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The role of carbonic anhydrase in acid secretion and calcium uptake by the chorioallantoic membrane of… Virta, Valerie J. 1982

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THE THE ROLE OF CARBONIC ANHYDRASE IN ACID SECRETION AND CALCIUM UPTAKE CHORIOALLANTOIC MEMBRANE OF THE CHICK EMBRYO by VALERIE J. VIRTA B.Sc, University of Texas at San Antonio, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Poultry Science) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF June, © Valerie J. BRITISH COLUMBIA 1982 V i r t a , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Valerie J. Virta Department of Poultry Science The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 August 441982 Date DE-6 (3/81) i i ABSTRACT The major source of calcium for the developing chick embryo is the eggshell. However, the actual mechanism of calcium s o l u b i l i z a t i o n from the s h e l l i s unknown. The temporal c o r r e l a t i o n of carbonic anhydrase a c t i v i t y to calcium movement implies that an aci d i c environment i s e s s e n t a i l . To c l a r i f y the role of carbonic anhydrase in calcium s o l u b i l i z a t i o n and uptake by the c h o r i o a l l a n t o i c membrane of the chick embryo treatment e f f e c t s on embryo development, carbonic anhydrase a c t i v i t y and calcium and pH leve l s were investigated. The treatments consisted of solutions of acid, base, calcium and strontium chlorides, and the enzyme inhibitor-acetazolamide.Experimental sampling was conducted from eleven to sixteen days of incubation. The treatments were administered d a i l y by dipping the eggs into a treatment solution with subsguent sampling on the following day. The acid treatment solution produced a s i g n i f i c a n t (P<0.05) increase in calcium s o l u b i l i z a t i o n and a decrease in carbonic anhydrase a c t i v i t y from the control l e v e l s . The base treatment solution produced a s i g n i f i c a n t decrease in calcium s o l u b i l i z a t i o n and an increase in carbonic anhydrase a c t i v i t y from the control l e v e l s . The calcium chloride treatment solution (providing a p a r t i a l source of a calcium non-carbonate compound) and the strontium chloride treatment solution (providing a p a r t i a l source of a non-calcium non-carbonate compound) showed no o v e r a l l e f f e c t on embryonic calcium concentrations. However, there was a decrease in carbonic anhydrase a c t i v i t y from that of the co n t r o l . This i i i decrease did not appear to be due to a calcium mediated process but was more sensitive to changes in bicarbonate or carbonate l e v e l s . Treatments with acetazolamide demonstrated that there was a decrease in carbonic anhydrase a c t i v i t y and also a decrease in calcium transported. However, when an acid treatment was combined with the acetazolamide treatment, calcium was transported even though carbonic anhydrase a c t i v i t y was greatly suppressed. These results confirm that carbonic anhydrase a c t i v i t y appears to be fimctioriing in calcium s o l u b i l i z a t i o n and maintenance of the embryonic acid-base balance. It does not appear that the enzyme functions primarily in the transport of calcium across the chorio a l l a n t o i c membrane. i v TABLE OF CONTENTS Page Abstract i i Table of Contents iv L i s t of Tables v i i L i s t of Figures ix L i s t of Appendix Tables x L i s t of Appendix Figures x i Acknowledgement x i i i INTRODUCTION 1 REVIEW OF LITERATURE 3 I. Avian Embryo Development 3 A. Gross Structural Development 3 B. Skeletal Development 5 C. Calcium and Phosphorus Metabolism 8 1 . Vitamin D 9 2. Calcium:Phosphorus Ratio 10 3. Calcium:Hormone Interaction 10 4. Calcium Regulators 11 D. Calcium and the Avian Embryo 12 II . Chorioallantoic Membrane 14 A. General 14 B. C e l l u l a r Composition 15 C. Calcium Transport 17 D. Calcium S o l u b i l i z a t i o n 23 E. Carbonic Anhydrase 25 V I I I . C a r b o n i c Anhydrase 27 A. Background 27 B. A v i a n D i s t r i b u t i o n 28 C. M e t a b o l i s m 28 D. A c t i o n 30 E. I n h i b i t o r s 32 F. E g g s h e l l C a r b o n i c Anhydrase 34 G. Embryonic C a r b o n i c Anhydrase 36 METHODS AND MATERIALS 37 I . D i p p i n g Procedure 37 I I . E x p e r i m e n t a l Scheme 39 I I I . Sample C o l l e c t i o n 44 IV. P r e p a r a t i o n and D e t e r m i n a t i o n of C a l c i u m 47 V. P r e p a r a t i o n and D e t e r m i n a t i o n of C a r b o n i c Anhydrase 48 V I . Data E x p r e s s i o n 50 V I I . S t a t i s t i c a l P r o c e d u r e s 50 RESULTS AND DISCUSSION 51 I . Experiment I : A c i d / B a s e E f f e c t s 51 I I . Experiment I l t C a l c i u m C h l o r i d e E f f e c t s 62 I I I . Experiment I I I : S t r o n t i u m C h l o r i d e E f f e c t s 70 IV. Experiment I V : A c e t a z o l a m i d e E f f e c t s 78 GENERAL DISCUSSION 89 CONCLUSION 93 BIBLIOGRAPHY ' 94 v i APPENDIX 105 I. Preliminary Studies 105 A. Acid-Base ConcentrationsrEmbryo Tolerance ...105 B. Radiotracer Studies:Calcium Movement 107 C. Acetazolamide Studies 114 v i i LIST OF TABLES Page I. The Effects of Acid and Base Treatments on Blood Calcium 52 II . The Eff e c t s of Acid and Base Treatments on Blood pH 54 II I . The Effects of Acid and Base Treatments on Al l a n t o i c F l u i d pH 55 IV. The Effects of Acid and Base Treatments on Al l a n t o i c F l u i d Calcium 57 V. The Eff e c t s of Acid and Base Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 59 VI. The Effects of Calcium Chloride Treatment on Blood Calcium 63 VII. The Effects of Calcium Chloride Treatment on Blood pH 64 VIII. The Effects of Calcium Chloride Treatment on A l l a n t o i c F l u i d Calcium 66 IX. The Effects of Calcium Chloride Treatment on A l l a n t o i c F l u i d pH 67 X. The Effects of Calcium Chloride Treatment on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 69 XI. The Effects of Strontium Chloride Treatment on Blood Calcium 71 XII. The Eff e c t s of Strontium Chloride Treatment on Blood pH 73 XIII. The Effects of Strontium Chloride Treatment on A l l a n t o i c F l u i d pH 74 XIV. The Effects of Strontium Chloride Treatment on A l l a n t o i c F l u i d Calcium 75 XV. The Effects of Strontium Chloride Treatment on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 77 XVI. The Effects of Acetazolamide and Acid/Base Treatments on Blood Calcium 79 vi i i XVII. The Ef f e c t s of Acetazolamide and Acid/Base Treatments on Blood pH 81 XVIII. The E f f e c t s of Acetazolamide and Acid/Base Treatments on A l l a n t o i c F l u i d pH 82 XIX. The E f f e c t s of Acetazolamide and Acid/Base Treatments on A l l a n t o i c F l u i d Calcium 84 XX. The E f f e c t s of Acetazolamide and Acid/Base Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 86 ix LIST OF FIGURES Gross Structural Development of the Early Embryo Skeletal Development C e l l u l a r Composition of the Chorioallantoic Membrane Calcium Transport and Uptake by the Chorioallantoic membrane The Action of Carbonic Anhydrase Structural Formula of Acetazolamide:A Carbonic Anhydrase Inhibitor General Scheme of Dipping and Sampling .... Experimental Scheme I. Effect of Acid, Base and Control Treatment on pH, Calcium Uptake, and Carbonic Anhydrase A c t i v i t y ... Experimental Scheme I I . Effect of a Non-Carbonate Calcium Source on Carbonic Anhydrase A c t i v i t y and Calcium Uptake Experimental Scheme I I I . Effect of a Non-Carbonate, Non-Calcium Source on Carbonic Anhydrase A c t i v i t y and Calcium Uptake .... Experimental Scheme IV. Effe c t of a Carbonic Anhydrase Inhibitor on Carbonic Anhydrase A c t i v i t y and Calcium Uptake .... Discussion Comparisons:Effects of Acid, Base and Control Treatments on Calcium Uptake and Chorioallantoic Carbonic Anhydrase A c t i v i t y Discussion Comparisons:Effects of Acetazolamide and Acid/Base Treatments on Calcium Uptake and Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane . X APPENDIX TABLES Page I. Acid-Base ConcentrationsrEmbryo Tolerance 106 I I . The E f f e c t s of Acid and Base Treatments on Blood Calcium: 8 5Calcium Uptake 108 I I I . The Ef f e c t s of Acid and Base Treatments on A l l a n t o i c F l u i d Calcium:* 5Calcium Uptake 109 IV. The Ef f e c t s of Acid and Base Treatments on s 5Calcium Uptake by the Chorioallantoic Membrane 110 V. The E f f e c t s of Acid and Base Treatments on * 5Calcium Uptake by the Whole Egg Contents 111 VI. The Mineral Content of the Chick at Hatch 116 APPENDIX FIGURES Page 1. Experimental Scheme. E f f e c t s of Acid, Base and Control Treatments on " 5Calcium Uptake. 113 2. Carbonic Anhydrase Inhibit i o n by Acetazolamide 115 3. Ef f e c t s of Acid, Base and Control Treatments on 0 5Calcium Uptake by the Blood 117 4. Eff e c t s of Acid, Base and Control Treatments on " 5Calcium Uptake by the All a n t o i c F l u i d 118 5. E f f e c t s of Acid, Base and Control Treatments on , 5Calcium Uptake by the Chorioallantoic Membrane 119 6. Eff e c t s of Acid, Base and Control Treatments on a 5Calcium Uptake by the Whole Egg Contents 120 7. Ef f e c t s of Acid, Base and Control Treatments on Blood Calcium 121 8. Eff e c t s of Acid, Base and Control Treatments on A l l a n t o i c F l u i d Calcium 122 9. Eff e c t s of Acid, Base and Control Treatments on Blood pH .123 1 0 . Effects of Acid, Base and Control Treatments on A l l a n t o i c F l u i d pH 124 11. E f f e c t s of Acid, Base and Control Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 125 12. Ef f e c t s of 0.25M Calcium Chloride, 0.50M Calcium Chloride and Control Treatments on Blood Calcium 126 13. Ef f e c t s of 0.25M Calcium Chloride, 0.50M Calcium Chloride and Control Treatments on Al l a n t o i c F l u i d Calcium 127 14. Ef f e c t s of 0.25M Calcium Chloride, 0.50M Calcium Chloride and Control Treatments on Blood pH 1 28 xii 15. E f f e c t s of 0.25M Calcium Chloride, 0.50M Calcium Chloride and Control Treatments on A l l a n t o i c F l u i d pH 129 16. E f f e c t s of 0.25M Calcium Chloride, 0.50M Calcium Chloride and Control Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 130 17. E f f e c t s of 0.25M Strontium Chloride, 0.50M Strontium Chloride and Control Treatments on Blood Calcium 131 18. E f f e c t s of 0.25M Strontium chloride, 0.50M Strontium Chloride and Control Treatments on A l l a n t o i c F l u i d Calcium 132 19. E f f e c t s of 0.25M Strontium Chloride, 0.50M Strontium Chloride and Control Treatments on Blood pH 133 20. E f f e c t s of 0.25M Strontium Chloride, 0.50M Strontium Chloride and Control Treatments on A l l a n t o i c F l u i d pH ..134 21. E f f e c t s of 0.25M Strontium Chloride, 0.50M Strontium Chloride and Control Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane 135 22. E f f e c t s of Aceto-Acid, Aceto-Base Aceto and Control Treatments on Blood Calcium 136 23. E f f e c t s of Aceto-Acid, Aceto-Base, Aceto and Control Treatments on A l l a n t o i c F l u i d Calcium 137 24. E f f e c t s of Aceto-Acid, Aceto-Base, Aceto and Control Treatments on Blood pH 138 25. E f f e c t s of Aceto-Acid, Aceto-Base, Aceto and Control Treatments on A l l a n t o i c F l u i d pH 139 26. E f f e c t s of Aceto-Acid, Aceto-Base, Aceto and Control Treatments on Carbonic Anhydrase A c i t i v i t y of the Chorioallantoic Membrane 140 x i i i ACKNOWLEDGEMENTS It i s a p r i v i l a g e f o r the author of t h i s thesis to express her sincere gratitude and appreciation to her advisor, Dr. R.C. Fitzsimmons. His guidance and patience throughout, the graduate program was i n d i s -pensible. His advice i n periods of indecision and despair was exc e l l e n t . And his constructive c r i t i c i s m s of th i s thesis have resulted in great improvements to i t s contents. The author would l i k e to extend a special thanks to the members of her graduate committee: Dr. D.B. Bragg, Dr. CR. Krishnamurti, Professor B. March, and Dr. J. Sim, for t h e i r excellent advice and suggestions during the graduate program. The author would also l i k e to acknowledge many of her friends who showed i n t e r e s t and offered support, e s p e c i a l l y : Betty Carlson, Barbara Johnstone, David Ehret and Anne White. It i s the author's wish to dedicate t h i s d i s s e r t a t i o n to her mother, P a t r i c i a Ann Yungmeyer, for her encouragement and help of many kinds throughout her daughters endeavor. V.J. V i r t a 1 INTRODUCTION Carbonic anhydrase plays a v i t a l role in respiration and acid-base balance. It catalyses the hydration of carbon dioxide and the dehydration of bicarbonate ions. Carbonic anhydrase occurs in many ion transporting e p i t h e l i a , but i t s role in them i s s t i l l uncertain. It i s present in acid transporting e p i t h e l i a of organs, such as the stomach and kidney, and also in certa i n bicarbonate transporting e p i t h e l i a , l i k e the e p i t h e l i a of the large i n t e s t i n e . Carbonic anhydrase was i n i t i a l l y discovered for i t s role in acid-base balance in the red blood c e l l s ; and since i t s discovery (Meldrum and Roughton, 1933) i t has been demonstrated in many ion transporting e p i t h e l i a , such as the chick embryo chori o a l l a n t o i c membrane. The c h o r i o a l l a n t o i c membrane, in embryonic development, is essen t i a l for calcium and other ions movement as well as respiration in the vascular plexus. Also, because the major calcium source for the chick embryo i s the eggshell carbonate; the c h o r i o a l l a n t o i c membrane i s a major transport epithelium of calcium. The c e l l s of the ch o r i o a l l a n t o i c membrane are f u l l y d i f f e r e n t i a t e d by the thirteenth or fourteenth day of incubation. At thi s time active mobilization of calcium i s i n i t i a t e d . The movement of calcium ions across the chori o a l l a n t o i c membrane st a r t s at the thirteenth or fourteenth day of ' incubation when the membrane i s f u l l y d i f f e r e n t i a t e d . This movement of calcium increases with time u n t i l approximately the 2 seventeenth day of incubation, where i t then declines s l i g h t l y u n t i l the nineteenth or twentieth day of incubation. Also, during t h i s time, one sees a similar increase in carbonic anhydrase a c t i v i t y . Carbonic anhydrase a c t i v i t y r i s e s s ubstantially by the fourteenth day of incubation. Its a c t i v i t y increases u n t i l approximately the seventeenth day of incubation. It then decreases s l i g h t l y . These s i m i l a r i t i e s in the age-a c t i v i t y p r o f i l e s , or the high temporal c o r r e l a t i o n , suggests that carbonic anhydrase functions in calcium s o l u b i l i z a t i o n and transport. The actual mechanism that results in the s o l u b i l i z a t i o n of the eggshell i s not known. The calcium carbonate of the eggshell provides over 100 mg of calcium to the embryo during development (80% of t o t a l body calcium). It has been suggested that calcium s o l u b i l i z a t i o n occurs when the ch o r i o a l l a n t o i c membrane secretes acids or protons onto the eggshell (Leeson and Leeson,1963; Terepka and Coleman,1972). The calcium liberated from the eggshell i s then transported across the ch o r i o a l l a n t o i c membrane and the carbonate/bicarbonate ions liberated are dealt with by carbonic anhydrase. Therefore, the major objectives of thi s thesis were to determine the role of carbonic anhydrase in 1) calcium s o l u b i l i z a t i o n from the eggshell, and 2) calcium transport across the chorio a l l a n t o i c membrane. 3 REVIEW OF LITERATURE I.AVIAN EMBRYO DEVELOPMENT A. Gross Structural Development From an embryological point of view, the early development of the avian embryo i s one of tissue d i f f e r e n t i a t i o n , organization and orientation. The embryo proper arises within the area p e l l u c i d a . The remainder of the blastoderm beyond the embryo i s extraembryonic and from i t arise the extraembryonic membranes known as the amnion, chorion and yolk sac. The a l l a n t o i s a rises as an outgrowth from the hindgut of the embryo and spreads within the extraembryonic body ca v i t y . 4 F i g . 