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A histochemical study of the distribution and differentiation of alkaline phosphatase in the gastro-intestinal… Prakash, Anand 1960

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A H1SI@CHEMI.CAL STUDY OP THE DISTRIBUTION AND DIFFERENTIATION OF ALKALINE PHOSPHATASE IN THE GASTRO-INTESTINAL TRACT OF STEELHEAD TROUT (Salmo g a i r d n e r i i g a i r d n e r i i ) by ANAND PRAKASH B.Sc. (Honours), U n i v e r s i t y of D e l h i , 1952. M.Sc, U n i v e r s i t y of Allahabad, 1954. M.A., U n i v e r s i t y of B r i t i s h Columbia, 1958. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, I960 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r ' agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s ' r e p r e s e n t a t i v e s . I t i s understood that'copying-or p u b l i c a t i o n o f • t h i s t h e s i s f o r f i n a n c i a l ' g a i n ' s h a l l not be allowed without my w r i t t e n permission. Department of ZOOLOGY The U n i v e r s i t y of B r i t i s h Columbia, Vancouver $, Canada. Date September 30, i960. G R A D U A T E STUDIES Field of Study: Fisheries Biology Comparative Physiology W . S. Hoar Quantitative Zoology P. A . Larkin Biological Oceanography R. F. Scagel Fisheries Technology F R B C Technological Station Staff Related Studies: Hydraulics Biometry Marine Benthonic Organisms Experimental Biology Anthropology P U B L I C A T I O N S 1954—Living Fossils. Zool. Soc. Bull., Univ. Ailahabad. India. No. IX: 25. 1958— Food as a factor affecting the growth of coho salmon off the east and west coast of Vancouver Island, B.C. Fish. Res. Bd. Canada, Prog. Rept. Pacific, 112: 7, (with D . J. Milne). 1959— Why are west coast coho so much bigger? Western Fisheries, 57(5): 20, (with D . J. Milne). 1960— Some observations on the distribution and p H dependence of the intestinal alkaline phosphatase of steelhead trout (Salmo gairdnerii gairdnerii). Canad. J. Zool., 38: 228. I960—Differential susceptibility of histochemical localization of alkaline phosphatase activity to p H changes and inhibitor concentrations. Proc. B.C. Acad. Sci., 14th Sci. Confr. 3: 9. Abstract. I960—A histopathological study of the intestinal lesions induced by Echniorhynchus lageniformis (Acanthocephala-Echinorhynch-idae) in the starry flounder. Canad. J. Zool. , 38, (with J. R. Adams). I960—The effect of starvation on the histochemical localization of alkaline phosphatase activity in the intestinal mucosa of the steelhead trout. Canad. J. Biochem. and Physiol. (In press). .. E. S. Pretious ... J. C. Sawyer R. F. Scagel J. Allardyce H . B. Hawthorn W\t Pttttiersiig of ^rit is l j (EoIumBra FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY °f ANAND PRAKASH B.Sc. (Honours), Delhi, India, 1952 M.Sc, Allahabad, India, 1954 M . A . , British Columbia, 1958 IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING FRIDAY, SEPTEMBER 30, I960 AT 4:00 P.M. COMMITTEE IN CHARGE D E A N F. A . S O W A R D , Chairman P. C O N S T A N T I N I D E S I. M c T . C O W A N C. V . F I N N E G A N P. F O R D W . S. H O A R W . N . H O L M E S C. C. L I N D S E Y A . R. P. P A T E R S O N External Examiner: F L O R E N C E M O O G Washington University A H I S T O C H E M I C A L S T U D Y O F T H E D I S T R I B U T I O N A N D D I F F E R E N T I A T I O N O F A L K A L I N E P H O S P H A T A S E I N T H E G A S T R O I N T E S T I N A L T R A C T O F S T E E L H E A D T R O U T (Salmo gairdnerii gairdnerii) A B S T R A C T The pattern of alkaline phosphatase activity in the gastro-intestinal tract of steelhead trout as revealed by Gomori-Takamatsu's Ca-CoS method indicates that this enzyme is intimately concerned with differentiation. During the early stages of development, the enzyme is widely distributed and localized mainly in the cytoplasm and nuclei of the gut primordium, later on, however, it becomes electively localized in those areas where active differentiation is taking place and disappears from other regions. Regional accumulation and loss of alkaline phosphatase throughout the period of gut differentiation points to the characteristic discontinuity in enzyme development. In the fully developed gut, phosphatase activity is localized in the gastric and intestinal regions in the lamina propria, brush border, and nuclei of the tunica mucosa. The probable role of phosphatase in these locations is discussed. Topographically variable patterns of enzyme activity obtained under different p H conditions suggest that phosphatases present in the brush border, lamina propria, and nuclei have different p H optima and are active over different p H ranges. The use of inhibitors in various concentrations in the incubating medium shows that the enzyme located in the brush border is more resistant to high concentrations of inhibitors than that in the lamina propria. Nuclear phosphatase, on the other hand, is very sensitive to lower inhibitor concentrations. Experimental evidence is presented to show that the reaction in the nuclei is due to the presence of intrinsic phosphatase. Following starvation, changes in the enzyme activity pattern are indicated in the intestinal mucosa, probable factors inducing these changes are discussed. -Differential patterns of phosphatase activity in tissue sections obtained under different substrates, p H values, inhibitor concentra-tions, and levels of starvation strengthen the belief of "enzyme plurality". ABSTRACT The p a t t e r n of a l k a l i n e phosphatase a c t i v i t y i n the g a s t r o - i n t e s t i n a l t r a c t of steelhead t r o u t as revealed by GorITlori-TakaInatsu,s cobalt s u l p h i d e method i n d i c a t e s that t h i s enzyme i s i n t i m a t e l y concerned with d i f f e r e n t i a t i o n . During the e a r l y stages of development, the enzyme i s widely d i s t r i b u t e d and l o c a l i z e d mainly i n the cytoplasm and n u c l e i of the gut primordium, l a t e r on, however, i t tends to become e l e c t i v e l y l o c a l i z e d i n those areas where a c t i v e d i f f e r -e n t i a t i o n i s t a k i n g p l a c e , and disappears from other r e g i o n s . Regional accumulation and l o s s of a l k a l i n e phosphatase through-out the p e r i o d of gut d i f f e r e n t i a t i o n p o i n t s to the c h a r a c t e r i s t i c d i s c o n t i n u i t y i n enzyme development. In the f u l l y developed gut, phosphatase a c t i v i t y i s l o c a l i z e d i n the g a s t r i c and i n t e s t i n a l regions i n the lamina p r o p r i a , brush border, and n u c l e i of the t u n i c a mucosa. The probable r o l e of phosphatase i n these l o c a t i o n s i s discussed. Topographically v a r i a b l e patterns of enzyme a c t i v i t y obtained under d i f f e r e n t pH c o n d i t i o n s suggest that phosphatases present i n the brush border, lamina p r o p r i a , and n u c l e i have d i f f e r e n t pH optima and are a c t i v e over d i f f e r e n t pH ranges. The use of i n h i b i t o r s i n v a r i o u s concentrations i n the i n c u b a t i n g medium shows that the phosphatase present i n the brush border i s more r e s i s t a n t to high c o n c e n t r a t i o n s of i n h i b i t o r s than that i n the lamina p r o p r i a . Nuclear phosphatase, on the other hand, i s remarkably s e n s i t i v e to lower i n h i b i t o r concentrations. Under moderate s t a r v a t i o n , an i n c r e a s e i n the enzyme _ a c t i v i t y i s i n d i c a t e d i n the brush border of the i n t e s t i n a l mucosa; acute s t a r v a t i o n brings about s t r u c t u r a l d e t e r i o r a t i o n of the mucosa and desquamation of the columnar e p i t h e l i a l c e l l s , l e a d i n g to d i f f u s e phosphatase r e a c t i o n . Probable f a c t o r s i n d u c i n g phosphatase accumulation f o l l o w i n g s t a r v a t i o n are discussed. D i f f e r e n t i a l patterns of phosphatase a c t i v i t y i n t i s s u e s ections obtained under d i f f e r e n t s u b s t r a t e s , pH v a l u e s , i n h i b i t o r c o n c e n t r a t i o n s , and l e v e l s of s t a r v a t i o n strengthen the b e l i e f of 'enzyme p l u r a l i t y ' . The nuclear and extranuclear phosphatases behave d i f f e r e n t l y and probably are not i d e n t i c a l . LIST OF P1JBL10ATIO&S I*. flAiASIf# A* " Living Fossils % Z$ei!U Sod. Bull*? Univ* Allanaoa&b. India,,,..Mb. IX s 2§?28# (I954). 2. FRAKASHp A. s and MILBS*. D.J. " Food as a factor affecting the growth of eoho salmon off the east and v;est coast of Vancouver Tslan&.Bs-O* -'nv Flstl* Rdg* B&* Cm. 9 Prog,. Rept** Pacific. 112 l 7*9* (1958). 3* PBAEASH^ A.« ®nd MILBEs 1 1 why are west coast coho so much Digger '? * Western Fl$her|0Bis 57 (5) t .£0*2.2$ (1959)* 4*-. PHAEASHf, A.. # goat© ©oservations on the distribution and pH dependence of the Intestinal alkaline phosphatase of steelhoad trout (Salmo ^ i r d n e r i l gairdnerii) 0ana&*. J*, Zool.* 38 1 228*229i Feb I960. " 5*- PRAIASH9; At " Differential susceptibility of Mstoohemicai localisation of alkaline phosphatase activity to pH changes and inhibitor concentrations % proc.. 1,0. Acad., sci. s 14th Sol. Cbnftv 3 t9 (abstract) Feb 1960* 6. PRAKASHi and^ADAlS?> JAMES* Ri. * A hietopathOlogical study of the intestinal lesions induced by EcMnorhynchus la&enifdrinis (Acantho* OGphala * EeMnorhyneMdaej in the starry flounder." eaaad. J.. Zdol*» 38•* Oct i960*. 7. PRAlASHj Ai " The effect of starvation on the histoohemical localization of alkaline phosphatase activity in the intestinal mucosa of the steelhead trout OanMi. j . Blochem & Physiol* 9 (in press) - i i i -TABLE OFi,CONTENTS ABSTRACT i INDEX OF TABLES v INDEX OF FIGURES . v i ACKNOWLEDGEMENTS i x INTRODUCTION 1 MATERIALS AND METHODS Histoohemical l o c a l i z a t i o n 5 Con t r o l s 6 Measurement of enzyme a c t i v i t y • 8 H i s t o l o g i c a l s t a i n i n g ••• 9 Developmental stages 9 EXPERIMENTAL RESULTS D i s t r i b u t i o n of a l k a l i n e phosphatase i n adu l t f i s h 11 D i s t r i b u t i o n of a l k a l i n e phosphatase i n developmental stages I . E a r l y prehatching 22 I I . Late Prehatching 22 I I I . E a r l y posthatching 28 IV. Late pos t h a t c h i n g 34 V. F r y 35 Fac t o r s a f f e c t i n g enzyme l o c a l i z a t i o n pH 1. Topographic l o c a l i z a t i o n 45 2. S t a b i l i t y of the enzyme.. 50 - i v -Page I n h i b i t o r s • •• 50 Substrates 53 The e f f e c t of moderate and acute s t a r v a t i o n ... 56 DISCUSSION Enzymatic patterns i n r e l a t i o n to d i f f e r e n t i a t i o n 64 Nuclear phosphatase 69 S t a r v a t i o n 74 Enzyme p l u r a l i t y 76 SUMMARY AND CONCLUSIONS 82 REFERENCES ". 86 V -INDEX OP TABLES Table Page I . E f f e c t of pH on a l k a l i n e phosphatase a c t i v i t y i n the v a r i o u s r e g i o n s of i n t e s t i n e and p y l o r i c caeca 46 I I . The e f f e c t of i n h i b i t o r concentrations on a l k a l i n e phosphatase a c t i v i t y i n the v a r i o u s regions of the i n t e s t i n a l mucosa 52 I I I . A l k a l i n e phosphatase a c t i v i t y i n the i n t e s t i n a l mucosa w i t h v a r i o u s substrates 54 IV. The e f f e c t of v a r y i n g periods of s t a r v a t i o n on a l k a l i n e phosphatase a c t i v i t y i n the i n t e s t i n e and p y l o r i c caeca 58 - VI -INDEX OF PLATES AND FIGURES Page PLATE I 14 S i t e s of a l k a l i n e phosphatase a c t i v i t y i n the v a r i o u s regions of the g a s t r o - i n t e s t i n a l t r a c t of adu l t f i s h under standard c o n d i t i o n s of i n c u b a t i o n . F i g . 1. T.S. of corpus stomach. F i g . 2. T.S. of p y l o r i c stomach. PLATE I I ... 17 S i t e s of a l k a l i n e phosphatase a c t i v i t y i n the v a r i o u s regions of the g a s t r o - i n t e s t i n a l t r a c t of adult f i s h under standard c o n d i t i o n s of i n c u b a t i o n . F i g . 1. T.S. of i n t e s t i n e . F i g . 2. T.S. of rectum. PLATE I I I 21 S i t e s of phosphatase a c t i v i t y at i n t r a c e l l u l a r l e v e l i n the adult f i s h . F i g . 1. Under standard c o n d i t i o n s o f i n c u b a t i o n . F i g . 2. The e f f e c t of prolonged i n c u b a t i o n . PLATE IV 25 S i t e s of phosphatase a c t i v i t y during l a t e prehatehing stage. F i g . 1. T.S. through •endodermal p l u g ' . F i g . 2. T.S. through esophageal r e g i o n . PLATE V 27 S i t e s of phosphatase a c t i v i t y d u r i n g l a t e prehatehing stage. F i g . 1. T.S. through g a s t r i c r e g i o n . F i g . 2. T.S. through i n t e s t i n a l r e g i o n . - V l l -Page PLATE VI 31 S i t e s of phosphatase a c t i v i t y d u r i n g e a r l y posthatching stage. P i g . 1. T.S. of the embryo through buccal r e g i o n . P i g . 2. T.S. of the embryo through 'endodermal p l u g * . PLATE V I I 33 S i t e s of phosphatase - a c t i v i t y d u r i n g e a r l y posthatching stage. P i g . 1. T.S. of the embryo through stomach and swim bladder. P i g . 2. T.S. of the embryo through i n t e s t i n a l r e g i o n . PLATE V I I I 37 S i t e s of phosphatase a c t i v i t y d u r i n g l a t e posthatching stage. . . . . P i g . 1. T.S. of the embryo through f o r e gut. P i g . 2. Magnified view of endoderm plug and gut w a l l . PLATE IX 39 S i t e s of phosphatase a c t i v i t y during l a t e posthatching stage. P i g . 1. T.S. of esophagus showing d o r s a l a c t i v i t y . . P i g . 2. T.S. of the embryo through stomach and swim bladder. PLATE X 42 S i t e s of phosphatase a c t i v i t y i n the gut of newly emerged f r y . P i g . 1. T.S. of f r y through p y l o r i c s p h i n c t e r . P i g . 2. T.S. of i n t e s t i n e showing developing v i l l i . PLATE XI 48 The e f f e c t of pH on the l o c a l i z a t i o n of phosphatase a c t i v i t y . P i g . 1. Incubation at pH 9.4. - v i i i -Page P i g . 2. Incubation at pH 9.2. P i g . 3. Incubation at pH 8.6. P i g . 4. Incubation at pH 8.2. PLATE X I I 49 The e f f e c t of pH on a l k a l i n e phosphatase l o c a l i z a t i o n i n terms of percent a c t i v i t y per 60 minutes of i n c u -b a t i o n . PLATE X I I I 60 The e f f e c t of s t a r v a t i o n on the l o c a l i z a t i o n of phosphatase i n the i n t e s t i n a l mucosa. P i g . 1. Moderate s t a r v a t i o n (12 days). P i g . 2. Acute s t a r v a t i o n (120 days). PLATE XIV 62 The e f f e c t of s t a r v a t i o n on the nuclear phosphatase of kidney tubules. P i g . 1. Normal f e d f i s h . P i g . 2. Starved f i s h . - i x ACKNOWLEDGMENTS This study was made p o s s i b l e i n great p a r t , by the a s s i s t a n c e and advice which I so generously r e c e i v e d from v a r i o u s persons. I am p a r t i c u l a r l y indebted t o Dr. P e t e r Ford and Dr'*i C.V.Finnegan f o r t h e i r guidance, encouragement, and h e l p f u l c r i t i c i s m s d u r i n g the course of t h i s study. Suggestions and advice on the v a r i o u s aspects of the present i n v e s t i g a t i o n were r e c e i v e d from Dr. JJBrachet, U n i v e r s i t e L i b r e de B r u x e l l e s , and Dr. J . F . D a n i e l l i , King's College,, U n i v e r s i t y of London, and are g r a t e f u l l y acknowle-dged. My.sincere thanks,and g r a t i t u d e go to.Dr. W.S.Hoar f o r h i s keen i n t e r e s t i n my work, o f f e r i n g c o n s t r u c t i v e c r i t i c i s m s and f o r p r o v i d i n g acquaria f a c i l i t i e s : . S e veral s t i m u l a t i n g d i s c u s s i o n s w i t h Dr.. A..R.P. P a t t e r s o n of the Dept.