitteilung. Expericntia (Basel) 2, 24--26 (1946).

, u. J. DRUEu Wirksamkeit schilddriisenhemmender Stoffe auf die Xenopusmetamor- phose. 2. Mitteilung. Experientia (Basel) 2, 26 27 (1946).

GOMORI, G.: Microscopic histochemistry: Principles and practice. Chicago: University of Chicago Press 1952.

HALEY, H. L., G.M. DEWS, and S. C. SOMMERS: A histochemical comparison of primary thyroid hyperplasia and adenomatous goitre. Arch. Path. 59, 635--640 (1955).

HALONEN, i., A. N. KUUSISTO, and P. KOSKELO: On the effect of strophanthin on thyroid activity. An experimental study with frog tadpoles. Cardiologia (Basel) 21, 727--734 (1952).

KABAT, E. A., and J. FURT~: A histochemical study of the distribution of alkaline phospha- tase in various normal and neoplastic tissues. Amer. J. Path. 17, 303--318 (1941).

JEON, K. W. : Simple method for staining and preserving epoxy resin-embedded animal tissue sections for light microscopy. Life Sci. 4, 1839--1842 (1965).

LII~DSAY, S. : Adenosine triphosphatase activity in the rat thyroid gland. A histochemical study. Arch. Path. 76, 257--262 (1963).

, and I. M. ARIco: Enzyme histochemistry of the human thyroid gland. Arch. Path. 75, 627--647 (1963).

- - , and P. R. JENKS: Enzymatic histochemistry of the rat thyroid gland. In: Advances in thyroid research (ed. R. PITT-RIVERS). New York: Pergamon Press 1961.

MILLER, F.: Acid phosphatase localization in renal protein absorption droplets. Proc. 5th. Internat. Congr. Electron Micr. vol. 2, Q--Z, (1962).

NEGRI, L . , e G . WEBER: L'at t ivi ta fosfatasica nella tiroide di ratto nella norme e in diverse situazioni funzionali sperimentali. Arch. de Vecchi 20, 253--276 (1953).

NIEUWKOOP, P. D., and J. FABER: Normal table of Xenopus laevis (DAUDIN). Amsterdam: North Holland Publ. Co. 1956,

NOWKOFF, A. B., and A. VORBRO1)T: Lysosomes and thyroid function. J. Cell Biol. 19, 53A (1963).

PADYKULA, H. A., and E. HERMAN: The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytocbem. 3, 170--183 (1955).

PAKDAMAN, P., A. A. STEIN, and J. C. MCCLINTOCK: Enzymatic histochcmical patterns in surgically resected thyroid glands. Surg. Gynec. Obstet. l l3 , 542 546 (1961).

PEARSE, A. G. E.: Histochemistry, theoretical and applied, 2nd. ed. London: J. & A. Chur- chill Ltd. 1960.

SELJELID, R. : Electron microscopic localization of acid phosphatase in rat thyroid follicle cells after stimulation with thyrotropic hormone. J. Histochem. Cytochem. 13, 687--690 (1965).

SOREL, H. J. : The localization of acid phosphatase activity in the rat pituitary and thyroid glands and its relation to secretory activity. Endocrinology 68, 801--808 (1961).

- - Relationship of three lysosomal enzymes to the Golgi zone and secretory activity in the rat pituitary and thyroid glands. Anat. Rec. 143, 389--393 (1962).

- - Phosphatases of rat thyroid and anterior pituitary glands during various phases of secre- tory activity. A cytochemical study. J. Endocr. 30, 323--336 (1964).

TRANZ~R, J. P. : Utilisation de citrate de plomb pour la mise en evidence de la phosphatase alcaline au microscope electronique. J. Microscopie 4, 409-412 (1965).

UHLENHUTH, E . : The function of the Bensley cell in the thyroid of the salamander, Am. blystoma opacum. Anat. Rec. 27, 223 (1924).

Phosphatases in Tadpole Goitres 43

WAO~STEI~, M., and E. MEISEL: Histochemistry of hepatic phosphatases at a physiologic pH. Amer. J. elin. Path. 27, 13--23 (1957).

WFTZEL, B. K., S. S. SPICFR, and S. H. WOLLMAN: 5~on-specific acid phosphatase activity in rat thyroid glands following TSH stimulation. J. appl. Phys. 34, 2525 (1963).

- - - - - - Changes in fine structure and acid phosphatase localization in rat thyroid cells following thyrotropin administration. J. Cell Biol. 25, 593--619 (1965).

WOLFF, J. : Transport of iodide and other anions in the thyroid gland. Physiol. Rev. 44, 45--90 (1964).

- - , and N. S. HALMI: Thyroidal iodide transport. V. The role of Na +, K+-activated, ouabain- sensitive adenosinetriphosphatase activity. J. Cell Biol. 288, 847--851 (1963).

WOLLMA~N, S.H., and S. S. SPICES: Intracellular colloid droplets following thyrotropin injection. In: Thyrotropin (ed. S.C. WERNER), p. 168--177. Springfield (Ill.): Ch. C. Thomas 1963.

, and M. S. BURSTONE: Loca]isation of esterase and acid phosphatase in granules and colloid droplets in rat thyroid epithelium. J. Cell Biol. 21, 191--202 (1964).

ZAKREWSKA-HENISZ, A.: Effect of Strophantin G on 131I trapping by the thyroid gland in vivo. Bull. Acad. pol. Sci. C1.2 14, 149--152 (1966).

ZAWlSTOWS~A, H., and S. ZAWlSTOWSKI: Histochemical study of the thyroid gland of the white rat (English summary). Folia morph. (Warszawa) 14, 183--194 (1963).

Prof. J. M. ]:)ODD Department of Zoology, The University Leeds 2/England