1. F o u r t h and n i n e t h day of i n c u b a t i o n . The embryo i s surrounded by the amnion. The a l l a n t o i s i s a sac which expands around the amnion and y o l k sac and i t s o u t e r w a l l i s f u s e d w i t h t h e c h o r i o n . A l b . , A l b u m i n ; C h o r . , C h o r i o n ; A l l . , A l l a n t o i s ; Y.S.;Yolk Sac; E c t . , E c t o d e r m ; Ent.,Entoderm; Mes.,Mesoderm. ( H a m i l t o n , 1 9 5 2 ) . N e u r a l and mesodermal ( s o m i t e s ) t i s s u e s a r e e a r l y t o form i n the a v i a n embryo. T i s s u e o r g a n i z a t i o n and o r i e n t a t i o n o c c u r i n p r e p a r a t i o n f o r o r g a n o g e n e s i s . The b r a i n , o p t i c v e s i c l e s , gut and h e a r t are the o b v i o u s e a r l y s t r u c t u r e s t o form. The o r i e n t a t i o n of c e l l s and t i s s u e s i s c o n t i n o u s l y c h a n g i n g i n a t h r e e d i m e n s i o n a l sense and t h e r e f o r e , d u r i n g t h i s p e r i o d of embryo growth, many d e v e l o p m e n t a l sequences a r e s p a t i a l l y o v e r l a p p e d . The embryonic v a s c u l a r system ( i n c l u d i n g e x t r a e m b r y o n i c s y s t e m s ) , i s c o n t i n u a l l y expanding and c h a n g i n g t h r o u g h o u t t h e d e v e l o p m e n t a l sequence. A l s o , d u r i n g 5 o r g a n o g e n e s i s , and embryonic e l o n g a t i o n , t h e r e n a l - r e p r o d u c t i v e system d e v e l o p s . Limb development p r o c e e d s i n sequence w i t h c h o n d r o g e n e s i s ( s t a r t i n g a t a p p r o x i m a t e l y two t o t h r e e days of i n c u b a t i o n ) . Sense organ and nervous system development i s almost c o m p l e t e a t t h i s t i m e . I t must be kept i n mind t h a t the c e l l s a r e not o n l y o r g a n i z i n g and m u l t i p l y i n g d u r i n g development. These c e l l s a r e a l s o u n d e r g o i n g c y t o d i f f e r e n t i a t i o n and c e l l s p e c i f i c i t y t o a c q u i r e b i o c h e m i c a l and m o r p h o l o g i c a l p r o p e r t i e s n e c e s s a r y t o p e r f o r m t h e i r s p e c i a l i z e d f u n c t i o n s . The mid-embryo development ( t e n t o f o u r t e e n days of i n c u b a t i o n ) , shows the d e c l i n e of c h o n d r o g e n e s i s and the onset of bone f o r m a t i o n , the o r g a n i z a t i o n of embryonic m u s c u l a t u r e and t h e f u r t h e r development of t h e i n t e g u m e n t a r y and s e n s o r y systems. B. S k e l e t a l Development The embryonic s k e l e t a l system i s o r i g i n a t e d from c e r t a i n a g g r e g a t i o n s of mesenchyme ( s c l e r o t o m e s and s o m a t o p l e u r e of the body w a l l and unsegmented c e p h a l i c mesenchyme-bones of the s k u l l ) . The p r e l i m i n a r y s t a g e of bone f o r m a t i o n b e g i n s when t h i s mesenchyme, of a l o c a l r e g i o n , p o l i f e r a t e s , e n l a r g e s and d i f f e r e n t i a t e s i n t o a compact c e l l u l a r p r e c a r t i l a g e . T h i s p r e c a r t i l a g e assumes th e same shape as the c a r t i l a g e t o be formed. 6 Cartilaginous bones pass through three stages in embryonic development: a membranous or prechondral stage; a ca r t i l a g i n o u s stage; and a stage of o s s i f i c a t i o n . The c a r t i l a g e bones replace the p r o v i s i o n a l cartilaginous skeleton. This intermediate stage i s advantageous because c a r t i l a g e i s the only s k e l e t a l tissue than can grow r a p i d i l y enough to match the fast growth periods in the embryo. Preliminary destruction of the c a r t i l a g e i s necessary before o s s i f i c a t i o n can proceed. Once the c a r t i l a g e is removed from a l o c a l region, o s s i f i c a t i o n occurs both within the eroded c a r t i l a g e (endochondral), and p e r i p h e r i a l l y beneath i t s perichondruim (periosteal or perichondral). In endochondral bone formation, the c e l l s in the center of the hyaline c a r t i l a g e multiply, a l i g n themselves, and enlarge. At t h i s time some lime i s deposited in th e i r matrix. The c a r t i l a g e c e l l s and part of the c a l c i f i e d matrix then disintegrate and disappear, thereby, bringing into existence primordial marrow c a v i t i e s . Vascular primary marrow tissue, a r i s i n g from the inner c e l l u l a r layers of the perichondrium, invades and occupies the c a v i t i e s . This tissue also gives rise to osteoblasts (bone forming c e l l s ) , as well as the vascular marrow. The osteoblasts deposit matrix at many points forming a c h a r a c t e r i s t i c spongy appearance. This replacement of ca r t i l a g e continues u n t i l eventually the main c a r t i l a g e mass i s superseded by spongy or cancellous bone. While these changes are occuring within the c a r t i l a g e , compact bone develops around i t , from the periosteum. Zone oj cartilage erosion ami endochondral ossification Zone of calcified cartilage (Cells swollen and in rows) Zone of unmodified cartilage Periosteum—1 YLf.Q t^ - —Marrow cavity v, .. Endochondral bone deposited % ' on remains of cartilage F i g . 2. C a r t l i a g e bone development. L o n g i t u d i n a l S e c t i o n . ( A r e y , l 9 5 4 ) . Other bones of the s k u l l , the c l a v i c l e , and the u n c i n a t e f o r m a t i o n . These bones a r e c a l l e d membranous or c o v e r i n g bones and o s s i f i c a t i o n t a k e s p l a c e d i r e c t l y i n the membrane (or w i t h i n the b l a s t e m a l or mesenchymal s h e e t s ) . Bone i s l a i d down t h r o u g h the a c t i v i t y of s p e c i a l i z e d mesenchymal c e l l s ( o s t e o b l a s t s ) . A s o f t p r e o s s e u s t i s s u e made up of f i b r i l l a e and a s t r u c t u r e l e s s ground s u b s t a n c e f i r s t d i f f e r e n t i a t e s . T h i s o s t e o i d t i s s u e impregnates w i t h l i m e s a l t s . As t h i s bone m a t r i x i s p r o g r e s s i v e l y l a i d down, some o s t e o b l a s t s become t r a p p e d and remain i m p r i s o n e d as bone c e l l s , l o d g e d i n spaces termed l a c u n a e . These membranous bones e x h i b i t t h e same t y p e of p e r i o s t e a l o s s i f i c a t i o n as seen i n the c a r t i l a g i n o u s bones. The e n t i r e p r i m o r d i u m becomes e n c l o s e d i n a p e r i o s t e u m (a f i b r o u s membrane condensed from the l o c a l p r o c e s s of the r i b s do not pass t h r o u g h the s t a g e of c a r t i l a g e 8 mesenchyme). Osteoblasts d i f f e r e n t i a t e on i t s inner surface and deposit p a r a l l e l plates (lamellae) of compact bone. Osteoblastic a c t i v i t y i s influenced by hormones formed by the hypophysis, parathyroids and thyroid. Deposition of calcium s a l t s i s regulated by an enzyme, a l k a l i n e phosphatase, formed by osteoblasts. Normal o s s i f i c a t i o n also depends on the proper proportions of vitamins and mineral s a l t s a v a i l a b l e . C. Calc ium and Phosphorus Metabolism Numerous inorganic constituents or mineral elements are found in the developing avian embryo. Their ions are involved in the movement of water for the maintenance of e l e c t r o l y t e balance within the embryo and i t s internal environment within the s h e l l . Some minerals aggregate as structural elements in specialized tissues such as bones. Trace elements occur in minute quantities and are often associated with various biochemical processes of the organism, such as cofactors in enzyme systems. The concentration of a l l mineral elements, with the exception of calcium, decreases from about the eleventh day of development u n t i l hatching. (See Appendix Table VII for embryo mineral content at hatch). Most of the mineral study work has been connected with post-hatch chicks, and was concerned with the calcium interaction with other minerals, vitamins, hormones and a n t i b i o t i c s . 9 1.-Vitamin D Vitamin D, a fat soluble s t e r o l compound, i s necessary for proper metabolism of calcium and phosphorus. It has been c a l l e d the " a n t i - r a c h i t i c " vitamin, although only one form, c h o l e c a l c i f e r o l (D3), i s e f f e c t i v e for r i c k e t prevention in chicks (Scott et al., 1969). It i s questionable whether vitamin D in the hens' diet influences bone ash of chicks hatched from these hens; but i t was found that vitamin D was transmitted quant i t a t i v e l y from the hen to the egg or chick (murphy et al.,1936). Work by Halloran and DeLuca (1980), using rats, showed that calcium transport in the intestine during early development was not mediated by vitamin D, but that a vitamin D-sensitive transport system developed late in the suckling period. Scott et a l . (1969), summarized the theories involving vitamin D in calcium metabolism: a) that i t plays a major role in calcium absorption across the gut wall, b) that vitamin D i s involved in the release of calcium ions from kidney mitochondria and c) that the vitamin i s necessary for the proper function of parathyroid hormone in calcium transport. Zinc i s another factor inte r a c t i n g with vitamin D. Vitamin D increased zinc mobilization regardless of dietary zinc and calcium. Also i f the calcium to phosphorus r a t i o i s at the optimal 3:1 to 4:1, there i s a decrease in the requirement for vitamin D. Another vitamin, vitamin C (ascorbic a c i d ) , has been shown to promote small but consistent increases in the growth rate of chicks (Briggs et al.,1944;March and Biely,l953). Vitamin C in 10 the maternal diet influenced the s k e l e t a l retention of calcium by r a c h i t i c chicks (Thornton et al.,1959), and the presence of vitamin C was associated with increased calcium deposition by the bone. Thornton (1968b), suggests that ascorbic acid has a bone sa l t mobilizing influence and that the administration of ascorbic acid rapidly influences the mobilization of calcium and phosphorus. Vitamin E also affects bone c a l c i f i c a t i o n . A depressing ef f e c t on bone c a l c i f i c a t i o n was noted when excess vitamin E was administered to chicks fed either a calcium or vitamin D d e f i c i e n t diet (March et al.,1973). 2. —Calcium:Phosphorus Ratio The r a t i o between the lev e l s of calcium and phosphorus in the chicks and b r o i l e r s diet has been considered to be of major importance in the diet and in bone development. An excess of one results in a depletion of the other. The ideal dietary calcium:phosphorus r a t i o in the avian species i s one that changes with the dietary l e v e l s of magnesium, phosphorus, calcium and other mineral and vitamin constituents. 3. —CalciumrHormone Interaction A vitamin D d e f i c i e n t d i e t , resulting in r i c k e t s and feather blackening, i s related to calcium metabolism and the thyroid gland (Glazener et al.,1946). It was found that 11 e s t r a d i o l injections resulted in an increased calcium level in the blood serum. It was also demonstrated that blood calcium levels could be increased by adding d i n e s t r o l diacetate to the diet (Riddle and Dotti,1941; Harms et . al.,1963; Harms et al.,1964; Jones et al.,1964). Hydrocortisone increased bone calcium and phosphorus when the diet was- calcium d e f i c i e n t . This indicated a compensation action of the hormone for the mineral deficiency. It appears that increasing the l e v e l of estrogenic hormones influenced calcium u t i l i z a t i o n and i t s interaction with vitamins and a n t i b i o t i c s . 4.—Calcium Regulators Anderson and Consuegra (1970), state that calcium homeostasis i s precisely c o n t r o l l e d interdependently by the parathyroid hormone and c a l c i t o n i n . Both act at the s i t e of calcium mobilization from bone. The ultimobranchial glands are the production s i t e s of c a l c i t o n i n in birds (Copp et al.,1967a,b; Tauber,1967). Both the thyroid and ultimobranchial glands have hypocalcemic a c t i v i t i e s . It i s postulated that the thyroid hormone must be active at the s i t e of bone formation or destruction. It must also be active in the kidney, in order for the parathyroid hormone or c a l c i t o n i n to properly act during calcium and phosphorus metabolism. Calcium metabolism in chickens i s under the primary control of the parathyroid hormone, c a l c i t o n i n , and d i r e c t or indi r e c t influence via thyroxine on bone calcium turnover rate and renal excretion. Simkiss and Dacke (1971) however, postulate that 12 c a l c i t o n i n ' s normal action may be to prevent overshooting of parathyroid regulation of plasma calcium l e v e l and i t s important function may be to protect the skeleton from excessive resorption. S a l i s and Holdsworth (1962) presented evidence that the adrenal cortex was involved in calcium metabolism through i t s role of changing vitamin Dg to i t s active c a r r i e r form. D. Calcium and the Avian Embryo The avian embryo provides a unique method of studying calcium movement and bone development. The embryo i s almost e n t i r e l y dependent on the eggshell for i t s calcium source. Early embryonic calcium (prior to ten days of development), i s derived largely from the yolk. After t h i s time, the s h e l l i s the main contributory source of calcium (Johnston and Comar.,1955; Crooks and Simkiss,1975). The bulk of calcium u t i l i z e d for embryonic bone formation i s transferred from the eggshell to the embryo between the thirteenth day of incubation and hatching (Terepka et al.,1969). During skeletal c a l c i f i c a t i o n the c h o r i o a l l a n t o i c membrane i s responsible for mobilizing the calcium from the eggshell into the embryonic c i r c u l a t i o n (Romanoff,1967; Tuan and Zrike,l978). The developing avian embryo i s immersed in amniotic f l u i d and i s in close association with the a l l a n t o i c membranes and the contents of the yolk and albumen. The inert matter of the egg 13 ( y o l k , albumen and subembryonic f l u i d s ) , a n d the extraembryonic membranes (the amnion and the a l l a n t o i s ) , c r e a t e an e s s e n t a i l p h y s i c a l environment. T h i s environment i s necessary f o r the chemical r e s y n t h e s i s and formation of the new l i v i n g t i s s u e s of the embryo and i t s membranes. The a v i a n extraembryonic membranes: the yolk sac, the a l l a n t o i s the c h o r i o n and the amnion, are temporary l i v i n g appendages of the embryo. They appear as f u n c t i o n a l organs, p a r t i c i p a t i n g i n metabolic a c t i v i t i e s of the embryo. They a l s o serve as i n t e r m e d i a r i e s between the embryo and i t s environment. These membranes develop e a r l y i n embryo development and t h e i r s p e c i a l i z e d f u n c t i o n s cease o n l y at the time of h a t c h i n g . The y o l k sac i s a c h i e f r e s e r v o i r of food n u t r i e n t s . A b s o r p t i o n occurs through the w a l l s of the.sac and the n u t r i e n t s are then t r a n s p o r t e d to the embryo v i a the v i t e l l i n e c i r c u l a t i o n . The a l l a n t o i s i s a v e h i c l e f o r the t r a n s p o r t a t i o n of r e s p i r a t o r y gases through i t s a l l a n t o i c c i r c u l a t i o n . The blood v e s s e l s of the a l l a n t o i s are c l o s e l y l i n e d to the inner s u r f a c e of the c h o r i o n and s h e l l . The a l l a n t o i s a l s o serves as a r e p o s i t o r y f o r the e x c r e t a from the c l o a c a . 14 II.CHORIOALLANTOIC MEMBRANE A. General The c h o r i o a l l a n t o i c membrane, of the avian embryo, shows carbonic anhydrase a c t i v i t y at approximately fourteen days of incubation. This membrane i s formed by the fusion of the ectodermal/mesodermal chorion and the mesodermal/endodermal a l l a n t o i c membrane. It i s the extraembryoinc membrane through which most of the respiratory exchange of the chick embryo occurs. Also, i t i s an epithelium that i s c a l l e d upon to transport large quantities of eggshell calcium to the embryo at a cert a i n stage in development. After an incubation period of eleven days, the chor i o a l l a n t o i c membrane completely l i n e s the inside of the eggshell. Its c e l l s are not f u l l y d i f f e r e n t i a t e d u n t i l fourteen days of incubation. By t h i s time, the pH of the a l l a n t o i c side of the membrane changes from 7.5 to 5.5. This suggests that the chorionic c e l l s may secrete acids or protons onto the inner surface of the eggshell (Crooks and Simkiss,1974,1975). 15 B. C e l l u l a r Composition The chorionic ectoderm of the c h o r i o a l l a n t o i c membrane i s composed of two c e l l types; sinus covering c e l l s (also c a l l e d c a p i l l a r y covering c e l l s ) , and the v i l l u s cavity c e l l s . The two c e l l types contain carbonic anhydrase. The v i l l u s cavity c e l l s contain higher concentrations of the enzyme, as demonstrated by l a b e l l e d i n h i b i t o r autoradiography (Reider et al.,1980). The endoderm of the c h o r i o a l l a n t o i c membrane, or the a l l a n t o i c surface, i s seen to develop two d e f i n i t i v e c e l l types. The majority are granular c e l l s with a smaller number of mitochondria r i c h c e l l s interspersed between them. Most c e l l contacts are of the desmosome type, although gap junctions are usually found at the a p i c a l ends of the c e l l s . The endodermal function i s to s e l e c t i v e l y reabsorb urinary excretory products (Coleman and Terepka,1972a). S e r i a l sectioning of the chorion shows the c e l l types described by Owezarzak (1971), Coleman and Terepka (1972a), and Narbaitz (1971). According to the nomenclature of Coleman and Terepka (I972a,b), the c a p i l l a r y covering c e l l s are i d e n t i f i e d by the presence of numerous microfilaments, many glycogen granules and the long c e l l processes lying between the blood sinusoids and the s h e l l membranes. These c e l l processes extend various lengths before contacting other c e l l processes and may reach the next intersinusoid space. The v i l l u s cavity c e l l s are e a s i l y recognized by their prominent m i c r o v i l l i and the space which separates them from the s h e l l membrane. 16 F i g . 3. Diagrammatic r e p r e s e n t a t i o n of t h e c h o r i o n i c e p i t h e l i u m showing the f o u r main c e l l t y p e s . From l e f t t o r i g h t , they a r e a dark v i l l u s c a v i t y c e l l (DVC), a v i l l u s c a v i t y c e l l ( VC), two c a p i l l a r y c o v e r i n g c e l l s ( C C ; a l s o c a l l e d s i n u s c o v e r i n g c e l l ) , and a dark c a p i l l a r y c o v e r i n g c e l l (DCC). B L , b a s a l l a m i n a ; C P , c e l l p r o c e s s of t h e CC c e l l ; D,desmosome; G , g o l g i complex; EN,endothelium; M,mitochondr i o n ; M F , m i c r o f i l a m e n t s ; NU,nucleus; P E , p e r i c y t e ; RER,rough endoplasmic r e t i c u l u m ; S M , s h e l l membrane; S U , s i n u s o i d . E r y t h r o c y t e i n d i c a t e d by dark a rrows and the DCC c e l l shown by t h e open a r r o w s . ( S a l e u d d i n e t a l . , 1976). 