- of Biochemistry and Dr. P..Const ant-i n i d e s : of the F a c u l t y of Medicine, have been p a r t i c u l a r l y h e l p f u l i n the p r e p a r a t i o n of t h i s t h e s i s f o r which I wish to extend my s i n c e r e thanks. To the other members of my D o c t o r a l committee, Drs.. I.. MoT.. Cowan,: W.N.Holm& and C.C.Lindsey who have extended a s s i s t a n c e and encouragement, I wish t o express my s i n c e r e a p p r e c i a t i o n -T e c h n i c a l advice i n the i n i t i a l stages, of t h i s i n v e s t i g a t i o n was-provided by Mr.. C F . C u l l i n g of the Dept. of Pathology, F a c u l t y of Medicine, f o r which I am grateful"; 1 - X -This study would not have been p o s s i b l e without a K-egular supply of l i v e eggs,, j u v e n i l e and a d u l t f i s h and f o r t h i s I am indebted to* the B r i t i s h Columbia, F i s h and Game Commiss-i o n , through the good o f f i c e of Mr.. S.B.Smith, f o r t h e i r generous help. Last: but not l e a s t , I am t h a n k f u l t o Miss. Verna Caunt who gave her v a l u a b l e time i n a s s i s t i n g me w i t h the manuscript. F i n a n c i a l support f o r t h i s research was r e c e i v e d from N a t i o n a l Research C o u n c i l of Canada, through g r a n t s - i n - a i d of research t o Dr. P e t e r Ford. INTRODUCTION During the e a r l y stages of development, the f u n c t i o n a l a c t i v i t i e s of the embryo are of a general type and p r i m a r i l y concerned with maintenance and s y n t h e s i s of protoplasm l e a d i n g to growth. Later on, however, the development i s c h a r a c t e r i s e d by d e f i n i t e morphological changes and e l a b o r a t i o n of s p e c i f i c chemical substances by d i f f e r e n t t i s s u e s . I t i s recognized that the processes of organogenesis and h i s t o g e n e s i s must i n v o l v e c e r t a i n b a s i c p h y s i c a l and chemical changes and there i s reason to b e l i e v e that these changes are brought about under the i n f l u e n c e of 'enzymes'. Although high a c t i v i t y of a number of enzymes seems to be c h a r a c t e r i s t i c a l l y associated w i t h p a r t i c u l a r organs, the r e l a t i o n s h i p between enzymatic c o n s t i t u t i o n of c e l l groups p a r t i c i p a t i n g i n the formation of the various organ primordia and f u n c t i o n a l d i f f e r e n t i a t i o n of p a r t i c u l a r organs i s f a r from understood. A proper understanding of the s p e c i f i c f u n c t i o n s of enzymes i n t i s s u e s and organs c a l l s f o r a c r i t i c a l examination of the p r o p e r t i e s and s p a t i a l d i s t r i b u t i o n of such enzymes at c e l l u l a r l e v e l . Recent advances i n c e l l physiology i n d i c a t e that the number of c e l l u l a r enzymes i s probably extremely l a r g e and s u c c e s s f u l a p p l i c a t i o n of p h y s i c a l and chemical techniques to study morphology and physiology of the c e l l s , t i s s u e s and organs can be expected to provide i n f o r m a t i o n on the p r o p e r t i e s and f u n c t i o n s of the v a r i o u s enzymes. A l k a l i n e phosphomonoesterases which are present i n very h i g h concentrations i n the i n t e s t i n a l mucosa and kidney tubules are h y d r o l y t i c enzymes and c a t a l y s e the general r e a c t i o n R - 0 - P — 0 + H o0 . R - OH + H,PO. "OH 2 3 4 at a pH optimum around 9.0. Although s e v e r a l suggestions have been advanced, the f u n c t i o n s of t h i s group of enzymes i n t i s s u e s and c e l l s are not f u l l y understood. Histochemical demonstration of the s i t e s of enzyme a c t i v i t y has been s u c c e s s f u l i n r e l a t i v e l y few cases, most of these s t u d i e s have been made on hydrolases and oxidoreductases because the end products of t h e i r r e a c t i o n can be v i s u a l i z e d as stained p r e c i p i t a t e s . F o l l o w i n g almost simultaneous but independent p u b l i c a t i o n of a h i s t o c h e m i c a l method f o r the demonstration of a l k a l i n e phosphatase by Gomori and Takamatsu r e s p e c t i v e l y i n 1939, a multitude of papers r e p o r t i n g the l o c a l -i z a t i o n of t h i s enzyme i n the various t i s s u e s have appeared. This method has been c r i t i c a l l y examined by D a n i e l l i (1946, 1953); L i s o n (1948); Ruyter and Neumann (1949); Cleland (1950); Gomori (1950, 1951); Leduc and Dempsey (1951); and Novikoff (1951), and i t s v a l i d i t y i n l o c a l i z i n g s i t e s of high a l k a l i n e phosphatase a c t i v i t y i n t i s s u e s has now been f i r m l y e s t a b l i s h e d . A l k a l i n e phosphatase lends i t s e l f v ery favourably to h i s t o c h e m i c a l manipulations and can be l o c a l i z e d q u i t e e a s i l y w i t h a high degree of p r e c i s i o n provided adequate care and c o n t r o l s i n experimental procedures are adopted. S e v e r a l h i s t o c h e m i c a l and biochemical s t u d i e s on the establishment of c o r r e l a t i o n between a l k a l i n e phosphatase content and f u n c t i o n a l s t a t e of the developing organ have appeared i n recent years i n chick (Moog, 1944; 1950. Hinsch, I960), i n mouse (Moog, 1951; 1953), i n guinea p i g (Moog and O r i t z , 1957), i n amphibia (Brachet, 1946; K r u g e l i s , 1950; K r u g e l i s , N i c h o l i s and Vosgian, 1952) and i n human ( R o s s i , - 3 -Pescetto and Reale, 1956)., S i m i l a r s t u d i e s of the f i s h have been r e l a t i v e l y few. Roche and B u l l i n g e r (1939) and Roche and C o l l e t (1940) e s t a b l i s h e d , by biochemical methods, a c o r r e l a t i o n between phosphatase content and development of s c a l e s , teeth and bones i n t e l e o s t f i s h e s . The e a r l i e s t h i s t o c h e m i c a l study of a l k a l i n e phospha-tase i n the e a r l y development o f f i s h i s that of 2 o r z o l i (1947) who gave a b r i e f account of the l o c a l i z a t i o n of the enzyme i n the development of Pundulus h e t e r o c l i t u s from l a t e cleavage to post hatching p e r i o d . Loreh (1949a, 1949b) made an extensive h i s t o c h e m i c a l study of a l k a l i n e phosphatase i n r e l a t i o n to c a l c i f i c a t i o n and o s s i f i c a t i o n i n developing t r o u t and i n S c y l i o r h i n u s c a n i c u l a . Recently, Ikeda (1959) attempted to c o r r e l a t e morphological and h i s t o c h e m i c a l changes i n the d e v e l -oping alimentary t r a c t and gave an account of the appearance of a l k a l i n e phosphatase i n the gut of Oryzias l a t i p e s . Other h i s t o c h e m i c a l s t u d i e s on a l k a l i n e phosphatase c h i e f l y from the p o i n t of view of d i s t r i b u t i o n of t h i s enzyme i n the d i g e s t i v e t r a c t o f f i s h are those of A l - H u s s a i n i (1949) on the m i r r o r carp, the roach and the gudgeon, and of Weinreb and B i l s t e d (1955) on the rainbow t r o u t . Though t e l e o s t f i s h share c e r t a i n s u p e r f i c i a l resemblances i n t h e i r development w i t h b i r d s , the p a t t e r n of the development of the gut i n these two groups i s q u i t e d i f f e r e n t . I n t e l e o s t f i s h , the b l a s t o d i s c spreads over the surface of the yol k and c o n s i s t s of three elements - the ectoderm, the mesoderm and the p e r i b l a s t . Due to the f a c t t h a t ^ e n t i r e endoderm i s used up i n gut for m a t i o n , the endoderm i n bony f i s h e s does not enclose the y o l k and u n l i k e b i r d s , the gut d i f f e r -e n t i a t e s p r i o r to yol k absorption. Moog (1950) i n her study on the d i f f e r e n t i a t i o n of a l k a l i n e phosphatase i n the duodenum of the ch i c k reported an increase i n enzymic a c t i v i t y soon a f t e r h a t c h i n g , which c o i n c i d e s with the time when the gut becomes f u n c t i o n a l . I n f i s h , however, the hatching of the embryo i s independent of the a c q u i s i t i o n of f u n c t i o n by the gut s i n c e the embryo hatches w i t h the y o l k sac s t i l l attached and the gut f u n c t i o n s only a f t e r the e n t i r e yolk i s absorbed. The purpose of the present study was (1 ) to l o c a l i z e s i t e s of a l k a l i n e phosphatase a c t i v i t y i n the g a s t r o - i n t e s t i n a l t r a c t of steelhead t r o u t (Salmo g a i r d n e r i i g a i r d n e r i i ) d u r i n g e a r l y and advanced stages of development, (2) to c o r r e l a t e the d i s t r i b u t i o n of a l k a l i n e phosphatase w i t h f u n c t i o n a l d i f f e r e n -t i a t i o n of the gut, and (3) to study the e f f e c t of f a c t o r s such as pH, enzyme i n h i b i t o r s , substrates and s t a r v a t i o n on the hi s t o c h e m i c a l l o c a l i z a t i o n of t h i s enzyme i n t i s s u e s e c t i o n s . MATERIALS AND METHODS HISTOCHEMICAL LOCALIZATION Pre-Incubation treatment; The most commonly used method to immobilize the enzyme i s to f i x the t i s s u e i n c h i l l e d 80% e t h y l a l c o h o l or c h i l l e d acetone. Comparison between acetone and e t h y l a l c o h o l f i x e d t i s s u e s i n t h i s study revealed that i f t i s s u e s are placed d i r e c t l y i n c h i l l e d acetone, d i s t o r t i o n or shrinkage occurred and c y t o l o g i c a l d e t a i l s were o b l i t e r a t e d . F i x a t i o n i n c h i l l e d 80$ e t h y l a l c o h o l or i n a mixture of equal p a r t s of c h i l l e d a l c o h o l and acetone gave s a t i s f a c t o r y r e s u l t s and c y t o l o g i c a l d e t a i l s were good. Tissues were removed from the f i s h immediately f o l l o w i n g a blow on the head, f i x e d f o r not l e s s than 12 hours, dehydrated i n c o l d absolute a l c o h o l , cleared i n benzene and embedded i n Tissuemat (52-54°C. m.p.). A l l s e c t i o n s were cut at 5 microns and mounted on albumen coated s l i d e s on the warming p l a t e at 37°C In few cases where the e n t i r e l e n g t h of the d i g e s t i v e t r a c t was cut i n a s e r i e s , thickness of the s e c t i o n s v a r i e d from 7-10 microns. S l i d e s were d r i e d at 37°C f o r not more than two hours. Unused s l i d e s were stored i n the r e f r i g e r -ator at 4°G f o r not over 48 hours as i t was found that consider-able l o s s of a c t i v i t y occurred i f storage time exceeded t h i s p e r i o d . Post Incubation treatment: A f t e r d e p a r a f f i n i z a t i o n and h y d r a t i o n , the s e c t i o n s were incubated i n an i n c u b a t i o n mixture at 37°C. The composition of the standard i n c u b a t i n g medium i s given below: 2% Sodium Beta glycerophosphate 20 ml 2% Sodium d i e t h y l b a r b i t a l 20 ml 2% Ga(N0 5) 2. 4H 20 . 10 ml 2% MgCl 2. 6H 20 2 ml D i s t i l l e d water .48 ml 100 ml The pH of t h i s medium i s 9.3 without adjustment. Routine i n c u b a t i o n was c a r r i e d on f o r 60 minutes at pH 9.3. However, varying lengths of time, d i f f e r e n t pH v a l u e s , d i f f e r e n t s u b s t r a t e s , i n h i b i t o r s e t c . were used i n d i f f e r e n t experiments. S p e c i f i c d e t a i l s of procedure used are giv e n under each experiment. A l k a l i n e phosphatase was demonstrated by Herman's (1951) m o d i f i c a t i o n of Gomori's o r i g i n a l technique. The enzymatic r e a c t i o n obtained a f t e r 60 minutes i n c u b a t i o n at 37°C i n the standard i n c u b a t i n g medium at pH 9.3 was used as a ba s i s f o r comparison w i t h that obtained i n d i f f e r e n t experiments. The phosphatase which i s immobilized i n s i t u i n the t i s s u e by the f i x i t i v e , s p l i t s phosphate from sodium glycerophosphate. This i s trapped immediately and p r e c i p i t a t e d at the s i t e of phosphatase a c t i v i t y as i n s o l u b l e white calcium phosphate which i s converted i n t o c o b a l t phosphate by the a c t i o n of cobalt n i t r a t e . The r e s u l t i n g cobalt phosphate i s converted i n t o black cobalt sulphide by the a c t i o n of ammonium sulphide. No co u n t e r s t a i n was used. C o n t r o l s A r t e f a c t s a r i s i n g i n _ h i s t o c h e m i c a l l o c a l i z a t i o n of a l k a l i n e phosphatase by Gomc-ri-Takamatsu method have been blamed f r e q u e n t l y f o r f a l s e l o c a l i z a t i o n of the a c t u a l s i t e s of enzymic a c t i v i t y i n t i s s u e s e c t i o n s . These a r t e f a c t s are mainly due t o e i t h e r the d i f f u s i o n of the enzyme I t s e l f from the region of i t s t r u e a c t i v i t y i n t o the surrounding t i s s u e , , or of calcium phosphate durin g the course o>£ f i x a t i o n , , incubation,, or staining.. To e l i m i n a t e any n o n - s p e c i f i c r e a c t i o n i n the t i s s u e sections,, p a r a l l e l c o n t r o l s were run by one of*' more of the f o l l o w i n g methods- and compared with s e c t i o n s incubated under the standard c o n d i t i o n s : 1.. I n c u b a t i o n I n the standard substrate w i t h i n h i b i t o r added'1. Potassium 1, cyanide and b e r y l l i u m sulphate were used! as: i n h i b i t o r s . 2. I n c u b a t i o n i n control, i n c u b a t i n g medium without sodium glycerophosphate f o r v a r y i n g lengths of time. 3. - I n c u b a t i o n i n c o n t r o l i n c u b a t i n g medium! w i t h substrate added but without Mg i o n s . 4.. Incubation of i n a c t i v a t e d s e c t i o n s i n the standard s u b s t r a t e . The enzyme was destroyed by one of the f o l l o w i n g methods : a. . IN HC1 - Sections were t r e a t e d w i t h IN HC1 f o r 5 minutes and-then washed thoroughly with, d i s t i l l e d water. b. Heat - Enzyme was* i n a c t i v a t e d by exposing the s e c t i o n s o t o temperatures as h i g h as 90 C. c. U l t r a - v i o l e t exposure - Two 15 watt UV lamps (General E l e c t r i c ) w i t h r a d i a t i o n peaks around 2537A were used t o destroy the enzyme* Wet s e c t i o n s were exposed f o r 15-30 minutes at a d i s t a n c e of about 9 inches from the source of r a d i a t i o n . 