17 C. Calcium Transport The c h o r i o a l l a n t o i c membrane-calcium transport a c t i v i t y and the accumulation of calcium by the embryo exhibit age-activity p r o f i l e s s imilar to that of the c h o r i o a l l a n t o i c membrane-carbonic anhydrase a c t i v i t y . The temporal c o r r e l a t i o n between these a c t i v i t i e s and carbonic anhydrase a c t i v i t y strongly suggests that carbonic anhydrase plays a role in calcium s o l u b i l i z a t i o n and transport. Both rn vivo (Johnson and Comar,l955), and i_n v i t r o (Terepka et al.,1969) investigations show that eggshell calcium i s being transferred to the embryo's c i r c u l a t i o n by the fourteenth day of incubation. The movement of t h i s calcium has been the subject of considerable research. Most of t h i s work has been concerned with discerning the role of the c h o r i o a l l a n t o i c membrane in the transport and s o l u b i l i z a t i o n of eggshell calcium. The rate of calcium transport by the c h o r i o a l l a n t o i c membrane, measured in v i t r o (Terepka et al.,1969; Terepka et al.,1971) and jjn vivo (Crooks et al.,1976), exhibits an age-dependent increase subsequent to fourteen days of incubation (See Figure 4). The transport c a p a b i l i t i e s of the c h o r i o a l l a n t o i c membrane have been extensively studied using radioactive tracers ( f t 5Calcium and 3H-Inulin). The flux rate of calcium JLn vivo (Borle/1970; Crooks and Simkiss,1974; Crooks and Simkiss,1975), at f i f t e e n days of incubation, was 116 nmole/cm2 membrane/hour. 18 0.104 0.084 10-11 12-13 14 15 16 17 18 19-20 Age of Embryo (Days) Figure 4. Transport of Calcium by the Chorioallantoic Membrane of Di f f e r e n t Embryonic Ages. [:'•>: -Transmembrane flux ffl -Membrane Uptake (Terepka et a l . , 1969) (Tuan and Zrike, 1978) 19 The flux rate i n ovo at f i f t e e n days of incubation for normal development, was 122 nmole/cm2 membrane/tour (Crooks and Simkiss, 1 974) . Therefore, the rates are rcomparable (Garrison and Terepka,1972). Possible calcium transport meclhanisms of the cho r i o a l l a n t o i c membrane include d i f f u s i o n , pinocytotic v e s i c l e formation and the a c t i v i t y of a s p e c i f i c calcium binding protein. The normal rate of transport does not account for the high i n t r a c e l l u l a r l e v e l (18 x 10"2 mole/liter)) of calcium that e x i s t s . This concentration i s several thousand times the level of accepted i n t r a c e l l u l a r calcium (1 x 10~6 m o l e / l i t e r ) . Therefore, a d e l i c a t e l y balanced sysitem with rigorous homeostatic control must be engaged for the imetabolic well being of the transporting c e l l s . High l e v e l s of ^intracellular calcium can uncouple oxidative phosphorylation in mitochondria or markedly i n h i b i t pyruvate kinase and pyruvate carboxylase a c t i v i t y (Terepka et al.,1969). Two of the four c e l l types seen in the ectoderm of the mature ch o r i o a l l a n t o i c membrane; the c a p i l l a r y covering c e l l s and the v i l l u s cavity c e l l s , form the -outermost surface and exist in ti g h t junction's near the s h e l l membrane facing surface. The c a p i l l a r y covering c e l l s are thought to be d i r e c t l y involved in the active calcium transport process (Terepka et al.,1969; Coleman et al.,1970). These c e l l s show changes involved with the onset of calcium transport. There i s a thinning of the cytoplasmic layer between the s h e l l membrane and the associated c a p i l l a r y , the loss of golgi membranes and rough endoplasmic 20 r e t i c u l u m from the r e g i o n and the appearance of many plasma membrane i n v a g i n a t i o n s and v e s i c l e s . The c a p i l l a r y c o v e r i n g c e l l s w i t h t h e i r l o n g c y t o p l a s m i c p r o c e s s e s , c o m p r i s e v i r t u a l l y the e n t i r e o u t e r s u r f a c e of the ectoderm. In mature t r a n s p o r t i n g membranes, the t h i n c y t o p l a s m i c l a y e r o v e r l y i n g the c a p i l l a r i e s i s so f i r m l y a t t a c h e d t o the i n n e r s h e l l membrane t h a t i t t e a r s away from the remainder of the c e l l when the c h o r i o a l l a n t o i c membrane and the s h e l l membranes are m e c h a n i c a l l y s e p a r a t e d . T h i s s t r i p p i n g does not appear t o a f f e c t t h e v i l l u s c a v i t y c e l l s (Coleman and Terepka,1972a,b; Ga r r u s o n and Terepka,1972). T h i s s e q u e s t e r i n g of c a l c i u m by the c a p i l l a r y c o v e r i n g c e l l s of the c h o r i o a l l a n t o i c membane has been r e p o r t e d by Coleman and Terepka (1972a,1972b);and Terepka e t a l . ( 1 969) . I t was found t h a t the c a l c i u m a c t i v e l y t r a n s p o r t e d from the o u t s i d e t o the i n s i d e of a b a t h i n g s o l u t i o n , d i d so w i t h l i t t l e change i n s p e c i f i c a c t i v i t y , s u g g e s t i n g t h a t the c a l c i u m t r a n s f e r r e d a c r o s s the membrane d i d not c o m p l e t e l y mix w i t h the t o t a l t i s s u e c a l c i u m . The e l e c t r o n i c probe s t u d i e s a l s o suggest t h a t t h e c a l c i u m was not f r e e t o f i l l t h e e n t i r e c a p i l l a r y c o v e r i n g c e l l c y t o p l a s m . I t would appear t h a t the c a l c i u m was c o m p a r t m e n t a l i z e d or r e s t r i c t e d w i t h i n the c e l l s and o n l y a s m a l l p r o p o r t i o n was c o n t a i n e d as i o n i c c a l c i u m w i t h i n the c y t o s o l (Coleman and Terepka,1972b). Many models, of the mechanism of c e l l u l a r c a l c i u m r e g u l a t i o n suggest t h a t the i n t r a c e l l u l a r c a l c i u m i o n c o n c e n t r a t i o n i s m a i n t a i n e d a t low c o n s t a n t l e v e l s by a c t i v e l y e x t r u d i n g c a l c i u m which e n t e r s p a s s i v e l y from the s u r r o u n d i n g 21 e x t r a c e l l u l a r f l u i d (Schachter et al.,1966; Borle,1967; Wasserman,1968; Talmage,1969; Lehninger,1970; Rasmussen,1970). Many of these models assign an important role to mitochondria and endoplasmic reticulum and that these i n t r a c e l l u l a r organelles buffer against high i n t r a c e l l u l a r calcium levels by a c t i v e l y sequestering calcium themselves. However, this does not seem to be the case. The electronic probe studies showed that sequestered calcium in the c a p i l l a r y covering c e l l s was not found in areas where mitochondria or endoplasmic reticulum were conspicuously present, but rather in the upper portions or extended arms of the c e l l s (Coleman and Terepka,1972b; Crooks et al.,1976). Therefore, neither mitochondria nor endoplasmic reticulum appear to be d i r e c t l y responsible for calcium sequestered in the c h o r i o a l l a n t o i c membrane. Electron Microscopy studies have shown that the upper portion and arms of the c a p i l l a r y c e l l s contain v e s i c l e s , bundles of fine f i b e r s and small granules t e n t a t i v e l y i d e n t i f i e d as glycogen. Also the c a p i l l a r y covering c e l l s become firmly attached to the s h e l l membrane and the external plasma membrane takes on the ' r u f f l e d ' appearance common to c e l l s carrying out endocytosis (Coleman and Terepka,1972a). The c h o r i o a l l a n t o i c membrane also showed Na*-K+ stimulated ATP-ases. In the chorion the Mg*2 ATP-ase (4-18 nmole Pj /mg protein/min) rose (20-56 nmole P-j/mg protein/min) when Na* and K* were present. The calcium ATP-ase a c t i v i t y of the c h o r i o a l l a n t o i s was always of a low o v e r a l l a c t i v i t y (0.20-1.0 nmole P^ /mg protein/min), and no stimulation was found by adding extra calcium ions to the incubation medium (Saleuddin et 22 a l . , 1976). The d i s t r i b u t i o n of 4 5Calcium in subcellular fractions shows that the nuclear, mitochondrial and microsomal fractions have a very low a c t i v i t y with approximately 70% of the transported , 5Calcium found in the supernatant f r a c t i o n . There was no s i g n i f i c a n t change in the d i s t r i b u t i o n a f t e r the membrane had been transporting calcium for one hour. This suggests that the organelles of t h i s tissue do not normally accumulate th i s calcium (Saleuddin et al.,1976). The study of sodium, potassium and calcium transfer across the c h o r i o a l l a n t o i c membrane i s complicated by the fact that a l l three ions are involved in c e l l u l a r homeostasis. The movement of calcium ions from the chorionic surface of the membrane into the blood i s a complex problem. It has been established that calcium i s a c t i v e l y transported across the c h o r i o a l l a n t o i s by a process which is oxygen dependent and thi s system exhibits saturation kinetics (Garrison and Terepka,1972a). These experiments show that v i r t u a l l y none of the cho r i o a l l a n t o i c membrane transported calcium i s associated with the microsomal or mitochondrial f r a c t i o n s . The rate of incorporation into the c h o r i o a l l a n t o i s , however, corresponds very c l o s e l y with the rate at which calcium i s transported into the embryo (Crooks and Simkiss,1975). Also, the very low l e v e l of calcium stimulated ATP-ase found in the membrane does not suggest that t h i s enzyme plays a major role in the transport of calcium across t h i s tissue (Crooks et al.,1976; Saleuddin et al.,1976). Therefore, i f the electron dense precipitates produced in the i n t e r c e l l u l a r spaces by the technique of Oschman 23 and Wall (1972), represent s i t e s of calcium binding, then this may demonstrate an avenue for calcium translocation outside the cytoplasm of the chorio a l l a n t o i c c e l l s . However, work by Tuan and Scott (1977) found a calcium binding active substance that was a soluble component of the cho r i o a l l a n t o i c membrane. This substance (a protein) was not associated with membranes or other p a r t i c u l a t e components. They also found the le v e l of t h i s substance to follow the rate of calcium movement; with low le v e l s of calcium binding protein a c t i v i t y occurring at twelve to thirteen days of incubation and increasing to maximum level s of a c t i v i t y by day nineteen of incubation. They found that the calcium binding protein was present only in the transporting c h o r i o a l l a n t o i c membrane and that i t had an 'approximate molecular weight of 100,000. Also i t is not associated with a calcium-dependent ATP-ase. Therefore, i t appears that an endocytic mechanism may play a prominent role in t r a n s c e l l u l a r calcium transport by the chorio a l l a n t o i c membrane (Terepka et al.,1969; Terepka et a l . , 1 9 7 1 ) . D. Calc ium S o l u b i l i z a t i o n The involvement of ch o r i o a l l a n t o i c membrane carbonic anhydrase in calcium s o l u b i l i z a t i o n has often been alluded to in in the l i t e r a t u r e . It has been suggested that carbonic anhydrase was involved in H + secretion or l o c a l i z e d a c i d i f i c a t i o n of the eggshell calcium carbonate. One c e l l type 24 of the chorionic ectoderm, the v i l l u s cavity c e l l s , contains very high concentrations of the enzyme, as shown by lab e l l e d i n h i b i t o r autoradiography (Reider et al.,1980). It has been postulated that the v i l l u s cavity c e l l s produce H* for s o l u b i l i z a t i o n of calcium carbonate from the eggshell (Owezarzak,1971; Coleman and Terepka,1972b; Reider et al.,1980). The v i l l u s cavity c e l l comprises only a small part of the t o t a l surface of the ectoderm. Its richness in mitochondria, elaborate microvillous apex, and p l e n t i f u l supply of v e s i c l e s and ribosomes mark i t as an active c e l l . Leeson and Leeson (1963) compare i t to a gastric p a r i e t a l c e l l because of several common st r u c t u r a l features. The eggshell provides over 100 mg of calcium to be transported into the embryonic c i r c u l a t i o n , by the c h o r i o a l l a n t o i c membrane, during embryonic development. To s o l u b i l i z e the calcium, i t i s necessary to form bicarbonate. For each mole of calcium liberated, two moles of hydrogen ions are required. The suggestion that the v i l l u s cavity c e l l s supply hydrogen ions and perhaps carbonic anhydrase, so that a continuous supply vof calcium ions can be transfered from the c a l c i t e eggshell to the calcium-transporting c e l l s of the ectoderm, needs to be further investigated (Coleman and Terepka,1972a). 25 E. Carbonic Anhydrase There i s another l i n e of evidence that involves carbonic anhydrase with blood bicarbonate. At fourteen days of incubation, the venous blood bicarbonate concentration more than doubles in the chick embryo (Tazawa et al.,1971). There i s a large influx of bicarbonate ions into the blood, which i s independent of the hemoglobin l e v e l (Dawes,1975). Its source appears to be from eggshell carbonate. There are also suggestions that carbonic anhydrase i s involved in the transport of bicarbonate ions into the c i r c u l a t i o n , as well as, the movement of calcium across the cho r i o a l l a n t o i c membrane (Gay and Mueller,1973; Reider et al.,1980). The hypothesis that carbonic anhydrase i s involved in calcium transport i s based on increases in enzyme a c t i v i t y in the chor i o a l l a n t o i c membrane at the same time that there i s calcium transport across the membrane. Inhibitors of carbonic anhydrase cause both a decrease in enzyme a c t i v i t y and a reduction in calcium transport across the cho r i o a l l a n t o i c membrane (Crooks et al.,1976; Tuan and Zrike,l978). However, th i s occurs without a decrease in the calcium binding a c t i v i t y of the cho r i o a l l a n t o i c membrane. A sp e c i f i c non-erythrocytic carbonic anhydrase is expressed in the chor i o a l l a n t o i c membrane during embryonic development, whose a c t i v i t y i s absent under non-transporting conditions (early stages of development), or in the presence of carbonic anhydrase i n h i b i t o r s . Three l i n e s of functional c o r r e l a t i o n between 26 calcium transport a c t i v i t y and carbonic anhydrase a c t i v i t y of the c h o r i o a l l a n t o i c membrane are apparent: O t h e two a c t i v i t i e s are expressed concomitantly during embryonic development, 2)both of the a c t i v i t i e s are i n h i b i t e d by s p e c i f i c carbonic anhydrase i n h i b i t o r s and 3 )carbonic anhydrase is associa ted with calcium transport in the ectodermal c e l l s ( c a p i l l a r y covering c e l l s ) of the c h o r i o a l l a n t o i c membrane. 27 III.CARBONIC ANHYDRASE A. Background The existence of carbonic anhydrase, was known in the early 1930's because of i t s role in carbon dioxide physiology. It was discovered by Roughton and Meldrum in 1933. By 1943, Roughton had worked out the k i n e t i c measurements and noted that sulfonamides acted as s p e c i f i c i n h i b i t o r s and that zinc was a constituent of the enzyme. In 1946 Davenport found that kidney, stomach and pancreas also contained carbonic anhydrase. Many other investigators have studied carbonic anhydrase and i t s reaction k i n e t i c s , chemical properties, s t r u c t u r a l information, p u r i f i c a t i o n and new detection techniques (Carter,1972). Most of the work on carbonic anhydrase i s derived from red blood c e l l s of various species. The red blood c e l l s are a concentrated and abundant source of the enzyme. In mammals, there are one to two grams of carbonic anhydrase per l i t e r of red c e l l s . Red c e l l carbonic anhydrases are zinc containing proteins of about 30,000 molecular weight with one mole of metal strongly bound per mole of protein. Carbonic anhydrases are r e l a t i v e l y stable, water soluble and retain a c t i v i t y over a pH range of 6 to 10, with maximum a c t i v i t y at pH 8.0. They have i s o e l e c t r i c points varying from about 5.5 to 7.5 (depending on the enzyme type). 