5.. Use of kidney s e c t i o n s along with those of the gut i n the - 8 -standard i n c u b a t i n g medium. MEASUREMENT OP ENZYME ACTIVITY No attempt has been made i n the present study to determine q u a n t i t a t i v e l y the amount of a l k a l i n e phosphatase present i n t i s s u e sections at one time by d i r e c t chemical assay. I n t e r p r e t a t i o n of the s t a i n i n g r e a c t i o n i s p u r e l y semiquantit-a t i v e and based on v i s u a l assessment of the s t a i n i n t e n s i t y . The degree of s t a i n i n g r e a c t i o n as a f u n c t i o n of varying i n c u b a t i n g time appears to be the best c r i t e r i o n of the c o n c e n t r a t i o n of phosphatase when other f a c t o r s l i k e sub-s t r a t e c o n c e n t r a t i o n , pH, a c t i v a t o r s e t c . are kept constant. In one case the s t a i n i n g r e a c t i o n was measured by means of photometric method. O p t i c a l density was measured w i t h • M i c r o s i x 1 exposure meter (P. Gossen & Co. G.m.b.H., Erlangen. Germany) d i r e c t l y from the microscope keeping the l i g h t i n t e n s i t y , t h i c k n e s s of the s e c t i o n , thickness of the s l i d e , and i n c u b a t i n g conditions constant. A measurement value s c a l e was c a l i b r a t e d w i t h reference to 'blanks' and ' c o n t r o l s ' and a l l measurement values were expressed as percentage a c t i v i t y per 60 minutes i n c u b a t i o n . I f p r o p e r l y executed, the photometric method can be used to obta i n at l e a s t rough e s t i m a t i o n of the i n t e n s i t y of r e a c t i o n and hence of enzyme co n c e n t r a t i o n . In a personal communication, D a n i e l l i (I960) has expressed doubts as to the v a l i d i t y of photometric method. The photometric r e s u l t s obtained i n the present study have been used only to supplement v i s u a l assessment of the s t a i n i n g r e a c t i o n , t h e r e f o r e , the discrepancy, i f any, does not appear to be s i g n i f i c a n t . - 9 -HISTOLOGICAL STAINING M a t e r i a l f o r h i s t o l o g i c a l study was f i x e d i n e i t h e r Eouin's f l u i d , Zenker - f o r m o l , or Formol - a l c o h o l f o r 24 hours. Tissues f i x e d f o r histochemical s t u d i e s were a l s o used f o r gross h i s t o l o g i c a l examination. Sections were cut at 5-10 microns and st a i n e d w i t h E r h l i c h ' s Haematoxylin or Heidenhain*s I r o n Haematoxylin and counterstained w i t h a l c o h o l i c e o sin. To o b t a i n contrast between muscle f i b e r s and c o l l a g e n , a few sec t i o n s were stained w i t h Weigert's Haematoxylin and Van Geison's p i c r o - a c i d f u c h s i n . The presence of mast c e l l s -i n tissues-sections was e s t a b l i s h e d by Bunting's method (Bunting, 1950) i n which a mixture of Toluidine blue and Thionin blue i s used to e x h i b i t metachromasia i n the granules. DEVELOPMENTAL STAGES Eggs and e a r l y developing stages of steelhead t r o u t were obtained from B r i t i s h Columbia Game Commission trout h a t c h e r i e s at Summerland and Cultus Lake, B r i t i s h Columbia. Eggs were a r t i f i c i a l l y f e r t i l i z e d and maintained i n the l a b o r a t o r y a q u a r i a at 11° - 14°C. The f o l l o w i n g s i x stages have been described i n the present study: Stage I E a r l y prehatehing. S i z e range 6.5 - 8.0 mm\ Eyes f a i n t l y pigmented. Stage I I Late prehatehing. S i z e range 8.1 - 11.0 mm. Eyes h i g h l y pigmented. - 10 -Stage I I I E a r l y posthatching. S i z e range 12.5 - 18.0 mm:. Immediately a f t e r hatching. Stage IV. Late posthatching. S i z e v a r i a b l e . Yolk sac s t i l l attached. Stage V F r y . S i z e v a r i a b l e . Yolk sac completely absorbed. Stage VI A d u l t . Parr marks disappear. Developmental stages e a r l i e r than Stage I of the above s e r i e s were d i f f i c u l t to o b t a i n f o r hi s t o c h e m i c a l purposes c h i e f l y because c h i l l e d 80% e t h y l a l c o h o l and acetone are poor f i x i t i v e of yo l k and make i t un s u i t a b l e f o r s e c t i o n i n g . Stages I and I I were obtained by c a r e f u l l y removing the outer chorion from the eggs and detaching the embryo from the under-l y i n g yolky mass. The alimentary t r a c t was removed and sectioned i n the case of f r y and adu l t f i s h . The e a r l i e r stages were sectioned i n s e r i e s . EXPERIMENTAL RESULTS •i DISTRIBUTION OP ALKALINE PHOSPHATASE IN ADULT PISH Before comparing the pat t e r n s of the development and d i s t r i b u t i o n of a l k a l i n e phosphatase between e a r l y and more advanced stages, a survey of the s i t e s of phosphatase a c t i v i t y throughout the e n t i r e length of the g a s t r o - i n t e s t i n a l t r a c t of the adult f i s h i s appropriate. The microscopic anatomy of the g a s t r o - i n t e s t i n a l t r a c t of a d u l t steelhead t r o u t i s s i m i l a r to that described by Weinreb and B i l s t e d (1955) f o r the rainbow tr o u t and needs no s p e c i f i c mention. The present account of the d i s t r i b u t i o n of a l k a l i n e phosphatase i n the v a r i o u s regions of the d i g e s t i v e t r a c t of adult steelhead t r o u t i s based on obser-v a t i o n s made upon 37 f i s h belonging to the same stock and approximately of the same s i z e and age. These f i s h were main-tained i n the l a b o r a t o r y a q u a r i a on a commercial trout d i e t . Buccal and Esophageal r e g i o n : The study of the s e r i a l sections t r e a t e d by Gomori-TakamatsU's Ca-CoS method revealed that a l k a l i n e phosphatase a c t i v i t y i n the buccal and esophageal r e g i o n i s lowest of any other r e g i o n of the g a s t r o - i n t e s t i n a l t r a c t . This a c t i v i t y i s confined mainly to the cytoplasm and n u c l e i of the s t r a t i f i e d e p i t h e l i u m . The i n t e n s i t y of the r e a c t i o n i s much greater i n the stratum germinativum than i n any other p a r t of the bucco-esophageal w a l l . No a c t i v i t y was n o t i c e a b l e e i t h e r i n the K i n d l y supplied by J.R. C l a r k & Co. S a l t Lake C i t y . Utah. cytoplasm or i n the n u c l e i of the esophageal glands. In the esophageal r e g i o n i t i s d i f f i c u l t to d i s t i n g u i s h between the submucosa and the lamina p r o p r i a except on the b a s i s of the presence of numerous round c e l l s (lymphocytes) and blood c a p i l l a r i e s which give a p o s i t i v e r e a c t i o n f o r phosphatase. The granule c e l l s i n the submucosa show high a l k a l i n e phosphatase a c t i v i t y confined to cytoplasmic granules, whereas, the f i b r o -b l a s t s i n the same r e g i o n show very l i t t l e or no a c t i v i t y . No r e a c t i o n was n o t i c e a b l e i n the muscularis externa and the serosa. G a s t r i c r e g i o n : The simple columnar e p i t h e l i a l c e l l s of the mucosa * l i n i n g the ' g a s t r i c rugae' i n the corpus and p y l o r i c regions are devoid of a l k a l i n e phosphatase a c t i v i t y . Intense a c t i v i t y i s , however, seen i n the lamina p r o p r i a which i s composed of numerous strands of loose a r e o l a r and dense r e t i c u l a r connective t i s s u e , smooth muscle f i b e r s , blood c a p i l l a r i e s , lymphatics and numerous small round c e l l s probably lymphocytes. A l a r g e number of glands are present i n the mucosa of the corpus stomach which penetrate the t i s s u e of the lamina p r o p r i a . The l a t t e r can be d i s t i n g u i s h e d towards the lumen as t h i n strands showing intense phosphatase a c t i v i t y . The glands appear to be phosphatase negative except f o r a very f a i n t r e a c t i o n i n the n u c l e i of the gland c e l l s ( P l a t e I , P i g . 1 ) . The lamina p r o p r i a i n the region of the p y l o r i c stomach Throughout the t e x t the term 'corpus' has been used i n s t e a d of ' c a r d i a c 1 . This term was suggested by Plenck (1932) and adopted by B a r r i n g t o n (1957) f o r f i s h . PLATE I . Adult F i s h Figure 1. Transverse s e c t i o n of corpus stomach showing a l k a l i n e phosphatase a c t i v i t y i n the lamina pro-p r i a . Note a c t i v i t y i n i n t e r - g l a n d u l a r spaces. X 125. SC, Stratum eompecturn, SM, Submucosa, ME, muscularis externa. Figure 2. Transverse s e c t i o n of p y l o r i c stomach showing a l k a l i n e phosphatase a c t i v i t y i n the lamina p r o p r i a . Reduction i n g a s t r i c glands i n the mucosa i s evident. X 125. 14 - 15 -i s a l s o phosphatase p o s i t i v e , and, u n l i k e corpus stomach, i t i s not broken up i n t o numerous compartments by the reduced number of mucosal glands ( P l a t e I , P i g . 2). The submucosa shows a f a i n t r e a c t i o n i n the granule c e l l s and f i b r o b l a s t s . No r e a c t i o n i s n o t i c e a b l e i n the stratum compactum, the muscularis externa or the serosa throughout the stomach. The d i s t r i b u t i o n of a l k a l i n e phosphatase i n the p y l o r i c s p h i n c t e r i s s i m i l a r to that described f o r corpus and p y l o r i c stomach. This r e g i o n i s devoid of glands i n the mucosa, and the lamina p r o p r i a i s represented as a t h i n strand of t i s s u e extending i n t o r e l a t i v e l y short mucosal f o l d s . I n t e s t i n a l r e g i o n : Of a l l the t i s s u e s and organs of the adult animal, a l k a l i n e phosphatase occurs i n highest concentrations i n the i n t e s t i n a l mucosa. The i n t e s t i n e and p y l o r i c caeca show s i m i l a r h i s t o l o g i c a l s t r u c t u r e and p a t t e r n of a l k a l i n e phosphatase d i s t r i -b u t i o n and as such w i l l be considered together i n t h i s study. The p a t t e r n of a l k a l i n e phosphatase d i s t r i b u t i o n i n the i n t e s t i n a l r e g i o n , as stud i e d by transverse and l o n g i t u d i n a l s e c tions shows in t e n s e r e a c t i o n i n the mucosal l a y e r . The l a t t e r forms the main element of the i n t e s t i n a l v i l l i . An i n t e n s e a c t i v i t y i n the s t r i a t e d or brush border at the f r e e end of the columnar e p i t h e l i a l c e l l s and i n the lamina p r o p r i a extending i n t o each v i l l u s as a core can be seen a f t e r only 2-5 minutes of i n c u b a t i o n . This r e a c t i o n becomes more inte n s e as the i n c u b a t i o n time i s increased ( P l a t e I I , P i g . 1 ) . Some a c t i v i t y i s n o t i c e a b l e i n the n u c l e i of the columnar c e l l s , p a r t i c u l a r l y i n the nuclear membrane and the n u c l e o l i ( P l a t e I I I , P i g . I ) . The c o n t i n u i t y of t PLATE I I . Adult F i s h Figure 1. Cross section of the i n t e s t i n e . Phosphatase a c t i v i t y i s v i s i b l e i n the brush border, lamina propria, nuclei of the columnar e p i t h e l i a l c e l l s , and granule c e l l s at the base of each v i l l u s . Goblet c e l l s are negative f o r phosphatase. X 125. Figure 2. Cross section through r e c t a l region. Phosphatase a c t i v i t y i s similar to that noticed i n the i n t e s t i n e . X 125. J-7 - 18 -the s t r i a t e d border as seen i n the h i s t o c h e m i c a l preparations i s broken or i n t e r r u p t e d at s e v e r a l places on the margin of the mucosal e p i t h e l i u m . This i s due to the presence of numerous goblet c e l l s i n between the columnar c e l l s which are devoid of a l k a l i n e phosphatase. The r e a c t i o n i n the lamina p r o p r i a i s p a r t i c u l a r l y i n t e n s e i n the e n d o t h e l i a l c e l l s of the blood c a p i l l a r i e s , r e t i c u l a r connective t i s s u e and lymphatics. At the base of v i l l i , f i b r o b l a s t s , lymphocytes, granule c e l l s and mast c e l l s show in t e n s e r e a c t i o n . No i n d i c a t i o n of any b i l a m i n a r d i s t r i -b ution of phosphatase a c t i v i t y i n the columnar c e l l s as reported by Johnson (1954) was found. S i m i l a r l y no r e a c t i o n i n the ^ o l g i zone which according to Bourne (1943) i s a f a m i l i a r phenomenon of the i n t e s t i n a l e p i t h e l i u m was seen. The p a t t e r n of a l k a l i n e phosphatase d i s t r i b u t i o n i n the lamina p r o p r i a of the i n t e s t i n a l v i l l u s i s of i n t e r e s t . Although, phosphatase a c t i v i t y i n the brush border of the i n t e s t i n a l mucosa and i n adjacent blood c a p i l l a r i e s has been shown by s e v e r a l workers i n d i f f e r e n t animals, there i s apparently l i t t l e or no mention of a d e f i n i t e a c t i v i t y i n the lamina p r o p r i a . Whether or not the r e a c t i o n i n the lamina p r o p r i a i s species s p e c i f i c , needs to be e s t a b l i s h e d . The i n t e s t i n a l w a l l l a c k s a submucosal l a y e r , i t s place being occupied by a w e l l developed wavy stratum compactum and a stratum granulosum. The former i s devoid of phosphatase a c t i v i t y but the l a t t e r shows an i n t e n s e r e a c t i o n i n the granule c e l l s . The p a t t e r n of d i s t r i b u t i o n of phosphatase i n the r e c t a l r e g i o n i s s i m i l a r to that described f o r the i n t e s t i n e and p y l o r i c caeca. The mucosa of t h i s r e g i o n i s thrown i n t o numerous f o l d s without any apparent p a t t e r n comparable to that seen i n the i n t e s t i n e or p y l o r i c caeca. Due to the presence of a l a r g e number of goblet c e l l s i n t h i s r e g i o n , the c o n t i n u i t y of the r e a c t i o n i n the brush border along the outer margin of the v i l l i i s very f r e q u e n t l y broken ( P l a t e I I , P i g . 2 ) . 1 PLATE I I I . Adult F i s h Figure 1. Longitudinal section of v i l l u s of p y l o r i c caeca showing i n t r a c e l l u l a r patterns of phosphatase a c t i v i t y under standard conditions of incubation. Note a c t i v i t y i n the nuclear membrane and n u c l e o l i . X 1250. BB3, Brush border; LP, Lamina propria; N, Nucleus; G, Goblet c e l l ; LC, lymphocyte. Figure 2. L.S. of the v i l l u s showing e f f e c t of prolonged incubation (12 hours) on histochemical l o c a l i z -ation of i n t r a c e l l u l a r a l k a l i n e phosphatase. Note negative reaction i n the brush border and d i f f u s i o n into the cytoplasm. X 1250. BB., Brush border; G, Goblet c e l l . P L A T E III F i g u r e 1 . F i g u r e 2 . 21 - 22 -DISTRIBUTION OP ALKALINE PHOSPHATASE IN DEVELOPMENTAL STAGES I . E a r l y prehatching stage, 6.5 - 8.0 mm' i n l e n g t h , approximately 10 - 15 days a f t e r f e r t i l i z a t i o n . Eyes f a i n t l y v i s i b l e through the chorion. V e n t r a l to the notochord, by a process of i n f o l d i n g , . the thickened endodermal l a y e r forms a more or l e s s s t r a i g h t gut tube. At t h i s stage the gut i s not d i s t i n g u i s h a b l e i n t o f o r e , mid and hind r e g i o n s . The c r a n i a l gut l a c k s a d e f i n i t e lumen, i n s t e a d a s o l i d mass of c e l l s forming the primordium of the f o r e gut i s present. P o s t e r i o r l y , however, a d e f i n i t e lumen can be seen l i n e d w i t h endodermal c e l l s . A l k a l i n e phosphatase a c t i v i t y i s extremely low at t h i s stage and i s confined to the cytoplasm of the endodermal c e l l s and surrounding u n d i f f e r e n t i a t e d mesenchyme c e l l s . The presence of any a c t i v i t y i n the n u c l e i of these c e l l s could not be e s t a b l i s h e d even a f t e r prolonged i n c u b a t i o n . The only s i t e s where a d i s t i n c t phosphatase a c t i v i t y of r e l a t i v e l y high i n t e n s i t y i s v i s i b l e are the n u c l e i and cytoplasm of mesonephric ducts and the n u c l e i of mesenchyme c e l l s near centers of bone formation. At these l o c i 30 minutes i n c u b a t i o n showed the r e a c t i o n c l e a r l y . I I . Late prehatching stage. 8.1 - 11.0 mm: i n l e n g t h , approximately 15 - 30 days a f t e r f e r t i l i z a t i o n . Eyes s t r o n g l y pigmented. The gut at t h i s stage i s roughly d i v i s i b l e i n t o f o r e , mid and hind regions on the b a s i s of r e l a t i v e abundance of mesen-chyme c e l l s surrounding the endodermal element. A d e f i n i t e lumen i s present throughout the e n t i r e l e n g t h of the gut except at the - ,23-a n t e r i o r end i n the v i c i n i t y of the d i f f e r e n t i a t i n g esophagus. P o s t e r i o r to the esophagus, the endodermal c e l l s form a s o l i d 'endodermal p l u g 1 which tempo r a r i l y blocks the f u t u r e lumen. A l k a l i n e phosphatase a c t i v i t y i s more in t e n s e than that seen i n stage I and i s confined l a r g e l y to the n u c l e i of the mesenchyme c e l l s which w i l l g ive r i s e to f u t u r e lamina p r o p r i a , submucosal and muscularis elements. Some cytoplasmic a c t i v i t y can a l s o be seen i n the 'endodermal p l u g ' ( P l a t e IV, P i g . 