28 B. A v i a n D i s t r i b u t i o n The a v i a n s p e c i e s has a d i s t r i b u t i o n of c a r b o n i c anhydrase s i m i l a r t o t h a t of mammals. In a d d i t i o n , the s p e c i a l organs c o n t a i n c a r b o n i c anhydrase: s a l t g l a n d ( 3 0 E U / g ; f u n c t i o n s i n NaCl s e c r e t i o n ) , o v i d u c t a l system (80EU/g;role i n e g g s h e l l f o r m a t i o n i n u t e r u s ; where i n h i b i t i o n l e a d s t o s o f t - s h e l l e d e g g s ) , p r o v e n t r i c u l u s (600EU/g) and p e c t e n ( q u a l i t a t i v e l y p r e s e n t ) . C a r b o n i c anhydrase i s a l s o p r e s e n t i n the a v i a n r e d b l o o d c e l l s (800EU/g), k i d n e y (225EU/g), u t e r i n e e p i t h e l i u m (200EU/g), p a n c r e a s (75EU/g), and s m a l l i n t e s t i n e (35EU/g) (Common,1941; Maren,1967). C a r b o n i c anhydrase has a l s o been r e p o r t e d i n s a l i v a , sperm, e g g s h e l l and a l l a n t o i c f l u i d . The r o l e of c a r b o n i c anhydrase i n t h e s e t i s s u e s i s not known and add t o the c o n f l i c t i n g t h e o r i e s about c a l c u l u s f o r m a t i o n i n v o l v i n g c a r b o n i c anhydrase (McCance and Widdowson,1960; Bachra and F r a u t z , 1 9 6 2 ) . C. M e t a b o l i sm C h e m i c a l l y , c a r b o n i c anhydrase may be thought of as an i d e a l i z e d l o w - m o l e c u l a r weight s o l u b l e m e t a l l o p r o t e i n . I t s t u r n o v e r r a t e appears t o be the h i g h e s t of any enzyme. There i s the u n u s u a l f e a t u r e t h a t the p h y s i o l o g i c a l r e a c t i o n c a t a l y z e d 29 (C0 2 <--carbonic anhydrase—> HCOg " ) , proceeds at very s i g n i f i c a n t rates without the enzyme. Both the active and i n h i b i t o r s i t e s are in intimate r e l a t i o n to the zinc atom (although the precise reactions are unknown). The enzyme appears to serve as a proton exchanger and a general acid-base c a t a l y s t . Carbonic anhydrase has, broadly , two physiological roles. One i s in the red c e l l s , where i t subserves the hydration of metabolic carbon dioxide in the tissue c a p i l l a r i e s and i t s dehydration in the c a p i l l a r i e s of the lung or g i l l . The second is concerned with the transfer or accumulation of hydrogen or bicarbonate ions in organs of secretion. In t h i s context, carbonic anhydrase may also have a role in the elaboration of a neutral f l u i d by i t s e f f e c t on i n t r a c e l l u l a r carbon dioxide e q u i l i b r i a . In secretory organs, the e f f e c t s of carbonic anhydrase i n h i b i t i o n can be blocked or mimicked by appropriate changes in acid-base balance. Generally, in tissues that secrete hydrogen or bicarbonate ions, the addition of the tissue product blocks i t s production through metabolic a l t e r a t i o n . And the addition of the antipodal ions w i l l mimic enzyme i n h i b i t i o n . Thus, for the kidney, metabolic acidosis blocks and metabolic a l k a l o s i s mimics. For the pancreas the opposite pattern holds. Therefore, the pattern of blocking and mimicking reveals the ionic balance within the c e l l that i s motivated by enzyme action (Maren,1967; Carter,1972). 30 D. Action The mechanism of action of carbonic anhydrase i s largely unknown. The existence of several isozymes of the enzyme has shown the dependence of c a t a l y t i c a c t i v i t y of a l l isozymes on the following: 1) the presence of the metal ion, 2) the i n h i b i t i o n by a variety of sulfonamides and metal-binding anions and 3) the existence of at least three reactions catalyzed by the enzyme. These are the hydration of carbon dioxide, the hydration of certain aldehydes, and the hydrolysis of several esters (Pocker and Meany,l965; Duff and Coleman,1966). Of the f i r s t t r a n s i t i o n and IIB metal ions, only Zn(II) and Co(II) have been shown to restore carbon dioxide hydration or esterase a c t i v i t y to apocarbonic anhydrase. The firm binding of the f i r s t t r a n s i t i o n and IIB metal ion to carbonic anhydrase i s mutually exclusive (Coleman,1967). A l l pH rate p r o f i l e s published for carbonic anhydrase are f a i r l y adequately described by a sigmoid curve representing a single i o n i z a t i o n . This appears to be true whether the substrate i s carbon dioxide, an ester, or an aldehyde (Kernohan,1965; Pocker and Meany,l965; Pocker and Stone,1965; Lindskog,1966). 31 The form of the enzyme, in the hydration and hydrolysis reactions, is a mixed enzyme-Zn-hydroxide complex. The co-ordinated -OH may be v i s u a l i z e d as attacking the C0£ carbon, giving intermediate (A). F i g . 5. Suggested mechanism of the action of carbonic anhydrase. (Coleman,1967). There may be additional interactions contribution to the binding of carbon dioxide. Displacement of the intermediate would l i b e r a t e bicarbonate, regenerate the metal-hydroxide at high pH, and favor the hydration and the hydrated species at low pH, which the dehydration reaction i s known to proceed best (Kernohan,1965). This mechanism would explain the following: 1) the lack of i n h i b i t i o n by weakly binding anions at high pH, 2) the reversion at high pH of the spectra of the cobalt enzyme action or sulfonamide complexes to that, t y p i c a l of the a l k a l i n e form of the enzyme and 3) the displacement of the pH rate p r o f i l e to higher pH in the presence of anions. A l l t h i s can be related to competition with -OH which, at high enough concentration, displaces the anions and generates the active enzyme (Coleman,1967). 32 E. Inhibitors of Carbonic Anhydrase Inhibitors of carbonic anhydrase have been reported to perturb carbonic anhydrase a c t i v i t y i_n ovo, causing gross malformation of the embryonic skeleton (Landauer,1964; Landauer and Wakasugi,1967). Because of the ubiquitous occurrence of carbonic anhydrase in the physiological buffering systems of many tissues, there i s no d i r e c t indication of the functional importance of the enzyme in the uptake of calcium by the cho r i o a l l a n t o i c membrane (Maren,l967; Carter,1972; Bundy,l977). It i s possible that the enzyme i s involved in either the s o l u b i l i z a t i o n of the eggshell c a l c i t e reserve through l o c a l i z e d a c i d i f i c a t i o n and/or the metabolic scavenging of the bicarbonate release from the eggshell. The discovery of aromatic sulfonamides, (R-SO -NH ), has shed l i g h t on the chemical and pharmacological action of carbonic anhydrase. The discovery that sulphonamides i n h i b i t carbonic anhydrase was made by Mann and K e i l i n (1940). The str u c t u r a l s p e c i f i c i t y for i n h i b i t i o n i s absolute, since removal of a proton from the nitrogen atom completely destroys a c t i v i t y . In addition a number of metals i n h i b i t carbonic anhydrase. These include copper, s i l v e r , mercury and zinc (Meldrum and Roughton,1933). Cupric ions at low concentrations (10~ 6 M) i n h i b i t 'high a c t i v i t y ' isoenzymes, while higher concentrations are needed to i n h i b i t the 'low a c t i v i t y ' isoenzymes in blood carbonic anhydrase. In blood systems, monodentate metal-binding anions have 33 been assumed to i n h i b i t carbonic anhydrase by adding to an open co-ordination s i t e of the metal ion or replacing a ligand already present. Hydrogen ion e q u i l i b r i a influences sulfonamide binding. The low d i s s o c i a t i o n constant for the Z n ( l l ) carbonic anhydrase-acetazolamide complex holds over a r e l a t i v e l y narrow pH range. Below pH 6.0 or above pH 8.0, the binding becomes weaker. The pH range over which one mole i s bound can be extended about a pH unit by increasing the free acetazolamide concentration (Coleman,1967). Acetazolamide has a lower pKfl than benzenesulfonamide, suggesting that the lower arm of the binding curve relates to the concentration of the anionic form of the i n h i b i t o r . Competition with -OH" or some other ligand for the central metal ion may explain the loss of binding at high pH. There may also be pH-induced changes in the protein molecule which influence the binding of parts of the sulfonamide not d i r e c t l y in contact with the cation. Acetazolamide i s an organic i n h i b i t o r of carbonic anhydrase. 0 N — N II ii II / CH,C-N-C C-S0,N<H9 H * F i g . 6. Structural formula of Acetazolamide. Acetazolamide has long been shown to also i n h i b i t eggshell formation in the hen (Wilbur and Jodrey,1955). Carbonic 34 anhydrase i n h i b i t o r s have been shown to p a r t i a l l y suppress the formation of HCl and reduce the active transport of chloride ions in the gastric mucosa of mammals (Hogben,1967). F. Eggshell Carbonic Anhydrase Carbonic anhydrase has also been reported in the mammillae of the hens eggshell (Diamantstein,1964). The hens egg consists of the inner and outer s h e l l membrane, the mammillae, the matrix and the c u t i c l e . The mammillae and the eggshell matrix are consequently incrusted with an inorganic substance, which consists mainly of calcium carbonate. In the hen, carbonic anhydrase i s predominatly situated at the a p i c a l pole of the e p i t h e l i a of the tubular glands. These glands release the calcium and bicarbonate ions into the lumen of the uterus. Robinson and King (1963) have histochemically demonstrated carbonic anhydrase in the region of the mammillae in the eggshell. They proposed that the mammillae of the eggshell contains mucopolysaccharides which consist of up to 40% chondroitin sulphate. These mucopolysaccharides bind cobalt ions and other divalent cations. The enzymatic a c t i v a t i o n of carbonic anhydrase i s stimulated by the cobalt ions which are bound to the mucopolysaccharides of the mammillae. Other work by Ferguson (1981), showed that the a l l i g a t o r eggshell consisted 35 of an outer densely c a l c i f i e d zone, a honeycomb zone and a mammillary zone, to which i s attached the eggshell membrane. The entire eggshell i s composed of small rhombohedral c r y s t a l s of c a l c i t e . The dense outer layer contains no organic matrix but the honeycomb zone contains a high percentage of organic matrix which creates a meshwork of vesicular holes between the c a l c i t e c r y s t a l s . These holes serve as interconnections with the egg contents via spaces between the mammillae and pores in the eggshell membrane. These spaces increase during incubation producing erosion craters, and numerous microorganisms produce aci d i c metabolites as a fermentation product of decaying nest vegetation. These acids in combination with carbonic acid dissolve the c a l c i t e c r y s t a l s in the outer densely c a l c i f i e d layers. E x t r i n s i c carbonic acid (pH 6 ) dripped onto the eggshell surface, produced concentrically stepped erosion craters i d e n t i c a l to those seen i_n vivo. This natural erosion of c a l c i t e by carbonic acid is necessary for hatching. Incubators did not provide t h i s e x t r i n s i c source of carbonic acid necessary for erosion and therefore the hatching of the a l l i g a t o r s . 36 G. Embryonic Carbonic Anhydrase During embryonic development of the chick and mouse, tissue carbonic anhydrase i s usually formed at an early stage (optic v e s i c l e s - r e t i n a ; t h i r d day of incubation). The enzyme does not appear in the blood u n t i l a r e l a t i v e l y l ate stage (day twelve of incubation). Therefore, the blood islands, blastoderm and endothelial l i n i n g of the vascular system are not regions where carbonic anhydrase is formed. It is probable that the enzyme i s confined to those c e l l s produced in the bone marrow (Clark,1951). Other tissues showing carbonic anhydrase a c t i v i t y in the chick embryo are: the retina (three days); the brain (f i v e days); and the kidney and lens (nine days) (Romanoff,1967). In the c h o r i o a l l a n t o i c membrane of the chick embryo, carbonic anhydrase i s present at about twelve days of incubation and increases in an age-dependent manner u n t i l nineteen days of incubaton. The enzyme a c t i v i t y i s of tissue o r i g i n and is not from erythrocytes (Crooks and Simkiss,1975). 37 MATERIALS AND METHODS F e r t i l i z e d White Leghorn eggs were obtained from the University of B r i t i s h Columbia Poultry Farm. These eggs were used in a l l of the experiments. The eggs were incubated under standard conditions for eleven days. Eleven to sixteen days (inclusive) of incubation constituted the da i l y sample periods. Note the general scheme of dipping and sampling (Figure 7). I. DIPPING PROCEDURE This dipping arrangement was designed to provide uptake of the treatment solution without greatly disturbing or a l t e r i n g the i n t e g r i t y of the egg contents or i t s s h e l l . A l l solutions were introduced into the preheated eggs by immediately dipping the incubated eggs into room temperature treatment solutions (25°C) for one minute. A pretreatment sample was obtained on day eleven of incubation and the remaining eggs were dipped into the treatment solution for one minute. The eggs were then allowed to dry and were placed back into the incubator. The following day a '1 s t dip sample' was removed and the remainder of the eggs were dipped again for one minute and replaced into the incubator. This procedure of dipping and one day post dip sampling was repeated u n t i l day sixteen of incubation, when the l a s t sample was taken. Days of Incubation 10 11 12 13 14 15 16 treatment-.-* xe-group-^ XS-group-^ X4-group-^ ^ - g r o u p - ^ X2 g r o u p - ^ Xl-group group A 1 day \ 2 day \ 3 day \ 4 day \ 5 day \ \ of eggs dip dip ] dip ) dnp ) dip X7 1 s t dip 2 n d dip 3 r d dip 4 t h dip 5 t h dip 6 t h dip treatment sample P sample sample sample sample sample sample ure 7. Experimental Scheme of dipping and sampling. 39 I I . EXPERIMENTAL SCHEME E s s e n t i a l l y four series of experiments were run to investigate the role of carbonic anhydrase a c t i v i t y in acid secretion and calcium uptake by the c h o r i o a l l a n t o i c membrane of the chick embryo. Preliminary studies on calcium uptake, acid and base concentration e f f e c t s and acetazolamide concentration e f f e c t s were run. (See Appendix Table I and Appendix Figure 1). These preliminary studies showed that an acid concentration over 0.12 M i s not well tolerated by the embryonic system. However, a basic solution of twice t h i s concentration i s well tolerated. The concentration of 0.12M was used in a l l of the experiments which involved an acid or basic dip for a treatment. Previous studies have demonstrated that the eggshell i s the major source of calcium for the developing chick. Also, that eggshell s o l u b i l i z a t i o n may occur through the secretion of acid, presumably as a function of carbonic anhydrase a c t i v i t y . A. Experiment I:Acid-Base Experiment The f i r s t of the four experiments, outlined in Figure 8, was designed to test the e f f e c t s of an externally applied acid(HCl) and base(NaOH) solutions on calcium uptake and carbonic anhydrase a c t i v i t y of the egg. The three experimental solutions were acid, base or d i s t i l l e d water^HgO). The acid and base solutions, as stated e a r l i e r , had a concentration of 0.12 M. Ninety-one eggs were assigned to each treatment with eggs Days of Incubation 10 11 12 13 14 15 16 treatment--<-> 78 eggs-»-»65 eggs-y-=> 52 eggs-y->39 eggs-y-> 26 eggs-T-* 13 eggs, group \ 1 day \ 2 day \ 3 day \ 4 day \ 5 day \ of 91 I dip dip dip dip dip pre 1 s t dip 2 n d dip 3 r d dip 4 t h dip 5 t h dip 6 t h dip treatment sample sample sample sample sample sample sample 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs F i g u r e 8. Experimental Scheme I. Ef f e c t of Acid, Base, and Control Treatments 1 on pH, calcium uptake, and Carbonic Anhydrase a c t i v i t y . 1 T h i s scheme followed for the three treatment groups: 0.12 M HCl; 0.12 M NaOH; Control-dH 20. 2Samples of blood and a l l a n t o i c f l u i d were col l e c t e d f o r calcium and pH determinations. Chorioallantoic membrane samples were c o l l e c t e d for carbonic anhydrase determinations. 41 t h i r t e e n eggs removed d a i l y f o r s a m p l i n g . Samples were o b t a i n e d d a i l y s t a r t i n g a t day e l e v e n of i n c u b a t i o n and c o n t i n u i n g u n t i l day s i x t e e n of i n c u b a t i o n . Samples of b l o o d and a l l a n t o i c f l u i d were o b t a i n e d f o r pH and c a l c i u m d e t e r m i n a t i o n s and the c h o r i o a l l a n t o i c membrane was o b t a i n e d f o r c a r b o n i c anhydrase d e t e r m i n a t i o n s . B. Exper iment 11:Calcium C h l o r i d e Experiment The second e x p e r i m e n t , o u t l i n e d i n F i g u r e 9, was d e s i g n e d t o t e s t the e f f e c t of an e x t e r n a l l y a p p l i e d non-carbonate c a l c i u m compound on c a l c i u m uptake and c a r b o n i c anhydrase a c t i v i t y . The t h r e e e x p e r i m e n t a l s o l u t i o n s c o n t a i n e d 0.25M C a C l 2 , 0.50M C a C l 2 or dH 20. The same d i p p i n g and sam p l i n g p r o c e d u r e was f o l l o w e d as i n the p r e v i o u s e x p e r i m e n t . C. Experiment I I I i S t r o n t i u m C h l o r i d e Experiment The t h i r d e x p e r i m e n t , o u t l i n e d i n F i g u r e 10, was d e s i g n e d t o t e s t the e f f e c t of an e x t e r n a l l y a p p l i e d n o n - c a r b o n a t e , non-c a l c i u m compound on c a l c i u m uptake and c a r b o n i c anhydrase a c t i v i t y . The t h r e e e x p e r i m e n t a l s o l u t i o n s c o n t a i n e d 0.25M S r C l 2 , 0.50M S r C l 2 or dH20. The same d i p p i n g and sa m p l i n g p r o c e d u r e was f o l l o w e d as i n the p r e v i o u s e x p e r i m e n t s . Days of Incubation 10 11 12 13 14 15 16 treatment--r->78eggs-y->65 eggs-T—>52 eggs-y^> 39 eggs-r-^26 eggs-^-^13 eggs group \ 1 day \ 2 day \ 3 day \ 4 day \ 5 day of 91 dip 1 dip I dip dip dip pre 1 s t dip 2 n d dip 3 r d dip 4 t h dip 5 t h dip 6 t h dip treatment sample sample sample sample sample sample 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs F i g u r e 9. Experimental Scheme I I . Ef f e c t of a Non-Carbonate Calcium Source 1 on Carbonic Anhydrase A c t i v i t y and Calcium Uptake . H h i s stheme followed for the three treatment groups: 0.50 M Calcium Chloride; 0.25 M Calcium Chloride; Control-dH 20. 