1 ) . Mesenchyme c e l l s surrounding the endodermal element i n the c r a n i a l r e g i o n of the foregut are not arranged i n a r e g u l a r p a t t e r n . P o s t e r i o r l y , however, the foregut has a d i s t i n c t lumen l i n e d by endoderm c e l l s and the outer mesenchyme c e l l s tend to become c o n c e n t r i c a l l y arranged. High a l k a l i n e phosphatase a c t i v i t y i s n o t i c e a b l e i n the mesenchyme c e l l s , whereas, the i n t e n s i t y of r e a c t i o n i n the endodermal l a y e r i s low ( P l a t e IV, P i g . 2 ) . The g a s t r i c r e g i o n i n l a t e prehatehing stage i s very short and c o n s i s t s of a columnar epit h e l i u m surrounded by a t h i c k l a y e r of c i r c u l a r l y disposed mesenchyme c e l l s . A l k a l i n e phosphatase a c t i v i t y i s very high i n the n u c l e i of the mesenchyme c e l l s ( P l a t e V, P i g . 1 ) . The endodermal c e l l s forming the g a s t r i c e p i t h e l i u m have dark a p i c a l ends which give the appearance of the presence of a c u t i c u l a r border r i c h i n phosphatase a c t i v i t y . Comparison w i t h c o n t r o l s e c t i o n s revealed that t h i s dark band i s phosphatase ne g a t i v e , the cytoplasm of the endodermal c e l l s , , however, showed l i t t l e phosphatase a c t i v i t y . The i n t e s t i n a l r e g i o n shows a high degree of d i f f e r e n t i a t i o n at t h i s stage. The surrounding mesenchymal l a y e r shows very l i t t l e or no r e a c t i o n PLATE IV. Figure 1. Late prehatching stage. Phosphatase a c t i v i t y i s in t e n s e i n the n u c l e i of the mesenchyme c e l l s (MC). Some cytoplasmic a c t i v i t y i s v i s i b l e i n •endodermal plug' (BP.).. X 562. Figure 2. Late prehatching stage. Esophageal r e g i o n showing high phosphatase a c t i v i t y i n mesenchyme c e l l s (MC) and low a c t i v i t y i n endoderm c e l l s (EC). X 562. P L A T E IV • F i g u r e 2 . MC 25 PLATE V. Fi g u r e 1. Late prehatching stage. T.S. of g a s t r i c r e g i o n showing a l k a l i n e phosphatase a c t i v i t y i n mesenchyme c e l l s (MC) and endoderm c e l l s (EC). X 562. Figu r e 2. Late prehatching stage. T.S. of i n t e s t i n a l r e g i o n showing in t e n s e phosphatase a c t i v i t y i n endoderm c e l l s (EC) p a r t i c u l a r l y at the d i f f e r -e n t i a t i n g brush border (BB). Mesenchymeal w e l l (MC) i s devoid of phosphatase a c t i v i t y . X 562. P L A T E V 2 Y f o r phosphatase hut an intense r e a c t i o n i s seen i n the cytoplasm and brush border of the e p i t h e l i a l l a y e r ( P l a t e V, P i g . 2). The brush border makes i t s f i r s t appearance at l a t e prehatehing stage and i s completely formed i n the a n t e r i o r p o r t i o n of the i n t e s t i n e but occurs i n patches towards the p o s t e r i o r end. I I I . E a r l y posthatching stage. 12.5 - 18.0 mmi i n t o t a l l e n g t h , immediately a f t e r h a t c h i n g , approximately 35 - 45 days a f t e r f e r t i l i z a t i o n . At the time of h a t c h i n g , the b a s i c morphological changes i n the d i f f e r e n t i a t i n g gut are s i m i l a r to that seen i n l a t e prehatehing stage except i n one f e a t u r e , i . e . the develop-ment of pneumatic duct and swim bladder. B r a n c h i a l or pharyngeal r e g i o n , esophagus, stomach and i n t e s t i n e can e a s i l y be d i s t i n g u i s h e d . P o s t e r i o r to the pharyngeal pouches, the d o r s a l and v e n t r a l s e c t i o n s of endoderm l i n i n g the buccal c a v i t y j o i n to form a tubular esophagus l i n e d w i t h squamous ep i t h e l i u m . The splanchnic mesoderm surrounds the gut on e i t h e r s i d e and continues d o r s a l l y as the d o r s a l mesentary. There i s no c l e a r demarkation between the corpus and p y l o r i c regions of the stomach which passes d i r e c t l y i n t o the i n t e s t i n e . The e p i t h e l i u m of the stomach i s thrown i n t o short blunt f o l d s g i v i n g the appearance of •microrugae'. Between the g a s t r i c and i n t e s t i n a l r e g i o n s , a c h a r a c t e r i s t i c bend develops but there i s no trace of p y l o r i c caeca. Near the j u n c t i o n of the esophagus and the stomach, a pneumatic d i v e r t i c u l u m a r i s e s from the dorsal w a l l , s l i g h t l y towards the l e f t . The pneumatic duct passes through the d o r s a l * 29 ^ mesentary and j o i n s the swim bladder primordium which i s surrounded by a t h i c k l a y e r o f mesenchymal c e l l s . The lumen of the pneumatic duct i s continuous w i t h that of the gut. A l k a l i n e phosphatase a c t i v i t y at t h i s stage i s very marked i n the n u c l e i of head mesenchyme c e l l s and at the s i t e s of o s s i f i c a t i o n near the angles of the jaws. High phosphatase a c t i v i t y i s a l s o v i s i b l e i n the lower jaw cl o s e to b a s i h y a l and i s confined to the primordium of den t a l lamina which subsequently w i l l form buccal d e n t i c l e s of the adult f i s h ( P l a t e V I , P i g . 1 ) . The d o r s a l w a l l of the buccal c a v i t y shows p o s i t i v e r e a c t i o n i n the c e l l s of the basal l a y e r , whereas, no r e a c t i o n i s v i s i b l e i n the s t r a t i f i e d e p i t h e l i u m . In the esophageal r e g i o n , the a c t i v i t y on the whole i s very low, only mesenchyme c e l l s showing p o s i t i v e r e a c t i o n i n the n u c l e i . A short distance a n t e r i o r to the place of emergence of the pneumatic duct, the 'endodermal p l u g 1 shows intense phosphatase a c t i v i t y i n the cytoplasm and n u c l e i ( P l a t e V I , P i g . 2 ) . Mesenchyme c e l l s around the p l u g are a l s o r i c h i n phosphatase a c t i v i t y and those at the periphery have s t a r t e d d i f f e r e n t i a t i n g i n t o c i r c u l a r muscle f i b e r s . Muscle c e l l s are devoid of any phosphatase a c t i v i t y . The r e a c t i o n i n the region of stomach and pneumatic duct i s very low and confined to those mesodermal c e l l s which l i e i n the immediate v i c i n i t y of the e p i t h e l i a l l a y e r . Primordium of the swim bladder shows p o s i t i v e r e a c t i o n i n the mesodermal c e l l s ( P l a t e V I I , P i g . 1 ) . In the i n t e s t i n a l r e g i o n , phosphatase a c t i v i t y i s seen only i n the brush border which by t h i s stage i s c l e a r l y n o t i c e a b l e ( P l a t e V I I , P i g . 2 ) . Some r e a c t i o n i s a l s o \ PLATE VI. Figu r e 1. E a r l y posthatehing stage. T.S. of the embryo showing a l k a l i n e phosphatase a c t i v i t y i n the primordium of dental lamina (DP) and s i t e s of o s s i f i c a t i o n . Some r e a c t i o n i n the mesenchyme c e l l s of d o r s a l buccal w a l l i s seen, whereas, buccal e p i t h e l i u m (BE) i s devoid o f phosphatase a c t i v i t y . X 125. Figure 2. E a r l y posthatehing stage. T.S. of the embryo showing in t e n s e phosphatase a c t i v i t y i n the endodermal plug (EP) and kidney tubules (K). X 125. 31 PLATE V I I . Pigure 1. E a r l y posthatching stage. T.S. of the embryo showing i n t e n s e phosphatase a c t i v i t y i n the primordium of swim bladder (SB). Note absence of a c t i v i t y i n the stomach (ST), D o r s a l mesentary (DM) and l i v e r d i v e r t i c u l u m (LD). X 125. Pigure 2. E a r l y posthatching stage. T.S. of the embryo showing phosphatase a c t i v i t y i n the i n t e s t i n a l r e g i o n (INT) confined to the brush border. X 125. P L A T E VII F i g u r e 1, INT £ F i g u r e 2 . 33 - 34 -v i s i b l e i n the c e l l s of the d o r s a l mesentary. IV. Late posthatching stage. Average le n g t h 23 mm. A l e v i n , y o l k sac attached. This stage i s marked by r a p i d d i f f e r e n t i a t i o n and growth of the a l e v i n and a c t i v e y o l k a b s o r p t i o n . Most of the morphological features of the developing gut which were not c l e a r l y d i f f e r e n t i a t e d i n the e a r l y posthatching stage, become qui t e evident at t h i s stage. Muscular l a y e r s become f i r m l y e s t a b l i s h e d , though the outer l o n g i t u d i n a l l a y e r i s s t i l l under-going d i f f e r e n t i a t i o n . Corpus and p y l o r i c regions of the stomach can now be recognized, and the mucosa of the l a t t e r i s thrown i n t o numerous f o l d s . The swim bladder acquires a d e f i n i t e lumen l i n e d w i t h low columnar e p i t h e l i a l c e l l s . P y l o r i c caeca are s t i l l undeveloped. The remaining morphological f e a t u r e s are s i m i l a r to stage I I I . Apart from the usual s i t e s of high a c t i v i t y , v i z . bone and kidney tubules, the a l k a l i n e phosphatase a c t i v i t y i n the g a s t r o - i n t e s t i n a l t r a c t i s markedly g r e a t e r than that seen i n the previous stages. Intense a c t i v i t y i s v i s i b l e i n the p r i m o r d i a of the buccal d e n t a l lamina. Mesenchyme c e l l s of the buccal and esophageal regions show a p o s i t i v e r e a c t i o n but high a c t i v i t y i s confined only to the d o r s a l w a l l adjacent to the endodermal l i n i n g ( P l a t e IX, P i g . 1 ) . The l a t t e r i s devoid of phosphatase a c t i v i t y and shows appearance of s e v e r a l goblet c e l l s . . The 'endodermal p l u g ' , which i s present at the j u n c t i o n of esophagus and corpus stomach, shows very l i t t l e phosphatase - 35 -a c t i v i t y i n the cytoplasm. The endodermal c e l l mass forming the 'plug', a c q u i r e s , by a process of v a c u o l a t i o n , a more or l e s s g l a n d u l a r appearance. The v a c u i t i e s i n the endodermal c e l l mass l a t e r on enlarge, coalesce and push the c e l l s to l i e alongside the submucosal l a y e r . This new l a y e r of endoderm c e l l s , w i t h c l e a r cytoplasm and w i t h n u c l e i occupying the ba s a l h a l f , forms the t u n i c a mucosa. The mesenchyme c e l l s i n the submucosa show very i n t e n s e phosphatase a c t i v i t y , whereas, the surrounding muscle c e l l s give a f a i n t cytoplasmic r e a c t i o n ( P l a t e V I I I , P i g s . 1 and 2). A few of the newly formed blood c a p i l l a r i e s can a l s o be recognized i n the submucosal re g i o n and show phosphatase a c t i v i t y i n the e n d o t h e l i a l c e l l s . Considerable a c t i v i t y i s seen i n the mesenchyme c e l l s of the stomach and swim bladder ( P l a t e IX, P i g . 2). Comparison w i t h the same s t r u c t u r e s i n stage I I I ( P l a t e V I I , P i g . 1) r e v e a l s that phosphatase concentration i s higher i n the present stage. Contrary to e x p e c t a t i o n s , the i n t e s t i n e does not show any appar-ent i n c r e a s e i n phosphatase a c t i v i t y from that seen i n stage I I I . There i s a l s o no i n d i c a t i o n of any major morphological change throughout the l e n g t h of the i n t e s t i n e . V. Pry stage. Yolk-sac completely absorbed. At t h i s stage the gut acquires most of the features seen i n the adult f i s h . In the buccal c a v i t y , buccal d e n t i c l e s are f u l l y formed. The esophageal and g a s t r i c regions have d i s t i n c t mucosal, submucosal and muscular elements. In the corpus stomach numerous glands make t h e i r appearance i n the mucosal l a y e r at t h i s stage. The p y l o r i c stomach i s elongated PLATE VIII. P i g u r e 1. Late posthatching stage. T.S. of the embryo through f o r e gut. Note intense phosphatase a c t i v i t y i n the submucosa surrounding the endodermal p l u g , and kidney tubules ( K ) . X 125» Pigure 2. Higher m a g n i f i c a t i o n of the i n s e t i l l u s t r a t e d i n P i g . 1. Note endodermal plug undergoing v a c u o l a t i o n (V). High phosphatase a c t i v i t y i n the n u c l e i of submucosal c e l l s (SM) i s seen. Some cytoplasmic a c t i v i t y i s v i s i b l e i n the endodermal c e l l s and muscularis externa (ME). X 562. C, newly formed blood c a p i l l a r y . P L A T E T i l l V PLATE IX. Pigure 1. Late posthatching stage. T.S. of esophageal r e g i o n showing.alkaline phosphatase a c t i v i t y i n d o r s a l w a l l near stratum germinativum (SG). X 562. Pigu r e 2. Late posthatching stage. T.S. of the embryo through g a s t r i c r e g i o n . Intense phosphatase a c t i v i t y i s seen i n the mesenchymal w a l l of the d i f f e r e n t i a t i n g stomach (ST). Some a c t i v i t y i s v i s i b l e i n the mesenchymal w a l l of the swim bladder (SB). Compare with P l a t e V I I , P i g . 1. X 125. P L A T E IX F i g u r e 39 - 40 -and continued i n t o a narrow and t h i c k - w a l l e d p y l o r i c s p h i n c t e r . The s l i g h t bend which appeared i n t h i s r e g i o n i n stage I I I becomes more pronounced and the e n t i r e s t r u c t u r e i s curved i n t o U or S shape. The f i r s t appearance of p y l o r i c caeca as outgrowths from near the p y l o r i c s p h i n c t e r occurs at t h i s stage. These caeca when seen i n transverse s e c t i o n s , are devoid of the c h a r a c t e r i s t i c mucosal v i l l i or f o l d s and have a more or l e s s smooth lumen. In the r e g i o n of the i n t e s t i n e , the mucosal l i n i n g i s thrown i n t o short and blunt f o l d s each with a c o n t i n -u a t i o n of submucosal elements forming the lamina p r o p r i a . The v i l l i i n the r e c t a l r e g i o n are r e l a t i v e l y l e s s developed and i n some cases completely absent. A s t r i k i n g change i n a l k a l i n e phosphatase a c t i v i t y i n the gut takes place soon a f t e r the emergence of f r y from a l e v i n . Esophagus shows no r e a c t i o n whatsoever and there i s no tr a c e of the presence of the 'endodermal p l u g 1 . The a c t i v i t y i n the corpus and p y l o r i c regions of the stomach i s confined to lamina p r o p r i a and i s s i m i l a r to that described e a r l i e r i n ad u l t f i s h . Intense r e a c t i o n i s , however, n o t i c e a b l e i n the p y l o r i c s p h i n c t e r e s p e c i a l l y near that r e g i o n from where p y l o r i c caeca bud out ( P l a t e X, P i g . 1). Phosphatase a c t i v i t y i n the brush border of the p y l o r i c caeca and the i n t e s t i n e i s very marked but elements of the lamina p r o p r i a are devoid of any a c t i v i t y ( P l a t e X, P i g . 