2Samples of blood and a l l a n t o i c f l u i d were c o l l e c t e d for calcium and pH determinations. Chorioallantoic membrane samples were co l l e c t e d for carbonic anhydrase determinations. Days of Incubation 10 11 12 13 14 15 16 group \ of 91 j eggs J day dip p r e 1 s t dip 2 n d dip 3 r d dip 4 t h dip 5 t h dip 6 t h dip treatment sample' sample sample sample sample sample 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs a eggs i g u r e 10. Experimental Scheme I I I . E f f e c t of a Non-Carbonate Non-Calcium 1 source on Carbonic Anhydrase A c t i v i t y and Calcium Uptake . ^ h i s scheme followed for the three treatment groups: 0.50 M Strontium Chloride; 0.25 M Strontium Chloride; and Control-dH 20. 2Samples of blood and a l l a n t o i c f l u i d were co l l e c t e d for calcium and pH determinations. Chorioallantoic membrane samples were co l l e c t e d for carbonic anhydrase determinations. 44 D. Experiment IV;Acetazolamide Experiment The fourth experiment,, outlined in Figure 11, was designed to test the ef f e c t of an externally applied s p e c i f i c i n h i b i t o r of carbonic anhydrase on calcium uptake and carbonic anhydrase a c t i v i t y . The four experimental solutions contained 20 u^M acetozolamide-acid (az-acid), 20 pM acetazolamide-base (az-base), 20 juM acetazolamide (az), and dl-^O.The same dipping and sampling procedure was followed as in the previous exper iments. I I I . SAMPLE COLLECTION Samples were c o l l e c t e d d a i l y , s t a r t i n g at day eleven of incubation (pre-treatment sample) and continued d a i l y u n t i l day sixteen of incubation (five day dip sample). Each day of sampling, consisted of three samples of each component, ( i e , blood, a l l a n t o i c f l u i d , e t c . ) , where each sample contained two aliquots(embryos). Three samples of blood per day were c o l l e c t e d for determination of calcium. Blood was co l l e c t e d by breaking open the eggshell to expose the a l l a n t o i c artery. The artery was pricked allowing blood to flow freely into unheparinized c a p i l l a r y tubes. Five tubes or 500 ul of blood constitute one aliq u o t . Two aliquots of blood were placed into a pre-weighed crucible for ashing. Total sample size was ten tubes or Days o f Incubat ion 10 11 treatment-group o f 91 12 13 14 15 16 - > 3 9 e g g s - * c - > 26 e g g s 1 3 eggs, 4 day \ 5 day d ip d i p pre 1 s t d ip 2 n d d ip 3 r d d ip 4 t h d ip 5 t n d i p 6 L n d ip t reatment samples samples samples samples samples samples 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs F i g u r e 11. Exper imental Scheme IV. E f f e c t o f a Carbonic Anhydrase I n h i b i t o r 1 on 2 Carbonic Anhydrase A c t i v i t y and Calcium Uptake . 1 T h i s scheme fo l lowed fo r the four t reatment groups: 0.12 M HCl -20 /JM Ace tazo lam ide ; 0.12 M Na0H-20 JJM Ace tazo lamide ; 20 juM Ace tazo lamide ; and C o n t r o l - d H 2 0 . 2 Samples o f blood and a l l a n t o i c f l u i d were c o l l e c t e d f o r ca lc ium and pH de te rm ina t i o C h o r i o a l l a n t o i c membrane samples were c o l l e c t e d f o r ca rbon ic anhydrase de te rmina t io 46 a p p r o x i m a t e l y 1000 pi (one ml o f b l o o d ) . T h r e e s a m p l e s p e r day of a l l a n t o i c f l u i d were c o l l e c t e d f o r d e t e r m i n a t i o n of c a l c i u m . A l l a n t o i c f l u i d was c o l l e c t e d by i m m e d i a t e l y d r a w i n g o u t a f i v e ml a l i q u o t , u s i n g a s y r i n g e . Two of t h e s e a l i q u o t s c o n s t i t u t e d one s a m p l e . The sample was t h e n p l a c e d i n t o a p r e - w e i g h e d c r u c i b l e f o r a s h i n g . T o t a l sample s i z e was t e n ml o f a l l a n t o i c f l u i d . T h r e e s a m p l e s p e r day o f t h e c h o r i o a l l a n t o i c membrane were c o l l e c t e d f o r d e t e r m i n a t i o n o f c a r b o n i c a n h y d r a s e . Two membranes c o n s t i t u t e d one s a m p l e . T h e s e membranes were o b t a i n e d by b r e a k i n g open t h e egg i n t o a p e t r i d i s h and q u i c k l y r e m o v i n g t h e membrane w i t h o u t l e a v i n g any t i s s u e a t t a c h e d a t t h e a b d o m i n a l j u n c t i o n . The membranes were t h e n p l a c e d i n t o a p r e -c o o l e d , p r e - w e i g h e d c o v e r e d t u b e . T h i s was p l a c e d i n t o t h e f r e e z e r (-20°C) t o be w e i g h e d a n d p r e p a r e d f o r c a r b o n i c a n h y d r a s e a c t i v i t y d e t e r m i n a n t i o n . A l l pH d e t e r m i n a t i o n s o f b l o o d and a l l a n t o i c f l u i d were o b t a i n e d by o p e n i n g t h e egg and p l a c i n g t h e B r o a d l y - J a m e s c o m b i n a t i o n f l a t membrane pH e l e c t r o d e a t t a c h m e n t e i t h e r d i r e c t l y i n t o t h e a l l a n t o i c f l u i d o r d i r e c t l y o v e r t h e f r e e f l o w i n g b l o o d of t h e a l l a n t o i c a r t e r y . S i x pH d e t e r m i n a t i o n s p e r t r e a t m e n t c o n s t i t u t e d one d a y s s a m p l i n g . 47 IV. PREPARATION AND DETERMINATION OF CALCIUM A. Preparat ion The calcium concentration of the blood and a l l a n t o i c f l u i d was determined on the dry ash samples (Perkin,E,1976; Pick et al.,1976). Samples were c o l l e c t e d and dried in an oven at 105°C overnight. They were placed in a cold muffle furnace and the temperature was brought to 600°C and maintained u n t i l a whitish grey ash was obtained. The crucibles were removed and cooled in a desiccator overnight. The dry ash sample weights were then recorded. The c r u c i b l e and l i d were rinsed with 10 ml of d i l u t e HCl Opart HC1:3 parts d^O). The cruvibles were placed in a water bath and evaporated to dryness. The residue was moistened with 2 ml of 36% HCl, covered and boiled for two minutes. Five ml of dH£0 was added and the mixture was brought to a b o i l again. It was then transferred to a volumetric flask and lanthanum chloride(5%) was added so that the f i n a l concentration would contain 1% LaClg. This was then d i l u t e d appropriately with dHgO. F i f t y ml volumetric flasks were used for the blood calcium samples and one-hundred ml volumetric flasks were used for the a l l a n t o i c f l u i d samples. The flasks had been previously acid washed and rinsed with d i s t i l l e d water to remove any mineral contaminants. 48 B. D e t e r m i n a t i o n The c a l c i u m c o n c e n t r a t i o n of the samples as d e t e r m i n e d by the J a r e l l - A s h Atomic S p e c t r o p h o t o m e t e r and the p e r c e n t absorbance was r e c o r d e d . The ug per ml of c a l c i u m was d e t e r m i n e d by use of the s t a n d a r d c u r v e , p r e p a r e d u s i n g 0,3,6,9,12 and 15 ug per ml of c a l c i u m . V. PREPARATION AND DETERMINATION OF CARBONIC ANHYDRASE A. P r e p a r a t i o n Two f r o z e n c h o r i o a l l a n t o i c membranes were homogenized w i t h a t i s s u e homogenizer ( t e f l o n p i s t o l and g l a s s t u b e s ) , i n an i c e b a t h . T h i s homogenate was c e n t r i f u g e d a t 5000 RPM f o r 5 minutes i n a S o r v a l GLC-l(room t e m p e r a t u r e ) . The s u p e r n a t a n t was c o l l e c t e d and used d i r e c t l y f o r d e t e r m i n a t i o n of c a r b o n i c anhydrase a c t i v i t y . V e r o n a l B u f f e r - ( c o n t a i n i n g 5 mg % bromothymol b l u e ) : 4 . 5 3 6 g sodium b a r b i t u r a t e added t o 950 ml dH 20. B a r b i t u r i c a c i d was added t o b r i n g the pH t o 8.15. S a t u r a t e d C 0 2 s o l u t i o n i C C u , b u b b l e d t h r o u g h c o l d dH 20 a t 0°C f o r one hour p r i o r t o use and c o n c o m i t a n t l y t h r o u g h o u t the ex p e r i m e n t . 49 Protein determinations were performed on aliquots of supernatant by the Lowry procedure. To 0.5 ml of sample, 2.5 ml of Reagent C was added (Reagent C: 50 ml 2% sodium carbonate in 0.1 M NaOH; 0.5 ml 2% potassium t a r t r a t e ; 0.5 ml 2% copper s u l f a t e ) . This was mixed and after ten minutes 0.25 ml of Reagent E was added (Reagent E: Phenol Reagent d i l u t e d by one half with water). This was mixed and timed for one hour. The absorbance was then read at 700 nm. The amount of protein in the samples was determined using Bovine Serum Albumin (BSA), for the standard graph (0,50,100,150,...600 pq BSA). B. Determinat ion This procedure is outlined by Wilbur and Anderson (1941). Two ml of iced Veronal buffer was pipetted into 17 x 100 mm polystyrene tubes (large size used to accommodate pH electrode). This was held in an ice bath. F i f t y ul of iced experimental solution (supernatant) or dH 20 (blank) was added to the test tube. This was mixed and iced for 30 seconds to equilibrate temperatures. Two ml of iced saturated CO2 (drawn into a cold glass syringe), was injected into the test tube, mixed with the pH electrode and the reaction was timed s t a r t i n g at pH 8.0. The reaction was timed to the end point determination at pH 6.3. 50 VI. DATA EXPRESSION A l l calcium concentrations were standardized (due to the variance in sample size and to cut down on t h i s individual v a r i a t i o n ) , as an expression of ug of calcium per mg of ash. This thesis d i s s e r t a t i o n uses the enzyme unit (EU) as defined by Roughton and Booth (1946). EU=to-t/t-1 where t 0=reaction time without the enzyme and t=reaction time with the enzyme (this calculates the enzymatic hydration of 0,0^ in moles/liter/sec allowing a period of one second for mixing). Carbonic anhydrase a c t i v i t y was expressed as enzyme units per mg of protein. VII. STATISTICAL PROCEDURES A l l s t a t i s t i c a l procedures were performed by programs from the UBC MFAV-Analysis of Variance/Covariance (Le,l978) and the UBC TRP-Triangular Regression Packages (Le and Tenisci,1978), on the University of B r i t i s h Columbia's Michigan Terminal System (MTS) Computer. Analysis of variance were always run to test treatment e f f e c t s . Mean separation techniques were employed from the orthogonal tests of individual degree of freedom te s t i n g . This was done to test s p e c i f i c treatment, day and between treatment e f f e c t s . A l l analyses for experiments were conducted at the 0.05 l e v e l of significance.. 51 RESULTS AND DISCUSSION A. Experiment I:Acid-Base E f f e c t s The f i r s t experiment tested the eff e c t of a basic dip (NaOH) on embryonic calcium uptake. If acid secretion by the cho r i o a l l a n t o i c membrane i s important for eggshell s o l u b i l i z a t i o n and consequent calcium uptake, th i s treatment should neutralize any acids produced and decrease calcium uptake. Conversely, calcium uptake might be f a c i l i t a t e d by an acid dip treatment(HCl). If carbonic anhydrase i s involved in acid production, then the acid treatment might be expected to suppress and the basic treatment to enhance carbonic anhydrase a c t i v i t y . The data obtained showed that blood calcium and pH levels were not s i g n i f i c a n t l y affected (P<0.05) by the acid and base treatments over that of the control (Tables I and II, res p e c t i v e l y ) . This is not unusual when one considers that the function of the embryo c i r c u l a t o r y system i s to s t r i v e towards a homeostatic maintenance of pH at a l l costs. With respect to day differences, the blood calcium concentration did not change s i g n i f i c a n t l y a f t e r the f i r s t day of incubation (Table I ) . The concentration of calcium in the blood tended to show a decrease from day eleven to thirteen (with the acid treatment being the exception). On day fourteen of incubation the concentration increased (except for the acid treatment). A second decline in the calcium concentration was noted from day fourteen to sixteen of incubation. The acid 52 o Table I. The E f f e c t s of A c i d and Base Treatments on Blood Calcium 1- 2 Treatments Days of ; 3. cl cl Incubation A c i d Base C o n t r o l -pg/mg ash-11 36.1±11.8a 37.8±l9.1a 38.8±7.5 a 12 I9.3±1.6a 16.4±2.5a 16.3±3.2a 13 20.2±6.8a 11.4±1.9a 11.8±2.3a 14 14.2±2.5a I9.8±4.3a I7.2±2.0a 15 I6.9±1.8a 13.9±1.5a 14.7±0.6a 16 14.2±6.3a 12.9±1.2a 12.7±2.5a 1Two embryos per sample:Mean±SEM of three samples; expressed i n micrograms of c a l c i u m per m i l l i g r a m of ash. 2 0 v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means t e s t e d by the use of the IDF t e s t . 53 treatment showed an alternating decrease and increase over t h i s time, with an ov e r a l l decrease in blood calcium concentration over time. The f i r s t decrease in the blood calcium concentration may be due to a general tissue demand for calcium by the embryo. This may trigger the a c t i v i t y of carbonic anhydrase (fourteen days of incubation) and the increase in blood calcium i s seen. The second decrease in blood calcium may be due to the large calcium demand needed for skel e t a l development. The blood pH showed inconsistent v a r i a b i l i t y of the between day differences with a general decrease in blood pH over time (Table I I ) . The a l l a n t o i c f l u i d showed a s i g n i f i c a n t treatment difference in pH from the acid treatment (Table I I I ) . A l l three treatments showed a decrease in a l l a n t o i c f l u i d pH with respect to time. However, s t a r t i n g at thirteen days of incubation, the acid treatment showed a lower pH l e v e l than seen in the base and control treatments. This lower acid treatment pH l e v e l continues and becomes s i g n i f i c a n t l y lower at f i f t e e n and sixteen days of incubation. The base treatment a l l a n t o i c f l u i d pH varied s l i g h t l y from the control but did not show any s i g n i f i c a n t changes u n t i l day sixteen of incubation. At day sixteen of incubation the pH was s i g n i f i c a n t l y higher than for the control or acid treated groups. The day ef f e c t s of the a l l a n t o i c f l u i d pH showed s i g n i f i c a n t differences between day twelve and thirteen of incubation and between day f i f t e e n and sixteen of incubation (Table I I I ) . There was an ov e r a l l decrease in a l l a n t o i c f l u i d pH, with time, in a l l of the treatments. 54 Table I I . The E f f e c t s of Acid and Base Treatments on Blood pH 1 , z Treatments Days of Incubation A c i d a Base a Control 11 7.78±0.l6 a 7.78±0.16 a 7.78±0.l6 a 12 7.52±0.08 a 7.66±0.07 a 7.66±0.14 a 13 7.68±0.03 a 7.70±0.11 a 7.54±0.04 a 14 7.43±0.09 a 7.53±0.09 a 7.54±0.04 a 15 7.50±0.02 a 7.62±0.07 a 7.6l±0.08 a 16 7.49±0.07 a 7.48±0.l2 a 7.56±0.03 a 1Six embryos per sample:Mean±SEM. 2 0 v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF test. 55 Table I I I . The Ef f e c t s of Acid and Base Treatments on A l l a n t o i c F l u i d pH1'2 Treatments Days of Incubation A c i d a Base b Control' 11 7. 79±0. a 18 7 .79±0. I8 a 7. 79±0. 18S 1 2 7. 29±0. a 07 7 .54±0. 09 a 7. 64±0. 12 a 13 6. 81 ±0. a 23 7 ,43±0. !5 b 7. 18±0. 64 1 4 6. 79±0. a 27 7 .07±0. 48 a 7.. 1 7±0. 2 5 a 1 5 6. 33±0. a 26 7 ,06±0. 24 b 7. 4 1 ±0. 3 l b 1 6 5. 7 1 ±0. 6 8 a 6 .98±0. 08 b 6. 33±0. 23° 1Six embryos per sample:Mean±SEM. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF te s t . 56 Table IV shows the e f f e c t s of the acid and base treatments on a l l a n t o i c f l u i d calcium. The a l l a n t o i c f l u i d calcium levels of the acid treatment showed a s i g n i f i c a n t treatment effect, over those of the base and control treatments. The values were variable u n t i l fourteen days of incubation, at which time the acid treatment started to show a greater concentration of calcium in the a l l a n t o i c f l u i d compared to the base and control treatments. At f i f t e e n and sixteen days of incubation the calcium concentration of the acid treatment was s i g n i f i c a n t l y higher than the base and control treatments. The a l l a n t o i c f l u i d calcium concentrations of the base treatment were more variable but tended to be close to or s i g n i f i c a n t l y lower than the control treatment (fourteen to sixteen days of incubation). The day e f f e c t s of the a l l a n t o i c f l u i d calcium showed s i g n i f i c a n t differences between eleven, twelve, thirteen and f i f t e e n days of incubation (Table IV). Day fourteen overlapped in s i gnificance with day twelve of incubation, and day sixteen overlapped with days thirteen and f i f t e e n of incubation. These results indicate that, although the treatments did not cause any s i g n i f i c a n t changes in blood pH and calcium concentration in blood, they did cause changes in the a l l a n t o i c f l u i d environment. This s h i f t towards changes in the a l l a n t o i c environment i s probably due to r i g i d homeostatic controls on the blood systems. This would be the l o g i c a l place for changes to occur due to the nature of the a l l a n t o i s and i t s f l u i d . One may expect, as the results allude to, that the a l l a n t o i s and i t s f l u i d not only absorb the acid or H + (lowering the pH) and maintain the embryo's natural pH balance, but also absorb any 57 Table IV. The Ef f e c t s of Acid and Base Treatments on A l l a n t o i c F l u i d Calcium 1' 2 Treatments Days of a b b Incubation Acid Base Control jug/mg ash a a a 11 9 .5±4 . 1 a 9.3±1.5 a 10.313.0 b 12 9 .8±0 .3 b 12.412.4 c 15.6+7.4 a 1 3 16 .6+2 .2 b 21 . 3+3.5 a 10.411.3 b 1 4 16 . 1±2 .6 b 10.616.7 a 14.815.6 a 15 22 .1 + 1 .2 c 14.9+1.0 a 16.114.3 b 16 21 .0±1 . 1 12.516.1 16.613.6 1Two embryos per sample:MeaniSEM of three samples expressed in micrograms calcium per milligrams of ash. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF te s t . 58 excess calcium due degredation of eggshell calcium carbonate. If the acid causes the breakdown of c a l c i t e , then one would expect that with the acid treatment, the production of HCO3" and the acid would suppress the a c t i v i t y of carbonic anhydrase. The resu l t s in Table V show that there was a s i g n i f i c a n t difference in carbonic anhydrase a c t i v i t y between a l l the treatments s t a r t i n g at thirteen days of incubation. There was a s i g n i f i c a n t decrease in the carbonic anhydrase a c t i v i t y of the acid treatment over those of the control and base treatments. The base treatment, on the other hand, showed a s i g n i f i c a n t increase in carbonic anhydrase a c t i v i t y over those of the control and acid treatments. The day eff e c t s of the cho r i o a l l a n t o i c membrane carbonic anhydrase a c t i v i t y showed no s i g n i f i c a n t difference u n t i l day fourteen of incubation (Table V). Days f i f t e e n and sixteen were s i g n i f i c a n t l y d i f f e r e n t from each other as well as from the other days of incubation. There was a general increase in ch o r i o a l l a n t o i c membrane carbonic anhydrase with time. Therefore, i t i s concluded that carbonic anhydrase a c t i v i t y of the c h o r i o a l l a n t o i c membrane i s influenced by the external application of acid or base to the embryonic system. Furthermore, carbonic anhydrase may be involved in s o l u b i l i z a t i o n of the eggshell carbonate or maintenance of thi s procedure. It would appear that acid secretion by certain c e l l s of the c h o r i o a l l a n t o i c membrane i s occurring. The basic solution not only s l i g h t l y increased the pH of the a l l a n t o i c f l u i d but also caused a decrease in the calcium accumulated by the f l u i d after thirteen days of incubation. On the other hand, 59 Table V. The Ef f e c t s of Acid and Base Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane1'2 Treatments Days of 3. b c Incubation Acid Base Control EU/mg protein 11 8 . 1 6 ± 0 . 0 6 a 8 . l 6 ± 0 . 0 6 a 8 . 1 6 ± 0 . 0 6 a 12 8 . 7 0 ± 0 . 7 8 a 9 . 2 3 ± 0 . 7 4 a 8 . 5 4 ± 0 . 5 l a 13 4 . 1 2 ± 0 . 4 1 a 1 1 . 2 4 1 0 . 3 1 ° 7 . 2 9 ± 2 . 8 l b 14 8 . 6 0 ± 1 . 0 8 a 1 6 . 5 4 ± 2 . 4 3 ° l 0 . 6 4 ± O . 9 6 b 15 1 5 . 6 4 ± 1 . 7 5 a 2 1 . 6 4 ± 1 . 2 5 b 1 6 . 5 3 ± 0 . 1 8 a 16 I 5 . 9 6 ± 0 . 2 7 a 2 9 . 1 1 ± 0 . 9 8 ° 2 0 . 3 0 ± 0 . 6 3 b 1Two ch o r i o a l l a n t o i c membranes per sample: Mean±SEM of three samples; expressed in enzyme units per milligram of protein. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF te s t . 60 the a c i d i c solution consistently decreased the pH of the a l l a n t o i c f l u i d and s i g n i f i c a n t l y increased calcium uptake by the a l l a n t o i c f l u i d after twelve days of incubation. This data suggests that acid enhanced eggshell calcium s o l u b i l i z a t i o n while a base treatment impeded the calcium s o l u b i l i z a t i o n system. Previous work by other investigators suggested that the temporal c o r r e l a t i o n of increasing carbonic anhydrase a c t i v i t y and calcium mobilization c l e a r l y proved the role of carbonic anhydrase in calcium uptake. The present work showed increasing calcium mobilization along with increasing carbonic anhydrase a c t i v i t y in the control treatment af t e r thirteen days of incubation. However, the acid treatment showed an increase in calcium mobilized at th i s time, with a s i g n i f i c a n t decrease in carbonic anhydrase a c t i v i t y . The base treatment resulted in a decrease in calcium mobilized at t h i s time with a s i g n i f i c a n t increase in carbonic anhydrase a c t i v i t y (Figures 12 a,b',c). Thus, i t appears from t h i s data that the temporal co r r e l a t i o n between carbonic anhydrase a c t i v i t y and calcium uptake is not indic a t i v e in determining the role of carbonic anhydrase in calcium uptake. Therefore, i t can be concluded that carbonic anhydrase i s not an integral function of calcium mobilization. But i t may be s i g n i f i c a n t for calcium s o l u b i l i z a t i o n . 61 n < 2 25. tr 3 e 2 0 ^ 10 u •o •H 3 tH Cu U •w O 4-1 c 10 15 ioH 5H a. 10 u •H 3 r-l - 3 0 - 2 5 S •20 45 4J o « - 1 5 S •10 >1 - 5 5 M 10 U 11 12 13 14 15 16 Days o f I n c u b a t i o n F i g u r e 1 2 a . A c i d T r e a t m e n t E f f e c t s on A l l a n t o i c F l u i d C a l c i u m ( ) and C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . 30M 25-20-15' 10H - 2 0 30 •25 S 4-> •15 -10 o C u m u 11 12 13 14 15 16 Days o f I n c u b a t i o n F i g u r e 1 2 b . B a s e T r e a t m e n t E f f e c t s on A l l a n t o i c F l u i d C a l c i u m ( ) and C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . _ 30' < ^25-3 I 2 0"l •H U rH u 15 •a -H 3 S io u - H - 3 0 •25 o n a. E •20 4J U - 1 5 -10 11 12 13 14 15 16 Days o f I n c u b a t i o n F i g u r e 1 2 c . C o n t r o l T r e a t m e n t E f f e on A l l a n t o i c F l u i d C a l c i u m ( ) and C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . 10 u •o £ C < c & u a u c t s 62 B. Experiment II:Calcium Chloride E f f e c t s The next experiment employed calcium chloride as a p a r t i a l calcium compound to compete with the carbonate f r a c t i o n of the c a l c i t e . This was done to c l a r i f y the role of carbonic anhydrase in regulating the metabolic fate of the bicarbonate ions l i b e r a t e d from the c a l c i t e s h e l l , after i t s s o l u b i l i z a t i o n and during active calcium transport by the ch o r i o a l l a n t o i c membrane. The data obtained showed that blood calcium was not affected s i g n i f i c a n t l y by the CaCl2 treatments over that of the control (Table VI). The day ef f e c t s of blood calcium showed s i g n i f i c a n t changes with time to an o v e r a l l decrease in blood calcium (Table VI). As seen in the acid-base experiment, there i s a decrease in the blood calcium concentration from eleven to thirteen days of incubation. At fourteen days of incubation there is an increase in concentration subsequent to the occurence of the second decline. The day e f f e c t s seem to r e f l e c t t h i s with a repeated pattern of s i g n i f i c a n c e . Days eleven and fourteen were s i g n i f i c a n t l y d i f f e r e n t from twelve and f i f t e e n ; which were s i g n i f i c a n t l y d i f f e r e n t from thirteen and sixteen. Days f i f t e e n and sixteen of incubation were also not s i g n i f i c a n t l y d i f f e r e n t from one another. The blood pH le v e l s did not show o v e r a l l s i g n i f i c a n t differences-between treatments (Table VII). However, there were inconsistant treatment differences at twelve and thirteen days 63 Table VI. The Ef f e c t of Calcium Chloride Treatment on Blood Calcium 1* 2 Treatments Days of a a a Incubation 0.50M CaCJ^ 0.25M CaCl 2 Control pq/mq ash 11 49.2113. a 5 a 49.2113.5 49. 211 3. a 5 1 2 29.51 3. a 8 26.7+ 5.4 a 19 . 91 3. a 0 1 3 16.0+ 3. a • 4 17.01 2.7 3 12. 11 1 . a 4 14 33.7112. a 2 46.9116.2a 43. 71 9. a 9 15 29 .91 5. a 5 25.51 9.0 a 15. 91 6. a 2 16 16.91 2. a 9 20.71 0.7 a 18. 7+ 4. a 0 1Two embryos per sample:MeansiSEM of three samples; expressed in micrograms of Calcium per milligrams of ash. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by use of the IDF te s t . 64 T a b l e V I I . The E f f e c t s of C a l c i u m C h l o r i d e Treatment on B l o o d pH 1' 2 Treatments Days of I n c u b a t i o n 0 . 5 0 M C a C l a O ^ M C a C l ^ C o n t r o l 3 11 7.83±0.06 a 7.83±0.06 a 7.83±0.06 a 12 7.80±0.02 a 7.88±0.03 C 7.86±0.02 b 13 7.77±0.07 b 7.76±0.09 a b 7.73±0.08 a 14 7.59±0.07 a 7.59±0.07 a 7.6l±0.03 a 15 7.59±0.l8 a 7.62±0.08 a 7.63±0.06 a 16 7.56±0.08 a 7.57±0.14 a 7.56±0.09 a 1 S i x embryos per smaple:Mean±SEM. 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by t h e same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 65 of incubation. The day e f f e c t s of blood pH showed s i g n i f i c a n t differences between days eleven and twelve; day thirteen; and days fourteen, f i f t e e n and sixteen. There was an overall decrease in blood pH with time. The a l l a n t o i c f l u i d calcium concentration showed no overall s i g n i f i c a n t treatment differences (Table VIII). Day f i f t e e n of incubation showed a s i g n i f i c a n t , but inconsistant, treatment e f f e c t . The day effects of the a l l a n t o i c f l u i d calcium showed s i g n i f i c a n t differences between days eleven through thirteen; day fourteen; and days f i f t e e n and sixteen. There tended to be an o v e r a l l increase in the calcium concentration of the a l l a n t o i c f l u i d over time with either a drop or a further increase at sixteen days of incubation. These non s i g n i f i c a n t o v e r a l l e f f e c t s were expected since the calcium source was not changing. The a l l a n t o i c f l u i d pH showed s i g n i f i c a n t treatment differences at fourteen, f i f t e e n and sixteen days of incubation as influenced by CaCl2 (Table IX). The high CaCl 2 treatment had s l i g h t l y lower pH leve l s than the contr o l . The day effects showed s i g n i f i c a n t differences between days eleven through thirteen; days thirteen through fourteen; days fourteen through f i f t e e n ; and day sixteen of incubation. There was a general trend to a decrease in pH over time. 6 6 T a b l e V I I I . The E f f e c t s of C a l c i u m C h l o r i d e Treatment on A l l a n t o i c F l u i d C a l c i u m 1 ' 2 Treatments Days of I n c u b a t i o n 0.50M CaCl/ 1 0.25M C a C l a C o n t r o l 11 7 . 2±1 . a 4 7 . 2 ± 1 . 4 A 7. 2±1 a . 4 1 2 8 . 5±1 . a 5 8 . 8 ± 1 . 4 A 8 . 2±1 . 6 A 1 3 8 . 1 ± 0 . a 3 7 . 2 ± 1 . 9 a 8 . 3±2 a . 4 14 13. 1±7. a 0 I 0 . 7 ± 2 . 0 A 12. 7±1 .9 a 1 5 1 8 . 9±3. b 3 1 1 . 5 ± 8 . I A 17. 2±7 b . 3 1 6 16. 5 ± 5 . a 5 1 9 . 7 ± 5 . 3 A 15. 4±3 . 3 A 'Two embryos per sample:Mean±SEM of t h r e e samples e x p r e s s e d i n micrograms c a l c i u m per m i l l i g r a m s of a s h . 2 0 v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 67 T a b l e IX. The E f f e c t s of C a l c i u m C h l o r i d e Treatment on A l l a n t o i c F l u i d pH 1* 2 Treatments Days of 3.C be -I n c u b a t i o n 0.50M C a C l 2 0.25M C a C l 2 C o n t r o l 11 7. 77±0. 1 6 s 7 ,77±0. I 6 a 7 .7710. I 6 a 1 2 7. 57±0. 1 4 a 7 •89±0. 2 2 a 7 .7510. 0 9 a 13 7. 4 0 ± 0 . 0 5 a 7 ,66±0. 14 a 7 .4810. 2 2 a 14 6. 8 4 ± 0 . 4 6 a 7 .03±0. 5 5 a b 7 .1810. 2 9 b 1 5 6. 07±0. 6 l a 6 .45±0. 6 0 b 6 .8410. 6 2 c 16 6. 42±0. 3 7 b 5 .8710. 4 7 a 6 .2110. 5 7 b 1 S i x embryos per sample:MeaniSEM. 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 68 Of the treatment e f f e c t s , the s l i g h t s h i f t in a l l a n t o i c f l u i d pH may be due to a chloride e f f e c t , where in the normal c a l c i t e s i t u a t i o n : C0 2 + H 20 <—carbonic anhydrase—> H 2C0 3 H 2C0 3 < > H + + HCO3" + CaC03—> Ca + +<bound or transported HCOg'rbasic environment However, when the chloride ion i s in competetion with the carbonate ion the following s i t u a t i o n occurs: C0 2 + H 20 <—carbonic anhydrase--> H 2CO3 H 2C0 3 < > H + + HC03 " + CaCL,—> Ca+*<bound or transported HCl;acidic environment The calcium chloride treatment affected the carbonic anhydrase a c t i v i t y depending on the embryo age (Table X). The resul t was a decreased a c t i v i t y over that of the control at fourteen and f i f t e e n days of incubation. However, at sixteen days of incubation the carbonic anhydrase a c t i v i t y had increased to control l e v e l s or greater. This may be due to a larger demand for calcium by sixteen days of incubation than seen in the early phases of calcium transport. This sudden increase of carbonic anhydrase a c t i v i t y may be due to the system f i n a l l y overcoming the ef f e c t s of the treatment. A possible explanation of t h i s surge, may be that the system suddenly demanded a large quantity of calcium. Therefore, the r a t i o of chloride to bicarbonate ions may s h i f t . This may result in a c t i v a t i o n of carbonic anhydrase, causing the sudden jump in a c t i v i t y seen at sixteen days of incubation. 69 Table X. The Ef f e c t s of Calcium Chloride Treatment on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic Membrane1'2 Treatments Days of Incubation 0 .50M CaCl 0 a 0 .25M CaCl b C o n t r o l 3 EU/mg protein 11 8 . 6 1 ± 3 . 0 6 d 8 . 6 l ± 3 . 0 6 a 8 . 6 1 ± 3 . 0 6 12 7 . 9 8 ± 1 . 6 4 a 6 . 7 3 ± 1 . 6 4 a 7 . 4 3 ± 1 . 4 6 13 9 . 2 9 ± 2 . 4 2 a 9 . 4 7 ± 0 . 8 7 a 6 . 9 7 ± 1 . 8 4 14 5 . 6 5 ± 1 . 0 6 a 5 . 9 9 ± 0 . 5 8 a 8 . 7 7 ± 1 . 1 8 15 6 .78 + 0.81 a 5 . 9 2 ± 0 . 7 6 a 1 7 . 3 8 ± 0 . 6 1 16 3 7 . 4 8 ± 5 . 3 0 b 2 4 . 5 0 ± 2 . 8 5 a 2 1 . 9 2 ± 0 . 6 5 'Two ch o r i o a l l a n t o i c membranes per sample: Mean±SEM of three samples; expressed in enzyme units per milligram of protein. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF te s t . 70 The day e f f e c t s of the calcium chloride carbonic anhydrase a c t i v i t y showed s i g n i f i c a n t differences between days eleven through thirteen; days twelve and fourteen; day f i f t e e n and day sixteen. The o v e r a l l a c t i v i t y of c h o r i o a l l a n t o i c membrane carbonic anhydrase was decreased in the CaC^ treatment over that of the contr o l . This was achieved without a f f e c t i n g the calcium concentrations in the embryo. It would appear that the chloride suppressed or f a i l e d to activate carbonic anhydrase a c t i v i t y . Therefore, t h i s experiment suggests that the l e v e l of carbonic anhydrase a c t i v i t y may be a function of the bicarbonate ion concentrat ion. C. Experiment III:Strontium Chloride Effects This experiment employed strontium chloride to investigate the e f f e c t of a non-calcium non-carbonate compound that competes with both the carbonate f r a c t i o n and the calcium fraction of the eggshell c a l c i t e . The data obtained showed that blood calcium concentrations were not s i g n i f i c a n t l y affected by the treatments, except for day twelve of incubation (Table XI). The day effects of strontium chloride treatments on blood calcium showed s i g n i f i c a n t differences between day eleven; day twelve; and days thirteen through sixteen of incubation. S i g n i f i c a n t differences were noted in the blood pH at 71 T a b l e X I . The E f f e c t s of S t r o n t i u m C h l o r i d e Treatment on B l o o d C a l c i u m 1 ' 2 Treatments Days of I n c u b a t i o n 0.50M S r C l 2 a 0.25M SrCl*. C o n t r o l yug/mg ash 11 9 . 7 ± 0 . 2 a 9 . 7 ± 0 . 2 a 9 . 7 ± 0 . 2 a 12 1 3 . 2 ± 0 . 3 b 1 1 . 4 ± 3 . 9 a 13.2±0.8 b 1 3 1 2 . 1 1 1 . 1 a 1 1 . 2 ± 1 . 1a 1 1 . 4 ± 0 . 1 a 14 1 2 . 1 ± 1 . 0 a I 2 . 3 ± 0 . 2a 1 1 . 3 ± 0 . 0 a 15 1 1 . 6 ± 0 . 4 a 1 1 . 1 ± 0 . 6a 1 0.8± 0 . 4 a 16 1 1 . 1 ± 0 . 3 a I 0 . 4 ± 0 . 6a 1 2 . 1 1 1 . 1 a 'Two embryos per sample:Mean±SEM of t h r e e samples; e x p r e s s e d i n micrograms of c a l c i u m per m i l l i g r a m s of a s h . 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 72 thirteen through f i f t e e n days of incubation (Table XII). The ph was generally higher in the high strontium chloride treatment at those days of incubation. The day ef f e c t s of strontium chloride treatment on blood pH showed s i g n i f i c a n t differences between day eleven; day twelve; day thirteen; and days fourteen through sixteen of incubation. A l l a n t o i c f l u i d pH level s showed some v a r i a b i l i t y at day thirteen of incubation (Table XIII). However, there was a s l i g h t decrease in the strontium chloride treatment pH over that of the control s t a r t i n g at fourteen days of incubation. The difference was s i g n i f i c a n t at f i f t e e n days of incubation. This is a pattern similar to that seen in the calcium chloride experiment. The day ef f e c t s of strontium chloride treatments on a l l a n t o i c f l u i d pH showed s i g n i f i c a n t differences between days eleven through thirteen; days fourteen through f i f t e e n ; and day sixteen of incubation. The a l l a n t o i c f l u i d calcium concentrations were generally increased as influenced by the strontium chloride treatment (Table XIV). S i g n i f i c a n t increases in calcium concentrations over the controls were seen at thirteen, fourteen, f i f t e e n and sixteen days of incubation. The 0.50M strontium chloride treatment showed consistantly higher a l l a n t o i c f l u i d calcium l e v e l s than the controls, with the exception for day -fifteen of incubation.The day e f f e c t s of strontium chloride treatments on a l l a n t o i c f l u i d pH showed s i g n i f i c a n t differences between days eleven through f i f t e e n ; and day sixteen of incubation. The strontium chloride treatment resulted in an ove r a l l s i g n i f i c a n t decrease in carbonic anhydrase a c t i v i t y compared to 73 T a b l e X I I . The E f f e c t s of S t r o n t i u m C h l o r i d e Treatment on B l o o d pH 1' 2 Treatments Days of a b b I n c u b a t i o n 0.50M S r C l 2 0.25M SrCL^ C o n t r o l 11 7 .98±0. 0 3 a 7 .9810. 0 3 a 7 .98 + 0 .0 3 a 12 8 .08±0. 0 9 a 8 .0610. 0 6 a 8 .0810 . 0 8 a 1 3 7 .88±0. 04 b 7 .7710. i o a 7 .8210 . 0 6 a b 1 4 7 .6610. i o b 7 .5510. I 5 a 7 .6210 . 0 9 b 15 7 .6610. 7 .5710. 0 8 a 7 .6010 . 0 2 a 16 7 .5410. 05 a 7 .5710. 0 6 a 7 .5210 .0 6 a 1 S i x embryos per sample:MeaniSEM. 