2 ) . The f i r s t appearance of a l k a l i n e phosphatase i n the lamina p r o p r i a occurred i n f r y which had commenced a c t i v e f eeding f o r over a week. This a c t i v i t y i s very low i n the beginning but g r a d u a l l y becomes i n t e n s e . Phosphatase a c t i v i t y i n the n u c l e i PLATE X. Pigure 1. T.S. of newly emerged f r y showing in t e n s e phosphatase a c t i v i t y i n p y l o r i c stomach (PST), p y l o r i c s p h i n c t e r (PS), p y l o r i c caeca (PC), and i n t e s t i n e (INT). X 125. Pigure 2 T.S. of i n t e s t i n e of f r y showing high a l k a l i n e phosphatase a c t i v i t y i n the brush border. No a c t i v i t y i n the lamina p r o p r i a i s v i s i b l e . X 5 6 P L A T E X - 43 -of the columnar e p i t h e l i a l c e l l s l i n i n g the i n t e s t i n a l v i l l i and i n granule c e l l s at the base of each v i l l u s can be l o c a l i z e d when the f r y are a.few weeks old. A comparison of the pattern of alkaline phosphatase a c t i v i t y as seen i n the adult and i n the developmental stages of steelhead trout reveals that (1) during early stages of development, the enzyme i s widely distributed and l o c a l i z e d i n the nuclei of mesenchyme c e l l s and the cytoplasm of endodermal c e l l s , (2) l a t e r on, however, the enzyme tends to become e l e c t i v e l y l o c a l i z e d i n those areas where active d i f f e r e n t i a t i o n i s taking place and disappears from other regions, (3) soon a f t e r the absorption of yolk-sac, when the gut becomes functional, high phosphatase a c t i v i t y i s confined only to the g a s t r i c and i n t e s t i n a l regions, (4) i n the f u l l y formed gut, phosphatase a c t i v i t y i s l o c a l i z e d i n the lamina propria, brush border, and nuclei of the tunica mucosa. - 44 -THE EFFECT OF pH, INHIBITORS AND SUBSTRATES ON LOCALIZATION OF THE ENZYME The v a l i d i t y of Gomori-Takamatsu's method f o r a l k a l i n e phosphatase has been questioned by some workers, p a r t i c u l a r l y w i t h reference to enzyme l o c a l i z a t i o n i n the n u c l e i . At the same time, some workers have i n d i c a t e d that the nuclear and extranuclear phosphatase i s not a s i n g l e enzyme but a s e r i e s of enzymes a c t i v e at c e r t a i n a l k a l i n e pH. In order to examine the v a l i d i t y of Ca-CoS method i n more d e t a i l and to i n v e s t i g a t e the proposed 'enzyme p l u r a l i t y * hypothesis more f u l l y , 5 microns t h i c k s e c t i o n s of p y l o r i c caeca, i n t e s t i n e and kidney ( c o n t r o l ) from over 30 f i s h belonging to the same stock were incubated and the e f f e c t of va r y i n g pH, i n h i b i t o r c o n c e n t r a t i o n s , and v a r y i n g substrates on the l o c a l i z a t i o n of phosphatase a c t i v i t y was observed. P r o t o c o l A. J J H : Two sets of experiments. were designed to i n v e s t i g a t e the e f f e c t of v a r y i n g pH on (1) the topographic l o c a l i z a t i o n of the r e a c t i o n i n the brush border, lamina p r o p r i a , and n u c l e i , and ( 2 ) the s t a b i l i t y of such l o c a l i z a t i o n s at nonoptimal (low or high pH values) c o n d i t i o n s . The e f f e c t of the pH of the i n c u b a t i n g medium on the a l k a l i n e phosphatase a c t i v i t y i n these experiments was observed by varying the pH both by using M i c h a e l i s ' s Veronal acetate-h y d r o c h l o r i c a c i d b u f f e r (pH range 7 . 0 - 9 . 2 ) and by adding M/lO h y d r o c h l o r i c a c i d to the i n c u b a t i o n mixture. pH values higher - 45 -than 9.2 were obtained by u s i n g 0..1N NaOH i n the i n c u b a t i n g medium..- In- each case pH was rechecked by a Pho t o v o l t model 85 ©leetronic pH meter before and a f t e r the i n c u b a t i o n . Experiment #1.. Topographic l o c a l i z a t i o n ; Transverse s e c t i o n s tof the i n t e s t i n e and p y l o r i c caeca fromi 16 ad u l t f i s h were incubated at each pH u n i t from pH 8%0 t o pH 22:0 as given i n Table 1. The p a t t e r n of a l k a l i n e phosphatase a c t i v i t y i n the brush border,, lamina p r o p r i a , and n u c l e i at these pH values i s summarised I n Table 1. P l a t e X II gives a s e m i q u a n t i t a t i v e o v e r a l l p i c t u r e i n terms of percentage a c t i v i t y per- 60 minutes of i n c u b a t i o n , as determined by the photometric method, i n these three l o c a t i o n s . From the r e s u l t s presented,; i t i s c l e a r that the a c t i v i t y i n the brush border p e r s i s t s over the range from pH H"..5 t o pH 8.2. The zone of maximum a c t i v i t y l i e s between pH 10.1 and 8.9 and the r e a c t i o n i s v i s i b l e throughout the len g t h of each v i l l u s . On e i t h e r s i d e s of the zone of maximum, a c t i v i t y , the s t a i n i n g r e a c t i o n i s p a r t i a l and p r o g r e s s i v e l y disappears towards the extreme (pH 12.0 and 8.1).. The r e a c t i o n i n the lamina p r o p r i a p e r s i s t s over a smaller range from pH 10.1 t o pH 8.7> w i t h maximum a c t i v i t y between pH 9.6 and 9':;'2V ...Nuclear phosphatase has a much more c l e a r l y defined. pH optima at pH 8.;6 and 8.7,• w i t h a c t i v i t y p e r s i s t i n g over the range from pH 9.3 t o .8...5.. ( P l a t e XI,-Figs.. 1,2,3>and 4 ) . - 46 -TABLE I. E f f e c t of pH on a l k a l i n e phosphatase a c t i v i t y i n the various regions of i n t e s t i n e and p y l o r i c caeca. P i n a l pH Brush border Lamina p r o p r i a N u c l e i 12.0 — — - - - -11.5 - - - - -11.0 + - — - - -10.7 - + - - -10.4 + - +• - - -10.1 + + + - - -9.9 + + + - - -9.6 + + + 4- - -9.4 + + + - -9.3 + + + + -9.2 + + + + + -8.9 + - + -8.7 + - + -8.6 + - - - + + 8.5 - - - -8.4 - - - - -8.3 - - - - -8.2 + - - - - -8.1 - - - - - -8.0 — m Note: + +, complete r e a c t i o n ; + -, p a r t i a l r e a c t i o n ; - -, no r e a c t i o n . PLATE XI Figure 1. Incubation at pH 9.4. Brush border and lamina p r o p r i a show complete r e a c t i o n . No r e a c t i o n i n the n u c l e i . X 562. Fi g u r e 2* Incubation at pH 9.2. Complete r e a c t i o n i n brush border and lamina p r o p r i a , p a r t i a l r e a c t i o n i n the n u c l e i . X 562. Figur e 3. Incubation at pH 8.6. Complete r e a c t i o n i n n u c l e i , p a r t i a l r e a c t i o n i n brush border, and no r e a c t i o n i n lamina p r o p r i a . X 562. Figur e 4. Incubation at pH 8.2. No r e a c t i o n v i s i b l e i n n u c l e i or lamina p r o p r i a . Only brush border shows p a r t i a l r e a c t i o n near the t i p of v i l l u s . X 562. P L A T E X I P L A T E X I I EFFECT OF p H ON ALKALINE PHOSPHATASE LOCALIZATION 49 - 50 -Experiment #2.. S t a b i l i t y of the enzyme : I n the preceeding experiment i t was n o t i c e d that no phosphatase a c t i v i t y occurred at e i t h e r extreme, I.e. at pH 12..0 and pH 8..0.. The purpose of the present experiment waa to* i n v e s t i g a t e whether the l o s s of a c t i v i t y at these pH values wasj due t o merely i n a c t i v a t i o n of the enzyme or due t o i t s i r r e v e r s i b l e d e s t r u c t i o n . Tissue s e c t i o n s were incubated at pH 12.0 and pH 8..0 r e s p e c t i v e l y from 1 - 2 hours. A f t e r Incubation, the s l i d e s were washed thoroughly w i t h d i s t i l l e d water and then reincubated f o r one hour I n standard substrate at pH 9.3* A c t i v i t y was a l s o t e s t e d by r e a d j u s t i n g the pH from 12 and 8 u n i t s t o 9.3 by adding KD/lO h y d r o c h l o r i c a c i d or 0..1N NaOH to> the i n c u b a t i o n mixture. F o l l o w i n g the treatment w i t h cobalt n i t r a t e and ammoniumi sulphide„ i t was found that there was a d e c i s i v e r e t u r n of the phosphatase a c t i v i t y i n the brush border and lamina propria.. I n another e x p e r i m e n t w h e n t i s s u e s e c t i o n s were t r e a t e d a above a f t e r an i n i t i a l i n c u b a t i o n at pH 13.0 and 7.0 r e s p e c t i v e l y , , there was no a c t i v i t y v i s i b l e anywhere i n the s e c t i o n s even a f t e r prolonged i n c u b a t i o n i n the standard i n c u b a t i n g medium at pH 9»3- H i s t o c h e m i c a l l y speaking,; the l o s s of a c t i v i t y at pH 13 and 7 appears to he due t o i r r e v e r s i b l e d e s t r u c t i o n of the enzyme. P r o t o c o l B. I n h i b i t o r s : The e f f e c t of KCN,, BeSO > a n d NaF, which are strong 4 i n h i b i t o r s of a l k a l i n e phosphatase was i n v e s t i g a t e d by adding these i n h i b i t o r s t o the i n c u b a t i n g medium.. Each of these - 51 -i n h i b i t o r s were used i n c o n c e n t r a t i o n s 0.1M,. 0.01M,., 0.001M, and O.OOOIM; these f i n a l c o n c e n t r a t i o n s expressed as Hols p e r l i t r e of the i n c u b a t i o n mixture 1.' T i s s u e s e c t i o n s were in c u b a t e d at 37 C f o r 60-120 minutess i n the i n c u b a t i n g medium with i n h i b i t o r added. The a d d i t i o n of i n h i b i t o r changes the pH of the i n c u b a t i n g medium considerably,. t h e r e f o r e , ; i n each case,, the pH was r e a d j u s t e d t o 9«3'. F o r each standard incubation,, p a r a l l e l c o n t r o l s were run without the i n h i b i t o r i n the medium. Table I I summarizes' the v a r i o u s i n h i b i t o r s used,, t h e i r molar concentrations,; and the p a t t e r n of phosphatase a u t i v i t y o b t a i n e d under each c o n d i t i o n i n the brush border,;, lamina p r o p r i a and n u c l e i . A l k a l i n e phosphatase a c t i v i t y was almost completely i n h i b i t e d by OvlM 0N~, Be and F i n a l l the r e g i o n s of the i n t e s t i n a l mucosa. 0.O1M CN~ and F produced p a r t i a l i n h i b i t i o n i n the brush border and lamina pr o p r i a ; : Be,, i n the same concentration,- produced p a r t i a l i n h i b i t i o n i n the brush border but completely i n h i b i t e d the a c t i v i t y i n the lamina p r o p r i a . Lower c o n c e n t r a t i o n s of the t h r e e i n h i b i t o r s g e n e r a l l y d i d not a f f e c t phosphatase a c t i v i t y i n the brush border and lamina p r o p r i a except 0..001M: Be which produced a p a r t i a l i n h i b i t i o n i n the l a t t e r . . Phosphatase a c t i v i t y i n the nucleus was comple-t e l y i n h i b i t e d by Be and F i n c o n c e n t r a t i o n s as low as O.OOOIM, whereas?,; ON" i n the same c o n c e n t r a t i o n caused a p a r t i a l i n h i b i t i o n . From the s e r e s u l t s i t appears: t h a t phosphatase a c t i v i t y i n the brush border as: compared t o t h a t i n the lamina p r o p r i a showed g r e a t e r r e s i s t a n c e t o h i g h c o n c e n t r a t i o n s of i n h i b i t o r s . N u clear phosphatase on the o t h e r hand,, was remarkably s e n s i t i v e t o lower c o n c e n t r a t i o n s . - 52 -TABLE I I . The effect of i n h i b i t o r concentrations on alkaline phosphatase a c t i v i t y i n the various regions of the i n t e s t i n a l mucosa. INHIBITOR L 0 C A T I F i n a l concentration i n incubation mixture 0 N 10" 4M 10~3M 10"2M 10" B 4 4 4- 4 - -CYANIDE L N + 4 + -4 4 4 -- -B + 4 4- 4 4 - — — BERYLLIUM L N + 4 4 -- -B + 4 + 4 4 - - -FLUORIDE L 4 4 + 4 4 - - -N - - - — - - - — Note: 4- 4 , normal reaction; 4 -, p a r t i a l i n h i b i t i o n ; - complete i n h i b i t i o n . B;, Brush border; L, lamina propria; N, n u c l e i . Based on the observations made on tissue sections obtained from 11 adult fish". - 53 -P r o t o c o l C Sub s t r a t e s ; Modified Gomori's cob a l t sulphide method as suggested by Herman (1951) was used to examine the e f f e c t of d i f f e r e n t s u b strates on the l o c a l i z a t i o n of phosphatase a c t i v i t y i n the i n t e s t i n a l mucosa. Tissue s e c t i o n s were incubated at 37°C f o r 60-120 minutes at pH 9.3 wit h sodium beta glycerophosphate"'', 2 3 muscle a d e n y l i c a c i d , glucose-l-phosphate , and f r u c t o s e 4 diphosphate as the substrates i n d i f f e r e n t experiments. The a c t i v i t y p a t t e r n obtained with sodium glycerophosphate was used as a c o n t r o l and as a b a s i s of comparison w i t h h i s t o c h e m i c a l p a t t e r n s obtained w i t h other s u b s t r a t e s . The r e l a t i v e i n t e n s i t y o f the s t a i n i n g r e a c t i o n i n the brush border, lamina p r o p r i a and n u c l e i , w i t h each of the f o u r substrates a f t e r an i n c u b a t i o n period of 60 minutes i s shown i n Table I I I . The a c t i v i t y i n the brush border was found to be ne a r l y of the same i n t e n s i t y w i t h sodium glycerophosphate, muscle a d e n y l i c a c i d , glucose-l-phosphate and f r u c t o s e diphos-phate. In the lamina p r o p r i a , however, the r e a c t i o n was l e s s i n t e n s e w i t h glucose-l-phosphate and f r u c t o s e diphosphate as compared to that obtained with sodium glycerophosphate. Blood c a p i l l a r i e s and connective t i s s u e of the lamina p r o p r i a showed l i t t l e or no a c t i v i t y w i t h muscle a d e n y l i c a c i d as the subs t r a t e . The g r e a t e s t e f f e c t of d i f f e r e n t s u b s t r a t e s i s seen i n the n u c l e i of the columnar e p i t h e l i a l c e l l s . While w i t h 1 - 4 , Obtained from N u t r i t i o n a l Biochemicals Corp. Cleveland. Ohio. - 54 -TABLE I I I . A l i a l i n e p h o s p h a t a s e a c t i v i t y i n the i n t e s t i n a l mucosa w i t h v a r i o u s s u b s t r a t e s . SUBSTRATE B r u s h b o r d e r L a m i n a p r o p r i a N u c l e i Na g l y c e r o p h o s p h a t e + + + + + + + + A d e n y l i c a c i d (AMP) + + + + + +• G l u c o s e -1 - p h o s p h a t e + + + + + P r u c t o s e - l - 6 - D i p h o s p h a t e + + + + + N o t e : + r e f e r s t o r e l a t i v e i n t e n s i t y o f r e a c t i o n . Based on the observations made on t i s s u e s e c t i o n s obtained from. 1 1 adult f i s h . sodium glycerophosphate and AMP a d e f i n i t e s t a i n i n g r e a c t i o n of low i n t e n s i t y was observed i n the n u c l e i , there was a complete absence of any nuclear r e a c t i o n with glucose-l-phos-phate and f r u c t o s e diphosphate. - 56 -THE EFFECT OF MODERATE AND ACUTE STARVATION ON ENZYME LOCALIZATION Q u a n t i t a t i v e measurements on t i s s u e homogenates of fed and starved animals have shown that the l e v e l of serum and i n t e s t i n a l a l k a l i n e phosphatase decreases during f a s t i n g c o n d i t i o n s , whereas resumption of feeding produces a s i g n i f i c a n t e l e v a t i o n of the enzyme l e v e l . Moog (1950), however, demon-s t r a t e d , both by biochemical and h i s t o c h e m i c a l methods, that f o l l o w i n g s t a r v a t i o n , the l e v e l of a l k a l i n e phosphatase i n the i n t e s t i n a l mucosa of the chick i n c r e a s e s . This obvious d i s p a r i t y between r e s u l t s obtained by Moog and other workers c a l l s f o r a c l o s e r examination of the e f f e c t of food d e p r i v a t i o n on the phosphatase a c t i v i t y i n the i n t e s t i n a l mucosa. Since f i s h are able to withstand prolonged s t a r v a t i o n as much as four to f i v e months, i t was p o s s i b l e to make a progressive comparison between the e f f e c t of moderate and acute f a s t i n g c o n d i t i o n s on the h i s t o c h e m i c a l l o c a l i z a t i o n of phosphatase a c t i v i t y . A stock of adult steelhead t r o u t of same age and approximately of same s i z e was maintained i n the l a b o r a t o r y on a commercial trout d i e t given r e g u l a r l y f o r two weeks. L a t e r , the f i s h were t r a n s f e r r e d to two tanks, one w i t h c o n t r o l s r e c e i v i n g the same food every day throughout the experimental p e r i o d , and the other w i t h f a s t i n g f i s h . F i s h were starved f o r 8, 12, 20, 50, 60, 80, 100, and 120 days. The i n t e s t i n e , p y l o r i c caeca and kidney were removed i n each case and f i x e d f o r his t o c h e m i c a l purposes. Under standard c o n d i t i o n s of i n c u b a t i o n , the usual s i t e s of a l k a l i n e phosphatase a c t i v i t y i n the s e c t i o n s of the i n t e s t i n e and p y l o r i c caeca of normal fed f i s h are the - 57 -brush border, lamina p r o p r i a , and n u c l e i ( P l a t e I I , P i g . 1). Table IV summarises the e f f e c t of v a r y i n g durations of food d e p r i v a t i o n on the s t a i n i n g r e a c t i o n i n the brush border, lamina p r o p r i a and n u c l e i of the i n t e s t i n a l mucosa. In the e a r l y stages of food d e p r i v a t i o n some changes i n o v e r a l l s t a i n i n g r e a c t i o n are n o t i c e a b l e , p a r t i c u l a r l y r e d u c t i o n or disappearance of nuclear r e a c t i o n and a s l i g h t i n c r e a s e i n the i n t e n s i t y of s t a i n i n the brush border. Such a c o n d i t i o n i s represented i n f i s h starved f o r 8 to 12 days ( P l a t e X I I I , P i g . 