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by t h e same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 74 T a b l e X I I I . The E f f e c t s of S t r o n t i u m C h l o r i d e Treatment on A l l a n t o i c F l u i d pH1>2 Treatments Days of I n c u b a t i o n 0 . 5 0 M S r C l a 0.25M S r C l a C o n t r o l 1 1 1 2 1 3 14 1 5 16 7.86±0.l9 a 7.86±0.l9 a 7.86±0.l9 a 7.54±0.15 a 7.78±0.45 a 7.78±0.27 a 7.55±0.28 b 7.25±0.12 a 7.44±0.36 a b 6.27±0.47 a 6.29±0.4i a 6.48±0.34 a 6.12±0.32 a 5.98±0.42 a 6.41±0.41 b 5.63±0.41 a 5.75±0.l9 a 5.86±0.39 a 1 S i x embryos per sample:Mean±SEM. 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 75 T a b l e XIV. The E f f e c t of S t r o n t i u m C h l o r i d e Treatments on A l l a n t o i c F l u i d C a l c i u m 1 ' 2 Treatments Days of ID c i fo I n c u b a t i o n 0.50M SrClg 0.25M SrCL, C o n t r o l 11 7 .411 . 2 a 7.411.2 a 7. 411 a .2 1 2 9 .610 . 9 a 7.910.6 3 9. on a . 1 1 3 1 1 .111 c ab . b 11.911.5 b 7. 9+1 a .6 1 4 17 .011 . 1 b I 2 . 5 i 0 . 9 a 14. 312 ab .7 1 5 1 3 .418 . 9 a 19.311.6 b 13. 511 a .0 1 6 24 .312 . 1 b ! 5 . 6 l 3 . 5 a 16. 414 a . 1 1Two embryos per sample:Mean+SEM of t h r e e samples e x p r e s s e d i n micrograms c a l c i u m per m i l l i g r a m s of a s h . 2 O v e r a l l t r e t m e n t , o v e r a l l day and between t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the IDF t e s t . 76 the control (Table XV). These results are similar to those observed in the calcium chloride experiment. The day eff e c t s of strontium chloride treatments on carbonic anhydrase a c t i v i t y showed s i g n i f i c a n t differences between days eleven through fourteen; day f i f t e e n ; and day sixteen of incubation. From t h i s experiment and from the calcium chloride experiment i t can be seen that several occurances are si m i l a r . In both experiments o v e r a l l blood calcium l e v e l s were not s i g n i f i c a n t l y changed due to the treatments. Also, the blood pH lev e l s were variable but not s i g n i f i c a n t l y d i f f e r e n t . A l l a n t o i c f l u i d calcium was only s l i g h t l y higher than the control in some of the days with the 0.50M calcium and strontium chloride treatments. Also, a l l a n t o i c f l u i d pH tended to be s l i g h t l y lower in the calcium and strontium treatments. The carbonic anhydrase a c t i v i t y of the calcium chloride treatment shows a decrease from that of the control over fourteen and f i f t e e n days of incubation. If this decrease in a c t i v i t y can be explained by the chloride mediated e f f e c t , (as discussed e a r l i e r ) , then a similar decrease in a c t i v i t y due to the chloride in the strontium treatment would also be expected. As the res u l t s indicate, there i s a decrease in carbonic anhydrase a c t i v i t y of the strontium chloride treatments in a manner si m i l a r to that seen in the calcium chloride experiment. It can be concluded that the decrease in carbonic anhydrase a c t i v i t y i s not due to a calcium mediated process, since the strontium (a calcium competetive ion) did not cause a further decrease in a c t i v i t y over that seen with calcium chloride. The decrease in carbonic anhydrase a c t i v i t y appears to be almost 77 T a b l e XV. The E f f e c t s of S t r o n t i u m C h l o r i d e Treatment on C a r b o n i c Anhydrase A c t i v i t y of the C h o r i o a l l a n t o i c Membrane V Treatments Days of I n c u b a t i o n 0.50M SrCL^ 0.25M S r C L ^ C o n t r o l b EU/mg p r o t e i n 11 1 1 . 5 7 ± 0 . 9 0 a 11.57±0.90 a 11.57±0.90' 12 9 . 4 2 ± 4 . 8 5 a 8 . 8 8 ± 0 . 2 5 a 12.34±6.54' 13 1 1 . 5 9 ± 0 . 6 i a 12.69±2.60 a 1 6 . 91 ±2 . 03' 14 1 1 . 2 5 ± 1 . 3 0 a I 3 . 4 0 ± 1 . 9 8 a 15.47+2.05 15 1 6 . 3 6 ± 2 . 0 i a I 6 . 5 9 ± 4 . 9 9 a 22.90±2.60 16 2 2 . 9 6 ± 3 . 0 4 a 16.47+1.\ t 4 6 . 0 7 ± 5 . 1 7 1Two c h o r i o a l l a n t o i c membranes per sample: Mean+SEM of t h r e e samples; e x p r e s s e d i n enzyme u n i t s per m i l l i g r a m of p r o t e i n . 2 O v e r a l l t r e a t m e n t , o v e r a l l day and between • t r e a t m e n t means not f o l l o w e d by the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. Means t e s t e d by the use of the I D F , t e s t . 78 t o t a l l y mediated by the bicarbonate or carbonate ion levels present. Therefore, the data suggests that the primary function of carbonic anhydrase i s not with calcium uptake and mobilization across the c h o r i o a l l a n t o i c membrane. D. Experiment IV:Acetazolamide E f f e c t s This experiment involved investigation of the effects of a carbonic anhydrase i n h i b i t o r , acetazolamide, on carbonic anhydrase a c t i v i t y . The data showed that blood calcium concentrations were not s i g n i f i c a n t l y affected by the treatments, except for days thirteen and fourteen of incubation (Table XVI). The acetazolamide-acid (az-acid) treatment tended to have a higher calcium concentration than the contr o l . The exception was day fourteen of incubation, where the az-acid treatment had a s i g n i f i c a n t l y lower value. The acetazolamide-base (az-base) treatment tended to have a lower blood calcium concentration than the c o n t r o l . This was s i g n i f i c a n t l y lower at days thirteen and fourteen of incubation. The acetazolamide (az) treatment, although not s i g n i f i c a n t l y d i f f e r e n t from the control, tended to have a s l i g h t l y lower blood calcium concentrat ion. The day e f f e c t s of acetazolamide and acid-base treatments on blood calcium showed s i g n i f i c a n t differences at day eleven; days twelve through fourteen and day sixteen; and days f i f t e e n and sixteen. There tended to be an o v e r a l l decrease in blood calcium over time 79 Table XVI. The E f f e c t s of Acetazolamide and Acid/Base Treatments on Blood Calcium 1' 2 Treatments Days of Incubation Az- Az-c l C c l c l 1 Acid Base Az Control yug/mg ash 11 28. 2±0 .2 a 28.2±0. 2 a 28.2±0. 2 a 28 .2±0 .2a 1 2 19. 7±0 .3 a 18.9±0. 2 a 17.7±2. 2 a 19 .7±0 .7 a 1 3 21 . 6±0 . o b 14.9±1. 4 a 19.9±0. 6 b 19 :8±o .8b 1 4 17. 2±0 . o a 19.5±0. c a b D 15.8±0. 7 a 21 .611 . 1 b 1 5 17. 3±0 • 4 a 14.2±0. 6 a 14.7±0. 3 a 16 .6±2 .4a 16 19. 0±0 .4 a 16.7±0. 8 a 15.5±0. 3 a 18 .410 .6 a 1Two embryos per sample:Mean±SEM of three samples; expressed in micrograms of calcium per milligrams of ash. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF te s t . 8 0 The blood pH varied s i g n i f i c a n t l y due to treatments. From Table XVII, one can see that the az-acid treatment resulted in a s i g n i f i c a n t l y lower pH than the az-base treatment starting at day twelve of incubation. The az-acid treatment was also lower than the az treatment; being s i g n i f i c a n t l y lower at a l l days of except at day thirteen. The az-acid was also s i g n i f i c a n t l y lower than the control at a l l days of incubation except at days thirteen and fourteen. Consequently, the trend was for the base treatment to show a higher blood pH than the az-acid, az and control treatments. The az treatment, therefore, tended to have blood pH l e v e l s intermediate between those of the az-base and con t r o l . This was expected because of the role carbonic anhydrase plays in maintaining the s t r i c t acid-base balance in the blood vascular system. The day e f f e c t s of acetazolamide and acid-base treatments on blood pH showed s i g n i f i c a n t differences between day eleven; days twelve, f i f t e e n and sixteen; day thirt e e n ; and day fourteen. Table XVIII shows the changes due to treatments on the a l l a n t o i c f l u i d pH. Unlike the blood pH, where the s h i f t in pH i s variable u n t i l thirteen to fourteen days of incubation; the a l l a n t o i c f l u i d pH showed a more dramatic change. However, a similar arrangement of treatment e f f e c t s i s seen with the a l l a n t o i c f l u i d pH, as was seen with the blood pH. At twelve days of incubation the az-acid treatment showed a s i g n i f i c a n t decrease in pH from that of a l l the other treatments. At day thirteen of incubation, the control showed a s i g n i f i c a n t l y lower pH l e v e l than the az-base and az treatments. However, i t showed a higher pH l e v e l than that found in the az-acid treatment. The 81 Table XVII. The E f f e c t s of Acetazolamide and Acid/Base Treatments on Blood pH'>2 Treatments Days of Incubation Az- Az-A c i d a Base d Az c Control"1 11 7.89±0.02a 7.89±0.02 a 7.89±0.02 a 7.8910.02* 12 7.64i0.04a 7.84±0.03 b 7.8l±0.02 b 7.82±0.04 13 7.l2±0.04a 7.94±0.08 b 7.86±0.05 a 7.82+0.03 14 7.76±0.04 a 7.93±0.04 c 7.88±0.02 7.79±0.03' 15 7.56±0.02 a 8.07±0.04 d 7.8510.03° 7.68±0.02 16 7.52±0.04 a 8.14±0.'05d 7.81±0.08° 7.65±0.03 'Six embryos per sample:Mean±SEM. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r at P<0.05. Means tested by the use of the IDF test. 82 Table XVIII. The E f f e c t s of Acetazolamide and Acid/Base Treatments on A l l a n t o i c F l u i d pH1»2 Treatments Days of Incubation Az- Az-A c i d a Base d Az c C o n t r o l b 11 8 .02±0 .02a 8 .02±0 a .02 8. 02±0. a 02 8 .02±0 a .02 1 2 7 .94±0 .05 a 8 .44±0 d .06 8. 10±0. b 07 8 . 17±0 c .03 1 3 7 . 12±0 .06 a 8 .76±0 d .03 8. 1 3±0. c 07 7 .98±0 b .05 1 4 6 .93±0 • 02 a 8 .50±0 d .03 8. 02±0. c 07 7 .52±0 b .03 1 5 7 .44±0 .03 a 8 .75±0 d .04 8. 16±0. c 02 7 .68±0 b .02 1 6 7 ,45±0 .07 a 8 .86±0 d .03 8. 15±0. c 02 7 .58±0 b .03 'Six embryos per sample:Mean±SEM. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n at P<0.05. Means tested by the use of the IDF test. 83 az treatment had s i g n i f i c a n t l y lower pH levels than the az-base and s i g n i f i c a n t l y higher l e v e l s than the control and az-acid. The az-base treatment had s i g n i f i c a n t l y higher a l l a n t o i c f l u i d pH l e v e l s than a l l of the other treatments. The day e f f e c t s of the acetazolamide and acid-base treatments on a l l a n t o i c f l u i d pH showed s i g n i f i c a n t differences between days eleven, f i f t e e n and sixteen; day twelve; days thirteen, f i f t e e n and sixteen; and day fourteen of incubation. The a l l a n t o i c f l u i d calcium concentrations, as influenced by acetazolamide and acid-base treaments, are shown in Table XIX. The results indicated that at fourteen days of incubation the az-acid treatment showed an increased a l l a n t o i c f l u i d calcium concentration over that of a l l other treatments. This increase was s i g n i f i c a n t in a l l cases excepting from the control at f i f t e e n and sixteen days of incubation and from the az-base treatment at thirteen days of incubation. The az-base treatment showed a decrease in calcium l e v i e s from those of a l l other treatments after thirteen days of incubation. It was s i g n i f i c a n t l y d i f f e r e n t from the control and not s i g n i f i c a n t l y d i f f e r e n t from the az treatment. The control a l l a n t o i c f l u i d calcium l e v e l s were intermediate between those of the az-acid and those of the az-base at fourteen to sixteen days of incubation. The day ef f e c t s of acetazolamide and acid-base treatments on a l l a n t o i c f l u i d calcium showed s i g n i f i c a n t differences between day eleven; day twelve; day thirteen; days fourteen through f i f t e e n ; and days f i f t e e n through sixteen. The carbonic anhydrase a c t i v i t y (Table XX) of the 84 Table XIX. The Ef f e c t s of Acetazolamide and Acid/Base Treatments on A l l a n t o i c F l u i d Calcium 1* 2 Treatments Days of Incubation Az- Az-cl b V) Acid Base Az Control 0 jug/mg ash 11 8.2±0 .3 a 8 .2±0 .3 a 8. 2±0.3 a 8. 2±0. 3 a 12 10.2±1 .0 b 7 .9±0 .1 a 12. 3±0.7 C 12. 5±0. 6 C 13 16.4±0 .9 b 17 .8±1 .8 b 9. 5+0.6a 10. 3±0. 6 a 14 21.8±2 .0 C 1 1 .7±1 .1 a 12. 1±0.4 a 15. 5±2. 9 b 15 16.8±0 .8 b 14 .4±0 .8 a 15. 6±0.7 a b 16. 4±0. 5 b 1 6 18.2±0 .7 b 1 4 . 1±1 .4a 15. 4±0.3 a 17. 0±0. 4 b 1Two embryos per sample:Mean±SEM of three samples expressed in micrograms calcium per milligrams of ash. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n at P<0.05. Means tested by the use of the IDF te s t . 85 acetazolamide treatment showed a s i g n i f i c a n t decrease in a c t i v i t y compared to that of the control by day twelve of incubation and continued through to day sixteen. The az-base treatment, although s i g n i f i c a n t l y lower than that of the con t r o l , was not as low as the az-acid treatment. The az-acid treatment showed the lowest carbonic anhydrase a c t i v i t y of a l l the treatments. These results are in accordance with the e a r l i e r acid-base experiment (Experiment I ) , where the acid suppressed and the basic treatment enhanced carbonic anhydrase a c t i v i t y . In t h i s experiment, i t appears that the acid or base with acetazolamide act independently and a d d i t i v e l y . In the case of the acid a 'double suppression* i s observed; where as, in the case of the base a ' p a r t i a l enhancement' i s observed. The day effects of acetazolamide and acid-base treatments on carbonic anhydrase a c t i v i t y showed s i g n i f i c a n t differences between days eleven through twelve; day thi r t e e n ; day fourteen; day f i f t e e n ; and day sixteen of incubation. The data showed that acetazolamide decreased calcium transport to the a l l a n t o i c f l u i d . This may indicate that carbonic anhydrase was involved in calcium transport. When th i s treatment was combined with a basic solution there was a further decrease in calcium transport to the a l l a n o i c f l u i d . However, when an acid (increases a l l a n t o i c f l u i d calcum) was combined with acetazolamide (decreases a l l a n t o i c f l u i d calcium), i t was shown that calcium was transported to the a l l a n t o i c f l u i d at a • rate comparable with that of the control (See Figures 13 a, b, c , d). It i s also notable that in the az-acid treatment, calcium 86 Table XX. The Eff e c t s of Acetazolamide and Acid/Base Treatments on Carbonic Anhydrase A c t i v i t y of the Chorioallantoic MembraneV Treatments Days of • Incubation Az- Az-a c b Acid Base Az Control 11 10 .97±3 a .97 10. 9713.97a 10.9713. a 97 10. 9713. a 97 12 9 .6311 a .91 1 1 . 2410.97C 10.0114. b 29 13. 5512. a 04 j 13 8 .0111 a .65 15. 3212.70° 11.8914. b 40 19. 3813. a 69 j 14 6 .8012 a .48 19. 9312.08° 12.0813. b 90 27. 6212. a 75 j 1 5 10 .7512 a .28 25. 0711.72° 14. 1410. b 67 27. 6212. a 83 1 6 10 .9411 a ;62 26. 8712.32° 19.0312. b 96 36. 1710. a 08 'Two ch o r i o a l l a n t o i c membranes per sample:MeanlSEM of three samples; expressed in enzyme units per milligram of protei 2 O v e r a l l treatment, overa l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n at P<0.05. Means tested by the use of the IDF te s t . 87 a u 22 21 20' 194 18 17 16 15 144 13 12 114 10 9 8 7 141 c o 36 £ 31 a 26 > -H 4J O < 421 0) in IS u •o 16 £ 4 n c id u 11 12 13 14 15 Days o f I n c u b a t i o n 16 F i g u r e 1 3 a . A z - A c i d T r e a t m e n t E f f e c t s on A l l a n t o i c F l u i d C a l c i u m ( ) a n d C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . a. 2 2 -2 1 -2 0 -1 9 -1 8 -1 7 -1 6 -1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -41 c o 436 0' e \ 4315 >, 4-1 •H 26 > o rt 2 1 s id M T) >i 16 * s o 11 1 1 12 13 14 15 Days o f I n c u b a t i o n 16 F i g u r e 1 3 b . C o n t r o l T r e a t m e n t E f f e c t s on A l l a n t o i c F l u i d C a l c i u m ( ) and C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . 11 12 13 14 15 Days o f I n c u b a t i o n F i g u r e 1 3 c . A z T r e a t m e n t E f f e c t s on A l l a n t o i c F l u i d C a l c i u m ( ) a n d C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . 01 rt & en a 21-1 20 19 1 8 , 17-1 16 15 14 13 12 11 10H 9 8 7 41 c oi •p o u 1-36 & 31 S 26 > •U u rt I"21 s 10 u •0 >1 •16 -S HI 11 12 13 14 15 Days o f I n c u b a t i o n 16 F i g u r e 1 3 d . A z - B a s e T r e a t m e n t E f f e c t s o n A l l a n t o i c F l u i d C a l c i u m ( ) and C h o r i o a l l a n t o i c Membrane C a r b o n i c A n h y d r a s e A c t i v i t y ( ) . 