1). In f i s h starved f o r over 20 days, a s l i g h t decrease i n s t a i n i n g r e a c t i o n i n lamina p r o p r i a i s seen i n a d d i t i o n to changes mentioned above. Advanced stages of s t a r v a t i o n as seen i n 60 to 100 days starved f i s h are c h a r a c t e r i s e d by a d i f f u s e r e a c t i o n i n the cytoplasm very c l o s e to the brush border, a p a r t i a l or no r e a c t i o n i n the lamina p r o p r i a , and a complete absence of any v i s i b l e nuclear r e a c t i o n . P i s h starved f o r over 100 days show a d e f i n i t e s t r u c t u r a l d e t e r i o r a t i o n of the i n t e s t i n a l mucosa due to desquamation of columnar e p i t h e l i a l c e l l s . The brush border i s a l s o a f f e c t e d and a l k a l i n e phosphatase a c t i v i t y at t h i s s i t e appears very d i f f u s e but apparently more intense as compared to e a r l y stages ( P l a t e X I I I , P i g . 2 ) . In order to s u b s t a n t i a t e the r e s u l t s obtained f o r the i n t e s t i n e and p y l o r i c caeca, p a r a l l e l c o n t r o l s e c t i o n s of kidney from fed and starved f i s h were incubated under standard c o n d i t i o n s . No a c t i v i t y i n the n u c l e i of the r e n a l tubules of starved f i s h was v i s i b l e , whereas the s e c t i o n s from f e d f i s h gave a p o s i t i v e nuclear r e a c t i o n . The a c t i v i t y i n the brush - 58 -TABLE IV. The e f f e c t of v a r y i n g periods of s t a r v a t i o n on a l k a l i n e phosphatase a c t i v i t y i n the i n t e s t i n e and p y l o r i c caeca. S t a r v a t i o n p e r i o d Brush border Lamina p r o p r i a N u c l e i (days) 0 + + + + + 8 + +• + + + -12 + + + + - - •-20 + + •f - - -30 + + +• - - -60 * + + + - - -80 * + + - - -100 + + + - - - -120 * + + + Note: + + +, intense r e a c t i o n ; + +, normal r e a c t i o n ; + p a r t i a l r e a c t i o n ; - -, no r e a c t i o n . * D i f f u s i o n i n t o the cytoplasm. PLATS X I I I Pigure 1. P i s h starved f o r 12 days. The i n t e s t i n a l v i l l u s shows inte n s e r e a c t i o n i n the brush border, p a r t i a l r e a c t i o n i n the lamina p r o p r i a and no r e a c t i o n i n the n u c l e i . X 562. Pigure 2. P i s h starved f o r 120 days. A l k a l i n e phosphatase a c t i v i t y i n the brush border appears very much d i f f u s e . No r e a c t i o n i n lamina p r o p r i a and n u c l e i . X 562. P L A T E XIII PLATE XIV. Pi g u r e 1. Transverse s e c t i o n of kidney tubules from a normal f e d f i s h . A l k a l i n e phosphatase a c t i v i t y i s v i s i b l e i n the brush border and n u c l e i of the tubules. X 562. Pigure 2 Transverse s e c t i o n of kidney tubules from f i s h starved f o r 60 days. Note disappearance of nuclear r e a c t i o n . X 562. P L A T E X I V border d i d not show any s i g n i f i c a n t change i n i n t e n s i t y between fed and starved animals ( P l a t e XIV., P i g s . 1 and 2 ) . - 64 -DISCUSSION ENZYMATIC PATTERN IN RELATION TO DIFFERENTIATION Evidence from experimental embryology ex p l a i n s development as e s s e n t i a l l y a sequence of chemical and p h y s i c a l changes which have modifying e f f e c t on the d i f f e r e n t i a t i n g c e l l s . The chemical d i f f e r e n c e s between the d i f f e r e n t i a t i n g and n o n - d i f f e r e n t i a t i n g elements during organogenesis and h i s t o g e n e s i s seem to a r i s e g r a d u a l l y and p r o g r e s s i v e l y l e a d i n g e v e n t u a l l y to morphological d i s t i n c t i o n s . Considerable recent work on enzyme development during embryogenesis comes from the l a b o r a t o r i e s of Brachet (1950, I960), B o e l l (1955) and Moog (1950, 1951, 1958). These workers have shown that the processes of organogenesis and h i s t o g e n e s i s and eventual a c q u i s i t i o n of f u n c t i o n by a p a r t i c u l a r organ seem to f i t i n t o an o r d e r l y p a t t e r n i n f l u e n c e d by a synthesis or l o s s of enzyme or enzyme systems. A monophasic i n c r e a s e i n the l e v e l of duodenal a l k a l i n e phosphatase at the time of hatching or b i r t h has been experimentally demonstrated i n chick by Moog (1950), and i n guinea p i g by Moog and O r i t z (1957). S i m i l a r l y a d i p h a s i c ( p r e n a t a l and p o s t n a t a l ) increase of phosphatase has been reported i n the case of mouse by Moog (1951). Of the two peaks of phosphatase accumulation i n mouse duodenum, Moog (1951) a t t r i b u t e d p r e n a t a l i n c r e a s e to c e l l u l a r d i f f e r e n t i a t i o n and p o s t n a t a l r i s e to enzyme sy n t h e s i s i n r e l a t i o n to a c q u i s i t i o n of f u n c t i o n by the duodenum. From these studies one important f a c t emerged, i . e . d i s c o n t i n u i t y i n enzyme development. - 65 -Present observations on the h i s t o c h e m i c a l l o c a l i z a t i o n of a l k a l i n e phosphatase i n the developing gut of steelhead t r o u t have c l e a r l y demonstrated t h i s discontinuous nature of enzyme development. Prom the very e a r l y to most advanced stages of development, r e g i o n a l v a r i a t i o n s i n the concentration of a l k a l i n e phosphatase throughout the developing gut become qu i t e obvious. Phosphatase a c t i v i t y i n e a r l y prehatehing stage i s very low throughout the gut except i n the hind r e g i o n .where the endodermal l i n i n g around the t i n y lumen gives a moderately p o s i t i v e r e a c t i o n . A s i g n i f i c a n t d i f f e r e n c e i n enzyme a c t i v i t y i s n o t i c e d i n l a t e prehatehing stage when n u c l e i of the mesen-chyme c e l l s surrounding the endodermal element give intense p o s i t i v e r e a c t i o n w i t h i n few minutes of i n c u b a t i o n . Endoderm c e l l s at the same l o c a t i o n show l i t t l e or no phosphatase a c t i v i t y . The p a t t e r n of phosphatase a c t i v i t y i n the i n t e s t i n a l r e gion appears to be d i f f e r e n t ; here mesenchyme c e l l s are devoid of any r e a c t i o n but endodermal l i n i n g g i ves an i n t e n s e cytoplasmic r e a c t i o n . The absence of phosphatase a c t i v i t y i n mesenchyme c e l l s i n the i n t e s t i n a l r e g i o n and i t s presence i n f o r e and mid gut regions appears to be r e l a t e d to e a r l y and l a t e d i f f e r e n t i a t i o n . Other i n s t a n c e s of such r e g i o n a l accumulations and disappearance of a l k a l i n e phosphatase i n r e l a t i o n to d i f f e r -e n t i a t i o n are found d u r i n g e a r l y and l a t e posthatching stages i n connection w i t h the development of swim bladder, corpus and p y l o r i c stomach. F i n a l l y , at the f r y stage, the d i f f e r e n t i a t i o n of the submucosa and lamina p r o p r i a i n the g a s t r i c r e g i o n and the formation of p y l o r i c caeca are a l l accompanied by an - 66 -increased phosphatase a c t i v i t y i n the d i f f e r e n t i a t i n g t i s s u e . Although no q u a n t i t a t i v e data on the l e v e l of a l k a l i n e phosphatase present at any one time i n the gut are a v a i l a b l e , the h i s t o -chemical observations have shown that d u r i n g development of the gut i n t r o u t , phosphatase concentration increases with advancing stages, though w i t h r e g i o n a l v a r i a t i o n s . No s i g n i f i -cant i n c r e a s e i n enzyme concent r a t i o n i s n o t i c e a b l e i n f i s h soon a f t e r hatching, although such an i n c r e a s e i s reported f o r the chick (Moog, 1950). In f i s h , the enzymic a c t i v i t y i n the g a s t r i c and i n t e s t i n a l regions i s very pronounced soon a f t e r the absorption of the yolk-sac when the gut i s about to become f u n c t i o n a l . Thus, the f r y at the time of emergence i s comparable to hatched stage of the chick described by Moog. Ikeda (1959) i n h i s study of the d i g e s t i v e system of a t e l e o s t f i s h , Oryzias l a t i p e s reported that during the i n i t i a l phases of d i f f e r e n t i a t i o n , a l k a l i n e phosphatase, carbohydrates, and RNA are present i n a l l t i s s u e s but t h e i r i n c r e a s e or decrease i n the various organs at prehatching and hatching periods i s l i n k e d with the time at which a p a r t i c u l a r organ begins to appear. His r e s u l t s are comparable to Moog's (1944) f i n d i n g s that' phosphatase plays a fundamental r o l e i n h i s t o -genesis and i t increases or decreases i n r e l a t i o n to whether the organ begins to f u n c t i o n e a r l y or l a t e i n the developing p e r i o d . The r e s u l t s obtained i n the present study are i n general agreement w i t h those of Moog's and Ikeda's and once again p o i n t to the r e l a t i o n between enzyme a c t i v i t y and f u n c t i o n a l d i f f e r e n t i a t i o n i r r e s p e c t i v e of the groups of animal i n v o l v e d . In s p i t e of the wealth of l i t e r a t u r e d e a l i n g w i t h - 67 -enzymes i n r e l a t i o n to development, our knowledge of f a c t o r s c o n t r o l l i n g enzymic a c t i v i t y i s f a r from p e r f e c t . Moog (1958) has e s t a b l i s h e d that phosphatase s y n t h e s i s i s under hormonal c o n t r o l and the prehatching and preweaning r i s e i n i n t e s t i n a l phosphatase i n chick and mouse i s due to increased s e c r e t i o n of c o r t i c a l hormones at that time. I t i s therefore conceivable that e l a b o r a t i o n of s p e c i f i c chemical substances during development i s s u s c e p t i b l e to i n t e r n a l or e x t e r n a l s t i m u l i -i n c r e a s i n g or decreasing according to type and threshold of the s t i m u l u s . The r i c h n e s s of phosphatase a c t i v i t y i n the d o r s a l w a l l , i n contrast to the v e n t r a l w a l l of the developing esophagus i s c h a r a c t e r i s t i c during e a r l y and l a t e posthatehing stages only. The exact s i g n i f i c a n c e of t h i s d i f f e r e n t i a l a c t i v i t y i s not f u l l y understood. Brachet (1947) reported that the roo f of the primary gut i n embryonic amphibia i s r i c h e r i n r i b o n u c l e o -p r o t e i n s than i t s v e n t r a l r e g i o n . On the b a s i s of a c l o s e and constant r e l a t i o n s h i p between a l k a l i n e phosphatase and nu c l e o p r o t e i n s y n t h e s i s as reported by Brachet and Jeener (1948), Moog (1946), and Dempsey and W i s l o c k i (1946), the richness of phosphatase a c t i v i t y i n the n u c l e i of the d o r s a l mesenchymal w a l l can probably be explained. S u f f i c i e n t experimental evidence has accumulated i n recent years to show that a l k a l i n e phosphatase p a r t i c i p a t e s i n morphogenesis. The i n t e r p r e t a t i o n of the s i g n i f i c a n c e of phosphatase l o c a l i z a t i o n i n adult t i s s u e s and p a r t i c u l a r l y i n the i n t e s t i n a l mucosa has been l a r g e l y s p e c u l a t i v e or based on c i r c u m s t a n t i a l evidence. Most of the spec u l a t i o n s concerning - 68 -f u n c t i o n of a l k a l i n e phosphatase i n the i n t e s t i n e and kidney o f a d u l t animal have revolved around Lundsgaard's (1933) and Verzar and McDougall's (1936) t h e s i s of phosphorylation -dephosphorylation processes i n sugar absorption. Followers o f t h i s hypothesis have i m p l i c a t e d a l k a l i n e phosphatase i n the i n t e s t i n a l and r e n a l absorption of glucose and maintain that the t r a n s f e r of glucose through the i n t e s t i n a l e p i t h e l i u m i n v o l v e s phosphorylation on one end of the c e l l and dephos-p h o r y l a t i o n at the other. Researches of Davidson and Garry (1939, 1940), Campbell and Davson (1948) and r e c e n t l y of Treherne (1958a, 1958b) have provided evidence against the phosphorylation theory. These workers maintain that some metabolic a l t e r a t i o n takes place i n the molecule of glucose to make i t chemically d i s t i n c t from blood glucose, thereby i n c r e a s i n g the d i f f u s i o n g r a d i e n t s and i n t h i s scheme phosphorylation does not appear to be i n v o l v e d . At the present time, no d e f i n i t e statement on the v a l i d i t y of the phosphorylation theory can be made u n t i l s u f f i c i e n t f a c t s are a v a i l a b l e . I f phosphatase i s i n v o l v e d i n c a t a l y s i n g glucose t r a n s f e r a c t i v i t i e s i n the i n t e s t i n a l mucosa, the co n c e n t r a t i o n of the enzyme at e i t h e r ends of the columnar c e l l s , i . e . the brush border and the lamina p r o p r i a , appears s i g n i f i c a n t . High a l k a l i n e phosphatase a c t i v i t y i n the g a s t r i c r e g i o n , p a r t i c u l a r l y , i n the i n t e r g l a n d u l a r t i s s u e of the lamina p r o p r i a , suggests that t h i s enzyme i s probably i n v o l v e d i n the s e c r e t o r y a c t i v i t i e s of the g a s t r i c glands. Another f a c t which emerges from the present study i s the high degree of s i m i l a r i t y i n h i s t o l o g i c a l elements and the - 69 -p a t t e r n of phosphatase d i s t r i b u t i o n between the i n t e s t i n e and p y l o r i c caeca. The l a t t e r have long been suspected as s i t e s of absorption (Greene, 1913). Prom the present observations i t appears that the s t r u c t u r a l s i m i l a r i t y between the i n t e s t i n e and p y l o r i c caeca i s r e l a t e d to f u n c t i o n a l s i m i l a r i t y . NUCLEAR PHOSPHATASE The presence of a l k a l i n e phosphatase as shown by his t o c h e m i c a l techniques i n both n u c l e i and cytoplasm i s of considerable i n t e r e s t because of c o n f l i c t i n g r e s u l t s obtained by s e v e r a l workers. This i n c o n s i s t a n c y i n r e s u l t s has l e d to a r e a p p r a i s a l of the v a l i d i t y of Gomori-Takamatsu's method. Chevremont and P i r k e t (1953) have adequately reviewed the subject of nuclear phosphatase. High a l k a l i n e phosphatase a c t i v i t y has been reported i n the n u c l e i of a number of t i s s u e s : i n l i v e r by Dounce (1943), i n kidney by D a n i e l l i (1946) and Loveless and D a n i e l l i (1949), i n bone by Lorch (1949a, 1949b) and Bodansky, Bakwin and Bakwin (1931), i n b r a i n by R i c h t e r and H u l l i n (1951), and i n the d i f f e r e n t i a t i n g t i s s u e during embryonic growth by Moog (1946), Brachet (1946), K r u g e l i s (1947), Brachet and Jeener (1948) and Hinseh (I960). Nevertheless, some workers have expressed doubts as to the v a l i d i t y of i n t r i n s i c nuclear phosphatase as demonstrated by h i s t o c h e m i c a l methods (Ruyter and Neumann, 1949; N o v i k o f f , 1951, 1952), and maintain that during h i s t o -chemical manipulations, e i t h e r the enzyme or the r e a c t i o n product i n the form of calcium phosphate i s p r e f e r e n t i a l l y adsorbed by the n u c l e i and as such Gomori-Takamatsu 1s method - 70 -can not be r e l i e d upon to show i n t r a c e l l u l a r s i t e s of a l k a l i n e phosphatase a c t i v i t y . N o v i k o f f (1951, 1952) based h i s doubts on the low a c t i v i t y i n the i s o l a t e d n u c l e i of r a t l i v e r c e l l s i n c o n t r a s t to high a c t i v i t y i n l i v e r s e c t i o n s as demonstrated by h i s t o -chemieal method. The i n c u b a t i o n times reported i n h i s s t u d i e s are rather longer than reported by other workers and those used i n the present study. I t i s therefore l i k e l y that the discrepancy between r e s u l t s obtained by i s o l a t i o n of i n t r a -c e l l u l a r f r a c t i o n s by d i f f e r e n t i a l c e n t r i f u g a t i o n and by h i s t o c h e m i c a l methods i s due c h i e f l y to the l e n g t h of i n c u b a t i n g p e r i o d . The subject of d i f f u s i o n of the enzyme or of the r e a c t i o n product has been e x t e n s i v e l y d e a l t with by L i s o n (1948), M a r t i n and Jacoby (1949), C l e l a n d (1950), F e i g i n , Wolf, and Kabat (1950), Yokoyama, S t o w e l l and Mathews (1951), Gomori (1951), Moe (1952) and D a n i e l l i (1946, 1953). These i n v e s t i g a t o r s have shown that d i f f u s i o n a r t i f a c t s are l i k e l y to a r i s e near the s i t e s of high a l k a l i n e phosphatase a c t i v i t y . The n u c l e i seem to have a s p e c i a l a f f i n i t y f o r the enzyme but w i l l a l s o adsorb calcium phosphate when present i n supersaturated amounts i n the s u b s t r a t e thereby g i v i n g a f a l s e p o s i t i v e r e a c t i o n w i t h Ca-CoS method. In the present study, u s i n g the v a r i o u s c o n t r o l s mentioned e a r l i e r , the p o s s i b i l i t y of d i f f u s i o n a r t i f a c t s has been reduced and nuclear s t a i n i n g could be seen w i t h i n 30-60 minutes of i n c u b a t i o n under standard c o n d i t i o n s . This i n c u b a t i o n time i s considerably s h o r t e r than most workers have used and a - 71 -very d e f i n i t e p o s i t i v e r e a c t i o n , p a r t i c u l a r l y i n the nuclear membrane and the n u c l e o l i can under the circumstances be a t t r i -buted to the presence of i n t r i n s i c phosphatase. Gomori (1951) concluded that only such l o c a l i z a t i o n s as can be demonstrated a f t e r a short i n c u b a t i o n can be accepted as genuine since prolonged i n c u b a t i o n i s l i k e l y to produce a r t e f a c t s . Moe (1952) a l s o concluded t h a t , i n the h i s t o c h e m i c a l demonstration of a l k a l i n e phosphatase a c t i v i t y , the time f a c t o r i s very important because long i n c u b a t i o n promotes d i f f u s i o n of the r e a c t i o n product and may change a p o s i t i v e r e a c t i o n to a negative one. The h i s t o c h e m i c a l p i c t u r e obtained by prolonged i n c u b a t i o n (12 hours) i n the i n t e s t i n a l mucosa confirms Moe's conclusions. The brush border was devoid of a d e f i n i t e r e a c t i o n , whereas, the cytoplasm of the columnar e p i t h e l i a l c e l l s and the n u c l e i showed i n t e n s e a c t i v i t y ( P l a t e I I I , P i g . 2 ) . Incubation of nondeparaffinized t i s s u e s e c t i o n s i n standard substrate i n order to avoid l a t e r a l d i f f u s i o n and subsequent adsorption of the r e a c t i o n product by the n u c l e i , as suggested by Goetsh, Reynolds, and Bunting (1952), a l s o showed p o s i t i v e r e a c t i o n i n the n u c l e i of the i n t e s t i n a l mucosa and kidney tubules. High a l k a l i n e phosphatase a c t i v i t y , p a r t i c u l a r l y i n the mesenchyme c e l l s of the d i f f e r e n t i a t i n g gut and i t s absence i n endoderm and muscle c e l l s i n the same v i c i n i t y , would not be expected i f d i f f u s i o n or adsorption were major f a c t o r s f o r nuclear r e a c t i o n . A complete absence of any nuclear r e a c t i o n i n c o n t r o l s e c t i o n s of the d i f f e r e n t i a t i n g gut exposed to UV r a d i a t i o n s a l s o strengthens the b e l i e f that nuclear r e a c t i o n i s due to i n t r i n s i c phosphatase. - 72 -A d d i t i o n a l evidence as to the v a l i d i t y of the presence of a l k a l i n e phosphatase i n the n u c l e i comes from the study of D a n i e l l i and Catcheside (1945) who observed high phosphatase a c t i v i t y i n t i s s u e c u l t u r e chromosomes. K r u g e l i s (1946) a l s o reported that g i a n t chromosomes of Dro s o p h i l a are r i c h i n a l k a l i n e phosphatase. In the chromosomes and other nuclear c o n s t i t u e n t s of plant c e l l s , the presence of t h i s enzyme has been reported (Ross and E l y , 1951). Studies on t i s s u e c u l t u r e , w i t h proper f i x a t i o n and l i t t l e r p o s s i b i l i t y of d i f f u s i o n a r t i f a c t s , show a high a l k a l i n e phosphatase a c t i v i t y i n the n u c l e i (Willmer, 1942; Wachstein, 1945; B i e s e l e , 1949; Chevremont and F i r k e t , 1949). Loveless and D a n i e l l i (1949), Yokoyama, S t o w e l l , and Tsuboi (1950), Yokoyama, S t o w e l l , and Mathews, (1951) and Gomori (1951), comparing cobalt sulphide method and azo--dye method of phosphatase l o c a l i z a t i o n found that the l a t t e r method gave very l i t t l e or no r e a c t i o n i n the n u c l e i . This has-been i n t e r p r e t e d by some workers as a d d i t i o n a l evidence i n favour of the absence of a l k a l i n e phosphatase i n the n u c l e i . Prom the r e s u l t s presented i n the present study and reported e a r l i e r (Prakash, 1960a, 1960b, 1960c), w i t h respect to the d i f f e r e n t i a l t s u s c e p t i b i l i t y of nuclear and nonnuclear phosphatase to pH changes, i n h i b i t o r c o n c e n t r a t i o n s , and d i f f e r e n t l e v e l s of s t a r v a t i o n , i t appears that the a l k a l i n e phosphatase i n the nucleus behaves d i f f e r e n t l y from that present i n the brush border and lamina p r o p r i a of the i n t e s t i n a l mucosa. The concen-t r a t i o n of the enzyme i n the n u c l e i i s always lower than that found i n the other two s i t e s , and i t i s p o s s i b l e that the s u b s t r a t e s used i n the azo-dye method are not s u f f i c i e n t l y hydrolysed by the nuclear phosphatase to demonstrate the enzyme. In c o n c l u s i o n , the preponderance of evidence i n d i c a t e s that a l k a l i n e phosphatase i s present i n the n u c l e i of most, i f not a l l , t i s s u e s and can be r e l i a b l y demonstrated by Gomori-Takamatsu's c o b a l t sulphide method, provided adequate c o n t r o l s are employed. There i s no d e n i a l of the f a c t that the d i f f u s i o n of the enzyme, or of the r e a c t i o n product, can, and does, occur g i v i n g a f a l s e - p o s i t i v e l o c a l i z a t i o n w i t h t h i s method, but proper f i x a t i o n "and a short i n c u b a t i o n time are the necessary concomitant of a v a l i d h i s t o c h e m i c a l l o c a l i z a t i o n of phosphatase a c t i v i t y i n the nucleus. Biochemical stud i e s have shown t h a t , from the q u a n t i t a t i v e p o i n t of view, the concentration of a l k a l i n e phosphatase i n the n u c l e i i s low and i s subject to v a r i a t i o n s according to the metabolic s t a t e of the t i s s u e . T h i s s t a t e changes during embryonic d i f f e r e n t i a t i o n , r e g e n e r a t i o n , and c e l l d i v i s i o n and thus one can expect some t i s s u e s to be r i c h and others poor i n t h e i r phosphatase content. The r o l e of nuclear phosphatase i s indeed obscure. Although some experimental evidence i s a v a i l a b l e to show that phosphatase a c t i v i t y i n c r e a s e s during m i t o t i c a c t i v i t y of the c e l l , the exact mechanism i s f a r from understood. Chevremont and P i r k e t (1953) have reviewed the evidence and hypotheses put forward i n connection w i t h the f u n c t i o n of nuclear phosphatase. Judging from the high con c e n t r a t i o n of t h i s enzyme i n the n u c l e o l i and the nuclear membrane, i t appears that a l k a l i n e phosphatase i s probably concerned w i t h hucleo-p r o t e i n metabolism i n the nucleus and eventual t r a n s f e r of - 74 -cytoplasmic RNA through the nuclear membrane. Although t h i s statement i s p u r e l y s p e c u l a t i v e and based on c i r c u m s t a n t i a l evidence alone, Brachet and Jeener (1948) have i n d i c a t e d a clo s e and constant r e l a t i o n s h i p between the nuclear a l k a l i n e phosphatase content and the rate of DNA turnover. According to Gaspersson (1947), the nucleus i s the c h i e f center of p r o t e i n s y n t h e s i s and the nu c l e o l u s and the nuclear membrane play a prominant r o l e i n the synthesis of the cytoplasmic r i b o n u c l e o p r o t e i n granules. The existence of a s t r i k i n g p a r a l l e l i s m i n the d i s t r i b u t i o n of RNA and a l k a l i n e phosphatase i n the nuclear and e x t r a n u c l e a r elements appears s i g n i f i c a n t . STARVATION Biochemical evidence i n d i c a t e s that the l e v e l of i n t e s t i n a l a l k a l i n e phosphatase i s s u s c e p t i b l e to d i e t a r y i n f l u e n c e s (Lawrie and Yudkin, 1949; Jackson, 1952; Tuba and Robinson, 1953; Tuba and D i c k i e , 1954, 1955; D i c k i e , Robinson and Tuba, 1955). The same i s true of serum phosphatase which has been regarded as of i n t e s t i n a l o r i g i n (Gould, 1944; F l o c k and Bollman, 1948; Madsen and Tuba, 1952). I t i s known a l s o that serum and i n t e s t i n a l phosphatase l e v e l s decrease consid-erably d u r i n g f a s t i n g c o n d i t i o n s , and that the feeding of c e r t a i n p r o t e i n s , carbohydrates and f a t t y a c i d s produces a s i g n i f i c a n t e l e v a t i o n of the enzyme l e v e l s . Observations made i n the present study suggest that s t a r v a t i o n has a marked i n f l u e n c e on the a l k a l i n e phosphatase concentration i n the i n t e s t i n a l mucosa of f i s h . During the e a r l y stages of food d e p r i v a t i o n nuclear phosphatase i s a f f e c t e d , w h i l e advanced stages of s t a r v a t i o n are c h a r a c t e r i s e d hy a r e d u c t i o n or absence of phosphatase a c t i v i t y i n the lamina pro-p r i a . The brush border shows an i n c r e a s e i n a l k a l i n e phosphatase a c t i v i t y soon a f t e r the commencement of f a s t i n g . I f the f a s t i n g p e r i o d i s prolonged, a d i f f u s e r e a c t i o n i n the cytoplasm i n c l o s e p r o x i m i t y to the brush border, a p a r t i a l r e a c t i o n i n the lamina p r o p r i a and a complete absence of any v i s i b l e r e a c t i o n i n the n u c l e i can be seen. F i n a l l y , a d e f i n i t e s t r u c t u r a l d e t e r i o r a t i o n of the i n t e s t i n a l mucosa and a more pronounced d i f f u s e h i s t o c h e m i c a l r e a c t i o n jaear the brush border i s seen. Tuba and Robinson (1953) demonstrated that i n t e s t i n a l a l k a l i n e phosphatase i n r a t s i s composed of an 'adaptive f r a c t i o n ' which v a r i e s with the d i e t and n u t r i t i v e stage of the animal, and a 'nonadaptive f r a c t i o n ' which i s not markedly a f f e c t e d by prolonged s t a r v a t i o n . The present observations appear to i n d i c a t e that the phosphatase present i n the n u c l e i and the lamina p r o p r i a may represent the adaptive f r a c t i o n , whereas, that present i n the brush border can be regarded as the nonadaptive enzyme. The accumulation of a heavier p r e c i p i t a t e i n the brush border f o l l o w i n g s t a r v a t i o n i s i n t e r e s t i n g . Moog (1950) reported s i m i l a r accumulation i n starved chicks and concluded that t h i s i n c r e a s e i n very young c h i c k s i s due to an a c t i v e enzyme s y n t h e s i z i n g mechanism. Although no attempt has been made i n the present study to i n v e s t i g a t e the mechanics of t h i s abnormal accumulation of phosphatase i n the brush border, two p o s s i b i l i t i e s are suggested: (1) an agreement w i t h Leblond and and Stevens' (1948) contention that the r e l e a s e of a l k a l i n e - 76 -phosphatase from the gut w a l l i s due to progressive desquamation of the e p i t h e l i a l c e l l s r a t h e r than s e c r e t i o n , and (2) that the i n c r e a s e i n phosphatase concentration f o l l o w i n g s t a r v a t i o n i s under hormonal c o n t r o l . Of these two p o s s i b i l i t i e s , the l a t t e r appears more a t t r a c t i v e since i t i s known that f o l l o w i n g s t a r v a t i o n blood glucose l e v e l decreases considerably and the animal i s i n a s t a t e of ' s t r e s s ' . At t h i s time p i t u i t a r y -adrenal i n t e r r e l a t i o n s h i p comes i n t o p l a y f o r the maintenance of normal l e v e l s of blood sugar. Adrenal c o r t i c o i d s , p a r t i -c u l a r l y 11-oxysteroids are probably r e l e a s e d at a higher r a t e which s t i m u l a t e gluconeogenesis and to some extent glycogeno-l y s i s . The r e l a t i o n between increased a d r e n o c o r t i c a l s e c r e t i o n and phosphatase s y n t h e s i s i s a g e n e r a l l y accepted t h e s i s (Moog, 1953, 1958). On t h i s b a s i s the i n c r e a s e i n a l k a l i n e phosphatase a c t i v i t y i n the i n t e s t i n a l mucosa during s t a r v a t i o n appears to be l i n k e d w i t h increased s e c r e t i o n of adrenal c o r t i c o i d s . Nevertheless, the exact s i g n i f i c a n c e of the ' s o - c a l l e d ' accumulation of phosphatase duri n g s t a r v a t i o n as shown by h i s t o c h e m i c a l methods and considerable decrease i n normal phosphatase l e v e l s during s t a r v a t i o n as determined by biochemical assay on t i s s u e homogenates needs f u r t h e r e l u c i d a t i o n . EN2YME PLURALITY Demonstration of t o p o g r a p h i c a l l y v a r i a b l e patterns of a l k a l i n e phosphatase a c t i v i t y i n t i s s u e s obtained by changing the s u b s t r a t e s , pH, adding i n h i b i t o r s and a c t i v a t o r s has been regarded by s e v e r a l workers as i n d i c a t i o n of - 77 -involvement of more than one enzyme. Several enzymes distinct from non-specific alkaline phosphatase have been recognized by histochemical methods primarily based on the substrate specificity and pH optima. Among notable examples are the histochemical demonstration of 5-Nucleotidase by Pearse and Reis (1952) at a pH optimum of about 7.0 - 8.0 using muscle adenylic acid (AMP), inosinic acid and possibly adenosinetri-phosphoric acid, as substrates, and of glueosa-6-phosphatase by Chiquoine (195'3) having a pH optimum of 6.5 - 6.7 and substrate specificity towards glueose-6-phosphate. There i s a possibility that many more such enzymes w i l l be demonstrated as soon as histochemical methods are perfected. There i s evidence that variations i n the pattern of hydrolysis of phosphoric acid esters by phosphatases are not only a species specific phenomenon but also occur in the various tissues of the same animal. Bodansky (1937) indicated that alkaline phosphatases of bone, kidney and intestine are chemically different. Burgos, Deane and Karnovsky (1955) provided histochemical and chemical evidence that in some rat and cat tissues separate enzymes hydrolyzing glycerophosphate and naphthylophosphate at alkaline pH can be detected. Dempsey and Deane (1946) concluded that the activity of the intestinal phosphatase of mouse against a number of substrates and over a range of pH values i s i n i t s e l f an indication of the presence of several enzymes. Brunei's (1946, 1950) observations on differential susceptibility of phosphatases to varying concentrations of KCN provided additional evidence that the enzymes of kidney and intestine are not only different, but i - 78 -i n t r a c e l l u l a r and e x t r a c e l l u l a r phosphatases of the i n t e s t i n a l e p i t h e l i u m a l s o show v a r i a b l e l o c a l i z a t i o n s . Loveless and D a n i e l l i (1949) reported a d i f f e r e n t i a l behaviour of the nuclear and extranuclear enzymes i n r a t kidney to phenolic and phosphate components. Newman, P e i g i n , Wolf and Kabat (1950) and more r e c e n t l y Hinsch (I960) obtained v a r i a b l e h i s t o c h e m i c a l patterns by changing the substrates and a d d i t i o n of d i f f e r e n t a c t i v a t o r s and i n h i b i t o r s . Prom the r e s u l t s obtained i n the present study and reported e a r l i e r (Prakash, 1960a, 1960b), i t appears that the pH of the in c u b a t i n g medium i s the most s i g n i f i c a n t s i n g l e f a c t o r a f f e c t i n g p a t t e r n s of a l k a l i n e phosphatase a c t i v i t y i n t i s s u e s e c t i o n s . The c a t a l y t i c a c t i v i t y of enzymes i s u s u a l l y confined to a r e l a t i v e l y narrow range of pH with maximum a c t i v i t y a t a c e r t a i n pH. The pH optima f o r phosphatase present i n the brush border, lamina p r o p r i a and n u c l e i of the i n t e s t i n a l mucosa have been found to be d i f f e r e n t but at the same time overlap to a considerable extent. The a c t i v i t y i n the brush border p e r s i s t s over a long range of pH v a l u e s , whereas nuclear phosphatase i s a c t i v e only w i t h i n a narrow range of pH values. Lamina p r o p r i a , on the other hand, shows p r o p e r t i e s intermediate between these two extremes. On the b a s i s of Gomori 1s (1950) c o n c l u s i o n , i t might be argued that i n t e n s e nuclear r e a c t i o n at pH 8.6 and 8.7 i s not due to the presence of i n t r i n s i c phosphatase but due to adsorption of calcium phosphate which becomes i n c r e a s i n g l y s o l u b l e at low pH v a l u e s i The absence of any nuclear r e a c t i o n below pH 8.6 and over pH 9.3 cannot be explained on the b a s i s of Gomori's - 79 -s o l u b i l i t y hypothesis because according to that one would expect an increased f a l s e r e a c t i o n i n the n u c l e i at lower pH v a l u e s . v A l k a l i n e phosphatase shown i n the n u c l e i of the columnar e p i t h e l i a l c e l l s of the i n t e s t i n a l mucosa i s comparable to group I I I enzymes described by Newman and coworkers (1950). According to them, three groups of phosphatases can be recog-n i z e d i n the animal t i s s u e s . Group I I I enzymes are slow i n t h e i r h y d r o l y z i n g a c t i v i t y as compared to group I and group I I enzymes and showed a c o n s i s t e n t nuclear s t a i n i n g towards the a c i d s i d e . L i k e w i s e , phosphatases present i n the brush border and lamina p r o p r i a are comparable i n some ways to Group I and I I enzymes r e s p e c t i v e l y . The e f f e c t of pH on the l o c a l i z a t i o n of a l k a l i n e phosphatase can be due to one or s e v e r a l f a c t o r s . I t may be due to changes i n the stat e of i o n i z a t i o n of the enzyme i t s e l f , or of the s u b s t r a t e , or of the enzyme-substrate complex as the hydrogen i o n conce n t r a t i o n changes. The c h a r a c t e r i s t i c f a l l i n a c t i v i t y on either, s i d e of the pH optimum may be due to decreased s a t u r a t i o n of enzyme brought about by decreased a f f i n i t y or i t may be due to i n s t a b i l i t y of enzyme at non-optimal pH. Experiments to observe the s t a b i l i t y of a l k a l i n e phosphatase i n t i s s u e s e c t i o n s , as reported i n the present study, r e v e a l that h i g h a l k a l i n e and a c i d i c pH i n a c t i v a t e s the enzyme r e a c t i o n but the r a t e of i n a c t i v a t i o n of enzyme appears to be f a s t e r towards a c i d i c than a l k a l i n e s i d e . pH 12 and pH 8 appear to be the l i m i t s where the enzyme i s merely i n a c t i v a t e d and not i r r e v e r i s b l y destroyed. S e c t i o n s incubated at pH 15 - 80 -and 7 r e s p e c t i v e l y f a i l e d to show any r e t u r n o f a c t i v i t y a f t e r r e i n c u b a t i o n at optimum pH thereby suggesting an i r r e v -e r s i b l e d e s t r u c t i o n of the enzyme. In general there i s a zone of maximum s t a b i l i t y and the i n a c t i v a t i o n i n c r e a s e s on e i t h e r s i d e s of t h i s zone up to a c e r t a i n l i m i t , a f t e r which the enzyme i s i r r e v e r s i b l y destroyed. With regard to topographic v a r i a t i o n s obtained under v a r y i n g concentrations of d i f f e r e n t i n h i b i t o r s , n uclear and extr a n u c l e a r phosphatases e x h i b i t d i f f e r e n t i a l s u s c e p t i b i l i t y . Newman and coworkers (1950) reported that (1) those t i s s u e s or s i t e s which e x h i b i t e d the highest c o n c e n t r a t i o n of enzyme were the l e a s t a f f e c t e d by a given c o n c e n t r a t i o n of i n h i b i t o r , and (2) the nuclear phosphatase shows a comparatively slower r a t e of h y d r o l y s i s and gr e a t e r r e s i s t a n c e to i n h i b i t o r s than the cytoplasmic phosphatase. Present observations are i n agreement w i t h ( 1 ) , as a l k a l i n e phosphatase a c t i v i t y i n the brush border shows greater r e s i s t a n c e to high concentrations o f i n h i b i t o r s than any other s i t e i n the i n t e s t i n a l mucosa, but d i f f e r c o n s iderably with (2) because nuclear phosphatase a c t i v i t y was found to be remarkably s e n s i t i v e to lower concentrations of i n h i b i t o r s . This discrepancy between r e s u l t s i s probably due to nonenzymatic nuclear s t a i n i n g r e s u l t i n g from e x c e s s i v e l y long i n c u b a t i o n p e r i o d (24 hours) used by Newman and coworkers as compared to 5 - 60 minutes used i n the present study. The p a t t e r n obtained w i t h d i f f e r e n t substrates are not as d i s t i n c t as obtained with d i f f e r e n t pH and i n h i b i t o r s . Although there i s some i n d i c a t i o n of substrate s p e c i f i c i t y i n the n u c l e i , the o v e r a l l p a t t e r n o f a c t i v i t y i n d i f f e r e n t - 81 -l o c a t i o n s i n the t i s s u e i s overlapping and i t i s d i f f i c u l t to e s t a b l i s h the occurrence of d i f f e r e n t enzymes i n the brush border, lamina p r o p r i a and n u c l e i . F i n a l l y , d i f f e r e n t i a l behaviour of the nuclear and nonnuclear phosphatase as seen i n s t a r v a t i o n experiments provides a d d i t i o n a l evidence i n favour of the enzyme p l u r a l i t y hypothesis. No d e f i n i t e statement about the nature and p r o p e r t i e s of the v a r i o u s enzymes supposed to be i n v o l v e d i n d i f f e r e n t l o c a t i o n s i n the i n t e s t i n a l mucosa can be made u n t i l these enzymes are p u r i f i e d , t e s t e d and compared. The evidence a v a i l a b l e i n the present h i s t o c h e m i c a l study leads us to conclude that (1) topographic v a r i a t i o n s i n a l k a l i n e phosphatase a c t i v i t y obtained under d i f f e r e n t s u b s t r a t e s , i n h i b i t o r c o n c e n t r a t i o n s , pH, and v a r i o u s l e v e l s of s t a r v a t i o n , are few of s e v e r a l evidences which strengthen the b e l i e f of enzyme p l u r a l i t y , (2) d i f f e r e n t i a l patterns of phosphatase a c t i v i t y i n t i s s u e sections obtained under v a r y i n g i n c u b a t i n g c o n d i t i o n s cannot at present be i n t e r p r e t e d i n terms of a s i n g l e enzyme d i f f e r i n g only i n c o n c e n t r a t i o n i n the brush border, lamina p r o p r i a and the n u c l e i , and (3) nuclear and e x t r a n u c l e a r phosphatases behave d i f f e r e n t l y and i n , a l l p r o b a b i l i t y are not i d e n t i c a l . - 82 -SUMMARY AND jCONCLUSIONS 1. Under standard c o n d i t i o n s of i n c u b a t i o n , the s i t e s of a l k a l i n e phosphatase a c t i v i t y i n the d i g e s t i v e t r a c t of adult steelhead t r o u t are: Buccal and esophageal region — cytoplasm and n u c l e i of the s t r a t i f i e d e p i t h e l i u m and the stratum germinativum. G a s t r i c r e g i o n — lamina p r o p r i a , granule c e l l s and f i b r o b l a s t s of the submucosa. I n t e s t i n a l and r e c t a l r egion - r r brush border, lamina p r o p r i a , n u c l e i of the columnar e p i t h e l i a l c e l l s , granule c e l l s , mast c e l l s and lymphocytes. 2. During developmental p e r i o d of the f i s h , phosphatase a c t i v i t y i n the d i f f e r e n t i a t i n g gut i s discontinuous showing r e g i o n a l accumulation and disappearance as f o l l o w s : E a r l y prehatehing stage — very low cytoplasmic a c t i v i t y i n the gut primordium. Late prehatehing stage — n u c l e i of mesenchyme c e l l s show inten s e a c t i v i t y , l i t t l e a c t i v i t y i n the cytoplasm of the 'endodermal p l u g * , moderately h i g h a c t i v i t y i n endodermal l i n i n g of the hind and mid gut. Early, posthatching stage — high a c t i v i t y i n the n u c l e i o f the mesenchymal w a l l , primordia of d e n t a l lamina and swim bladder, cytoplasm of the 'endodermal plug' and the brush border. Late posthatching stage — a c t i v i t y on the whole i s much higher than that i n the previous stage and i s confined to mesenchyme c e l l s i n the dorsal w a l l of bucco-esophageal - 83 -r e g i o n and i n the submucosa. A c t i v i t y i n the brush border i s the same as i n the previous stage. Intense a c t i v i t y i s seen i n the primordium of d e n t a l lamina and i n the stomach w a l l . Pry stage — f u r t h e r increase i n phosphatase a c t i v i t y i s n o t i c e a b l e i n s i t e s described above, a d d i t i o n a l s i t e s of high a c t i v i t y are the p y l o r i c s p h i n c t e r and p y l o r i c caeca. I n t e s t i n a l v i l l i begin to form and the brush border shows high phosphatase a c t i v i t y compared to e a r l i e r stages. Lamina p r o p r i a g i v e s no r e a c t i o n at t h i s stage. 3. These data suggest that (a) during e a r l y stages of develop-ment, a l k a l i n e phosphatase i s widely d i s t r i b u t e d i n the n u c l e i and cytoplasm of the gut primordium, (b) at l a t e r stages the enzyme becomes e l e c t i v e l y l o c a l i z e d i n those areas where a c t i v e d i f f e r e n t i a t i o n i s t a k i n g place and disappears from other regions, and (c) the r i s e i n phosphatase a c t i v i t y i n the d i f f e r e n t i a t e d gut a f t e r the absorption of y o l k i s probably concerned with f u n c t i o n a l adaptation. 4. The phosphatases demonstrable i n the brush border, lamina p r o p r i a , and n u c l e i of the i n t e s t i n a l mucosa have d i f f e r e n t pH optima. The a c t i v i t y i n the brush border p e r s i s t s over a longer pH range than that i n the lamina p r o p r i a or n u c l e i . The r a t e of i n a c t i v a t i o n of enzyme i s f a s t e r towards a c i d than a l k a l i n e pH. 5. The use of v a r i o u s concentrations of BeSO A, KCN, and NaP - 84 -i n the incubating medium indicated that phosphatase i n the brush border i s more resistant to high concentrations of i n h i b i t o r s than that i n the lamina propria. Nuclear phosphatase, on the other hand, i s remarkably sensitive to lower concentrations. 6. The pattern of a c t i v i t y obtained with Na glycerophosphate, AMP, glucose-6-phosphate and fructose-6-phosphate was more or le s s s i m i l a r and no d e f i n i t e statement about the d i f f e r e n t i a l behaviour of enzyme present i n the brush border, lamina propria, and nuclei can be made on this basis. 7. Histochemical l o c a l i z a t i o n of alkaline phosphatase a c t i v i t y i s very much affected by the n u t r i t i v e state of the animal. In moderately starved f i s h , enzyme reaction increases i n the brush border of the i n t e s t i n a l mucosa but disappears i n the lamina propria and n u c l e i . Acute starvation induces desquamation of the columnar e p i t h e l i a l c e l l s and extensive diffused phosphatase a c t i v i t y i n the v i l l i . The probable factors inducing phosphatase accumulation following starvation are discussed. 8. S u f f i c i e n t experimental evidence i s presented to show that the reaction i n the nuclei i s due to the presence of i n t r i n s i c phosphatase. 9. Topographically variable patterns of enzyme a c t i v i t y obtained under d i f f e r e n t substrates, pH values, i n h i b i t o r concentrations, and levels of starvation are probably i n d i c a t i v e of the involvement of more than one alk a l i n e phosphatase. The nuclear and extranuclear phosphatases behave d i f f e r e n t l y and i n a l l p r o b a b i l i t y are not i d e n t i c a l . - 86 -REFERENCES A l - H u s s a i n i , A.H. 1949 On the f u n c t i o n a l morphology of the alimentary t r a c t of some f i s h i n r e l a t i o n to d i f f e r e n c e s i n t h e i r f e e d i n g h a b i t s . Cytology and physiology. Quart. Jour. Micros. S c i . 90: 323-354. B a r r i n g t o n , E.J.W. 1957 The alimentary canal and d i g e s t i o n . I n the physio-logy of f i s h e s , ed. M.E. Brown, v o l . 1: 109-161. Academic Press. B i e s e l e , J . J . 1949 Phosphatases of the m i t o t i c apparatus i n c u l t u r e d , • normal and malignant mouse c e l l s . Proc. Nat. Cancer. Confr.: 34. Bodansky, A., Bakwin, R.M., and Bakwin, H. 1931 The d i s t r i b u t i o n of phosphatase i n the t i s s u e s of t e l e o s t s and elasmobranchs. Jour. B i o l . Chem. 94: 551-560. Bodansky, 0. 1937 Are the phosphatases of bone, kidney, i n t e s t i n e and serum i d e n t i c a l ? The use of b i l e a c i d s i n t h e i r d i f f e r e n t i a t i o n . Jour. B i o l . Chem. 118: 341-362. B o e l l , E.J. 1955 Energy exchange and enzyme development duri n g embryogenesis. I n a n a l y s i s of development, ed. Willmer, Weiss and Hamburger. W.B. Saunders Co. P h i l a d e l p h i a . Bourne, G. 1943 The d i s t r i b u t i o n of a l k a l i n e phosphatase i n various t i s s u e s . Quart. Jour. E x p t l . P h y s i o l . 32: 1-19. Brachet, J . 1946 L o c a l i z a t i o n de l a phosphatase a l c a l i n e pendent l e developpment des Bat r a c i e n s . E x p e r i e n t i a . 2: 143. Brachet, J . 1947 N u c l e i c acids i n the c e l l and embryo. Symp. Soc. Exp. B i o l . 1: 207-224. Brachet, J . 1950 Chemical embryology. Translated by L.G. Barth. I n t e r s c i e n c e P u b l i s h e r s . N.Y. Brachet, J . 1960 The biochemistry of development. Pergamon Press. N.Y. - 87 -Brachet, J . and Jeener, R. 1948 Reeherches sur l e r o l e de l a phosphatase a l c a l i n e des noyaux. Biochim. Biophys. Acta. 2: 423-430. Bunting, H. 1950 The d i s t r i b u t i o n of aci d mucopolysaceharide i n mammalian t i s s u e s as revealed by h i s t o c h e m i c a l methods. Ann. N.Y. Acad. S c i . , 52: 977-982. Burgos, M.H., Deane, H.W., and Karnovsky. 1955 Histochemical and chemical evidence f o r more than one a l k a l i n e phosphomonoesterase. Jour. Cytochem. Histochem. 3: 103-121. Campbell, P.N. and Davson, H. 1948 Absorption of 3-methyl-glucose from the small i n t e s t i n e of the r a t and c a t . Biochem. Jour. 43: 426. Caspersson, T. 1947 The r e l a t i o n between n u c l e i c a c i d and p r o t e i n s y n t h e s i s . Symp. Soc. E x p t l . B i o l . 1: 127-151. Chevremont, M. and P i r k e t , H. 1949. 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Z o r z o l i , A. 1947 A l k a l i n e phosphatase i n the e a r l y development of Fundulus h e t e r o c l i t u s . B i o l . B u l l . , 93: 211. ( A b s t r a c t J . ~ 

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