88 was being transported to the a l l a n t o i c f l u i d while the carbonic anhydrase a c t i v i t y was suppressed. Therefore, i t seems apparent that carbonic anhydrase i s not d i r e c t l y involved in the transport of calcium across the c h o r i o a l l a n t o i c membrane and into the a l l a n t o i c f l u i d . However, i t i s c l e a r l y involved in the acid-base balances of the avian embryo and calcium s o l u b i 1 i z a t ion. 89 GENERAL DISCUSSION The results of t h i s thesis confirm those of Owezark (1971), Coleman and Terepka (1972b), and Reider et a l . (1980), who suggested the necessity of cho r i o a l l a n t o i c membrane carbonic anhydrase in s o l u b i l i z a t i o n of calcium. The present results showed that acid suppressed c h o r i o a l l a n t o i c membrane carbonic anhydrase a c t i v i t y while a base enhanced carbonic anhydrase a c t i v i t y . However, the acid treatment increased calcium s o l u b i l i z a t i o n , while the base caused a decrease. The results also showed that the decrease in carbonic anhydrase a c t i v i t y did not necessarily result in a decrease in calcium movement across the ch o r i o a l l a n t o i c membrane (into the a l l a n t o i c f l u i d ) . This i s contradictory to the hypothesis proposed by Gay and Mueller (1973), Crooks et a l . (1976), Tuan and Zrike (1978) and Reider et a l . (1980), that carbonic anhydrase i s involved in calcium transport. The hypothesis proposed by these authors, as discussed in the l i t e r a t u r e review ( H E ) , was ^based on: 1)the temporal c o r r e l a t i o n between the increased c h o r i o a l l a n t o i c membrane carbonic anhydrase and calcium transported; and 2)that i n h i b i t o r s of carbonic anhydrase caused both a decrease in cho r i o a l l a n t o i c carbonic anhydrase a c t i v i t y and a reduction in calcium transport. The f i r s t part of the hypothesis deals with the temporal c o r r e l a t i o n between increased chorioallanoic membrane carbonic anhydrase and calcium transport. One can see that t h i s i s not necessarily indicative of carbonic anhydrase involvement. As 90 shown by many authors, the ectoderm of the ch o r i o a l l a n t o i c membrane consists of several c e l l types (Narbaitz,1971; Owezark,1971; Coleman and Terepka,1972a,b; Saleuddin et al.,1976; Reider et al.,1980).This was shown with l o c a l i z a t i o n and radiotracer studies. The results proposed that c a p i l l a r y covering (or sinus covering) c e l l s function in active calcium transport while v i l l u s cavity c e l l s , which are high in carbonic anhydrase a c t i v i t y , function in hydrogen ion secretion. If these are two functional c e l l types, then i t may not necessarily follow that a decrease in carbonic anhydrase a c t i v i t y results in a decrease in calcium transported, as long as the treatment does not a f f e c t the c a p i l l a r y covering c e l l s or the homeostasis of the embryonic system. The second hypothesis; that i n h i b i t o r s of carbonic anhydrase decreased the ch o r i o a l l a n t o i c carbonic anhydrase a c t i v i t y and reduced calcium transport was considered. The results of t h i s thesis showed that the carbonic anhydrase i n h i b i t o r did decrease carbonic anhydrase a c t i v i t y and that there was a decrease in calcium transported across the cho r i o a l l a n t o i c membrane (into a l l a n t o i c f l u i d ) . But thi s again may not be t o t a l l y i n d i c a t i v e of carbonic anhydrase a c t i v i t y in calcium transport. The results obtained also showed that when an acid treatment was combined with the acetazolamide treatment, there was a decrease in carbonic anhydrase a c t i v i t y . However, there was an increase in calcium transported across the cho r i o a l l a n t o i c membrane (into the a l l a n t o i c f l u i d ) , over that of the c o n t r o l . This would suggest that carbonic anhydrase was 91 not d i r e c t l y involved in calcium transport. The decrease in calcium transported with the acetazolamide treatment may, therefore, not be a di r e c t e f f e c t . But, i t may be due to the decreased hydrogen ion release, a consequent decrease in calcium s o l u b i l i z e d and, therefore, a decrease in calcium available for transport. When these results are combined with the data concerning calcium and carbonate competition; one can see that carbonic anhydrase i s primarily involved with calcium s o l u b i l i z a t i o n and acid-base homeostasis. When calcium chloride was used as a treatment, the hypothesis was that the chloride f r a c t i o n of the treatment would compete with the carbonate f r a c t i o n of the eggshell causing a decreased carbonic anhydrase a c t i v i t y . When strontium chloride was used as a treatment, the hypothesis was that the chloride fract i o n of the treatment would compete with the carbonate f r a c t i o n of the eggshell causing again the decreased carbonic anhydrase a c t i v i t y . Also, the strontium f r a c t i o n of the treatment would cause a further decrease in carbonic anhydrase a c t i v i t y , i f carbonic anhydrase was involved in calcium transport. The results showed that in both cases, the chloride f r a c t i o n showed a decrease in carbonic anhydrase a c t i v i t y over that of the contro l . However, the strontium chloride treatment did not show a further decrease in enzyme a c t i v i t y over that seen in the calcium chloride treatment. One could only assume, therefore, that the carbonic anhydrase a c t i v i t y was sensitive to changes in the bicarbonate/carbonate f r a c t i o n but appeared to be 9 2 i n s e n s i t i v e to changes occurring in the calcium f r a c t i o n of the c a l c i t e . In both cases, calcium was transported across the cho r i o a l l a n t o i c membrane (into the a l l a n t o i c f l u i d ) in concentrations comparable to those seen in the contro l . Another factor that may have affected previous work with the ch o r i o a l l a n t o i c membrane, was that the procedures of administering treatments either exposed the membrane (to apply treatments via an annulus), or removed the membrane altogether. In exposing or removing the membrane, there may have been changes in pH, pressure differences or f l u i d s h i f t s . The procedure used in thi s study employed dipping the eggs. This has proven to be economical with respect to time, causing less chance of contamination and error, where the i n t e g r i t y of the system i s not disrupted. 93 CONCLUSION It i s concluded from t h i s study that c h o r i o a l l a n t o i c carbonic anhydrase functions in the s o l u b i l i z a t i o n of eggshell calcium, and in the regulation and s t a b i l i z a t i o n of acid-base balances in the avian embryo. This i s apparent from the ch o r i o a l l a n t o i c membrane carbonic anhydrase results which show that enzyme a c t i v i t y i s stimulated by a basic environment and suppressed by an ac i d i c environment. Further support i s given in the results which showed a s l i g h t suppression in enzyme a c t i v i t y when a chloride treatment competed with the eggshell carbonate f r a c t i o n . Carbonic anhydrase was not, however, involved d i r e c t l y in calcium ion transport across the ch o r i o a l l a n t o i c membrane. 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Acid/Base Concentrations: Embryo Tolerance expressed in % Mortality HCl -Acid Concentration Days of 0. 18M 0.24M Incubation O.OM 0.06M 0. 12M 1 1 0 0 0 0 O1 12 0 < 0 0 31 46 13 0 * 0 0 15 38 14 0 0 0 15 38 15 0 0 8 1 5 31 16 0 0 0 23 23 17 0 0 0 31 38 NaOH-Base Concentration Days of 0, 1 8M 0.24M Incubat ion O.OM 0.06M 0.12M 1 1 0 0 0 0 8 1 2 0 0 0 0 0 13 0 0 0 0 0 14 0 0 0 8 8 1 5 0 0 0 0 0 1 6 0 0 0 0 0 17 0 0 0 0 0 1 Embryo tolerance expressed in % mortality of thirteen embryos per day per treatment. From t h i s table one can see that the acid tolerance l e v e l of the embryo diminishes between 0.12-0.18M HCl. The base tolerance l e v e l of the embryo i s much greater showing only a sl i g h t decrease at 0.18-0.24M NaOH. The l e v e l of 0.12MHC1 and NaOH was used. This concentration was not so high as to cause treatment e f f e c t s due to stress and embryo death and was not so low that the treatment e f f e c t was l o s t . Therefore, a l l experimental HCl acid and NaOH base concentrations used were 0.12 Molar. 107 B. R a d i o t r a c e r S t u d i e s The movement and uptake of c a l c i u m p e r t i n e n t t o t h i s t h e s i s was i n v e s t i g a t e d . B l o o d c a l c i u m showed no s i g n i f i c a n t (P<0.05) d i f f e r e n c e i n uptake of * 5 C a l c i u m (Appendix T a b l e I I ) . However, the a l l a n t o i c f l u i d showed a s i g n i f i c a n t l y h i g h e r uptake of 0 5 C a l c i u m i n t h e a c i d t r e a t m e n t than t h a t of the c o n t r o l . The base t r e a t m e n t was not s t a t i s t i c a l l y d i f f e r e n t from t h e c o n t r o l (Appendix T a b l e I I I ) . The uptake of , s C a l c i u m by the c h o r i o a l l a n t o i c membrane i s shown i n Appendix T a b l e IV. The a c i d t r e a t e d c h o r i o a l l a n t o i c membrane showed the l o w e s t uptake of a 5 C a l c i u m . The base and c o n t r o l t r e a t e d c h o r i o a l l a n t o i c membranes showed s i g n i f i c a n t l y (P<0.05) h i g h e r uptakes of * 5 C a l c i u m . However, when the whole egg c o n t e n t s ( a l l of the e g g ; e x c l u d i n g s h e l l and s h e l l membranes) were a s s a y e d f o r 0 5 C a l c i u m c o n t e n t ; t h e r e was no s i g n i f i c a n t change i n uptake of the r a d i o t r a c e r due t o t r e a t m e n t (Appendix T a b l e V ) . T h e r e f o r e , s i n c e the same c o n c e n t r a t i o n of a 5 C a l c i u m was a v a i l a b l e , r e g a r d l e s s of t r e a t m e n t , ( i e ; t h e a c i d or base was not b l o c k i n g , p l u g g i n g , e n l a r g i n g or changing e g g s h e l l p r o p e r t i e s t o an e x t e n t where the t r e a t m e n t d i f f e r e n c e s c o u l d be s a i d t o be due t o a m e c h a n i c a l problem of e g g s h e l l s t r u c t u r e ) , i t would appear t h a t the a c i d t r e a t m e n t a l l o w e d the c a l c i u m p r e s e n t t o move more f r e e l y a c r o s s the c h o r i o a l l a n t o i c membrane and i n t o the a l l a n t o i c f l u i d than the base or c o n t r o l t r e a t m e n t s . The base t r e a t m e n t s l i g h t l y d e c r e a s e d movement of * 5 C a l c i u m under t h a t of the a c i d t r e a t m e n t . The c o n t r o l showed a s i m i l a r movement of a 5 C a l c i u m , i n the a l l a n t o i c f l u i d and i n 108 Appendix Table I I . The E f f e c t s of Acid and Base Treatments on Blood Calcium:' 5Calcium Uptake 1' 2 Treatments Days of a a Incubation Acid Base Control dpm a a a 11 31 . 3±1 4 .8 26 .9± 3. 7 27 .71 2. 7 1 2 29 .61 5 a .5 31 .21 5. a 7 26 .31 6. a 4 1 3 29 . 7± 1 0 a .5 28 .21 4. a 2 27 .8115. a 1 14 31 • 4± 3 a .3 31 .91 7. a 9 51 .5128. a 8 1 5 51 . 9± 1 4 a .2 48 .7142. a 4 23 .71 1 . a 81 6 64 . 6±1 8 a . 1 80 .1118. a 6 84 .9126. a 7 1Two embryos per sample:MeaniSEM of three samples expressed in dpm of * 5Calcium uptake. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means tested by the use of the IDF test. 109 Appendix Table I I I . The E f f e c t s of Acid and Base Treatments on A l l a n t o i c F l u i d Calcium ^Calcium Uptake 1' 2 Treatments Days of Incubation 0 5 C a - A c i d b " 5Ca-Base a 4 5 C a - C o n t r o i a dpm 11 20.1± 2.2 a 19.3± 2.0 a 16.8± 8.3 a 12 77.1± 33.3 a 100.5± 33.3 & 19.2± 2.3 & 13 169.8± 49.9 a 28.2± 2.2 a 21.1± 5.2 a 14 140.6± 62.5 a 36.6± 26.1 a 35.9± 3.7 a r 15 1308.51514.8b 46.2± 22.2 a 28.1± 9.3 3 16 1100.41116.5b 293.51359.3a 83.4162.0 a 1Two embryos per sample:MeaniSEM of three samples expressed in dpm of a 5Calcium uptake. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means Means dtested by the use of the IDF t e s t . 1 1 0 Appendix Table IV. The E f f e c t s of A c i d and Base Treatments on a 5 C a l c i u m Uptake by the C h o r i o a l l a n t o i c MembraneV Treatments Days of Incubation fl5Ca-Acida « 5 C a - B a s e b ft5Ca-Control dpm 11 2 5 . 5± 6 . a 2 2 6 . 9± 1 7 . 8a 2 7 . 0± 2 . 8 a 1 2 1 4 4 . 1± 7 8 . 5 a 7 7 . 5± 3 2 . Ia 1 0 5 . 5± 39 . 7 a 1 3 1 0 0 . 0± 2 6 . 9 a 2 4 0 . 1± 9 5 . 3 a 291 . 11114 . 4a 14 2 9 . 6± 3 9 . Ia 2 9 9 . 3 ± 1 4 5 . l a b 5 4 0 . 11157 . 5b 1 5 2 1 2 . 2± 1 2 . 8a 7 7 5 . 0 ± 2 2 6 . 9 b 3 6 6 . 0 1 1 8 0 . oa b 16 2 9 6 . 2 ± 2 0 6 . 2a 8 9 0 . 8 + 5 5 1 . 3 b 1 5 0 5 . 21324 . o c 1Two c h o r i o a l l a n t o i c membranes per sample:Mean+SEM of three samples; expressed i n dpm of " 5 C a l c i u m uptake. 2 O v e r a l l treatment, o v e r a l l day and between treatment fmeans not fo l l o w e d by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. Means t e s t e d by the use of the IDF t e s t . I l l Appendix Table V. The E f f e c t s of Acid and Base Treatments on ft5Calcium Uptake by the Whole Egg Contents 1- 2 Treatments Days of Incubation « 5Ca-Acid a ft5Ca-Basea * 5Ca-Control a dpm 11 20. 9± 5. 3 a 20. 9± 5 . 3 a 17 .6± 0. 6 a 1 2 558. 5± 212. 7 a 224. 1 ± 24 . 5 a 222 •6± 41. 3 a 1 3 1838. 3± 516. 2 a 279. 5± 101 . 9 a 495 .5± 446. 9a 14 5507. 5± 1 685. 4 a 887. 0 + 227 . 8 a 624 .31 219. 2 a 1 5 9416. 5± 4868. 3 a 1003. 7± 149 . 6 a 1 408 .31 562. 3 a 16 10859. 5± 1 162. 2^b 5094. 0±4147 . 3 a 25957 .211025. 2 b 1MeantSEM of three samples of whole egg contents. Whole egg contents includes everything except the eggshell and the s h e l l membranes; expressed in dpm of S 5Calcium uptake. 2 O v e r a l l treatment, o v e r a l l day and between treatment means not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e at P50.05. Means tested by the use of the IDF test. 112 the c h o r i o a l l a n t o i c membrane, to the base treatment. Therefore, i t would appear that 0 . 1 2 M HCl, 0 . 1 2 M NaOH, and control treatment solutions affected ft5Calcium movement in a way by which the acid treatment increased calcium movement to the a l l a n t o i c f l u i d over that of the control and base treatments. This was done without any of the treatments or experimental methods causing s i g n i f i c a n t mechanical or s t r u c t u r a l changes in eggshell i n t e g r i t y , porosity or t o t a l * 5Calcium uptake. Appendix Figure 1, i s an outline of the experimental scheme followed where the procedures followed are outline in the materials and methods. Days o f Incubat ion 10 11 12 13 14 15 16 t r e a t m e n t - ^ 78 eggs- i—>65 e g g s - v - > 5 2 e g g s - y - > 3 9 e g g s - y ^ 26 e g g s - y - > 1 3 eggs N group o f \ 1 day \ 2 day \ 3 day \ 4 day \ 5 day \ 91 eggs d ip d i p d i p d ip d i p \ pre 1 s t d i p 2 n d d ip 3 r d d i p 4 t h d i p 5 t h d i p 6 t h d i p t reatment sample sample sample sample sample sample 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs 13 eggs Appendix F igure 1. Exper imental Scheme. Uptake o f ^ C a l c i u m 1 : b y : t h e : b l o 6 d , a l l a n t o i c , f l u i d , c h o r i o a l l a n t o i c membrane, and t o t a l contents o f the av ian e g g . : Hhis scheme f o l l owed f o r the three t reatment groups: 4 5 C a l c i u m - A c i d ( 0 . 4 5 uCi 4 5 C a l c i u m - 0 . 1 2 M H C l ) ; 4 5 C a l c i u m - B a s e (0 .45 uCi 4 5 C a l c i u m - 0 . 1 2 M NaOH); C o n t r o l - d H 2 0 . Samples o f b l o o d , a l l a n t o i c f l u i d , c h o r i o a l l a n t o i c membrane and t o t a l whole egg contents were ashed by procedure i n Methods and M a t e r i a l s and then counted i n 10 mis B i o f l u o r (New England Nuc lear ) 114 C. Acetazolamide Studies A preliminary study was conducted using various concentrations of acetazolamide to find a concentration that would not eliminate but decrease carbonic anhydrase a c t i v i t y by 20-25%. Twenty micromolar acetazolamide was used and was found to decrease carbonic anhydrase a c t i v i t y by approximately 23%. Forty micromolar acetazolamide decreased carbonic anhydrase a c t i v i t y by approximately 79% and sixty micromolar acetazolamide resulted in too many deaths to give adequate r e s u l t s . (Appendix Figure 2 ) . The acetazolamide was introduced d a i l y into the egg by the standard dipping procedure. The cho r i o a l l a n t o i c membrane was prepared as previously described and the carbonic anhydrase a c t i v i t y was determined as outlined in the materials and methods. 115 Acetazolamide Concentration (uM) Appendix Figure 1. Carbonic Anhydrase I n h i b i t i o n by Acetazolamide 1. Expressed i n : A) Carbonic anhydrase a c t i v i t y , Enzyme Units (EU)/mg Protein and B) Carbonic anhydrase a c t i v i t y , ^remaining a c t i v i t y . Carbonic Anhydrase from two c h o r i o a l l a n t o i c membranes of fourteen day incubated eggs. Mean+SEM of three samples per concentration. 116 Appendix Table VI. Mineral and Trace Mineral Content at Hatch Mineral Content (mg) Calcium 129.3 Phosphorus 117.1 Sulpher 57.2 Sodi um 41.4 Potassium 38.4 Chloride 19.2 Iron 1.5 Magnesium 0.8 Trace Mineral Content (jjg) Lead 49.0 Copper 48.4 Manganese 9.0 (Romanoff, 1967) 117 Blood. 118 1,400 1,200 1,000 4 800 6001 CD 1. 400 X J E •r— u 10 o LO «3-200 11 12 13 14 15 Days o f Incuba t ion 16 Appendix F igure 4. The E f f e c t s o f Ac i d ' 1 45 , Base and Cont ro l E3 Treatments on 4 5 C a l c i u m Uptake by the A l l a n t o i c F l u i d , 1 1 9 1,800 + 1,500 f 1,200 | 900 to cn E E CL "D 6 0 0 in 300 11 "3 M 12 13 14 15 Days o f Incubat ion 16 Appendix F igure 5. The E f f e c t s o f A c i d t S a , Base and Cont ro l B Treatments on 4 5 C a l c i u m Uptake by the C h o r i o a l l a n t o i c Membrane. 120 30,000 t 25,000 20,000 15,000 IA cn E i . 10,000 E 3 u re S 5.000 11 12 13 14 15 16 Days o f Incubat ion Appendix F igure 6. The E f f e c t s o f Ac id L±ii , Base and Cont ro l L J Treatments on Calc ium Uptake by the Whole Egg Con ten ts . 121 50 12 Days of Incubation The E f f e c t s of Acid Appendix Figure 7 and Control S Treatments on Blood Calcium Base 122 123 11 12 13 14 Days of Incubation Appendix Figure -.9. The E f f e c t s of Acid and Control E3 Treatments on Blood pH 124 125 30 25 11 12 13 14 15 Days o f Incubat ion Appendix F igure 11. The E f f e c t s o f Ac id , Base , and Contro l 13 Treatments on Carbon ic Anhydrase A c t i v i o f the C h o r i o a l l a n t o i c Membrane. 126 60 t 50 f Blood Calcium. 127 A l l a n t o i c Fluid Calcium. 128 8 .04 7.9 ac o. •o o o cc 16 Days of Incubation Appendix Figure 14. The E f f e c t s of 0.25 M Calcium Chloride 0.50M Calcium Chloride Blood pH. and Control t. Treatments on 129 8.0J A l l a n t o i c F l u i d pH. 130 50 I 11 12 13 14 15 lb Days of Incubation Appendix Figure 16. The Effects of 0.25M Calcium Chloride H , 0.50M Calcium Chloride H , and Control E3 Treatments on Carbonic Anhydrase Activity of the Chorioallantoic Membrane. 131 14 Days o f Incuba t ion Appendix F igure 17. The E f f e c t s o f 0.25M St ron t ium C h l o r i d e 0.50M St ron t ium C h l o r i d e S3, and Cont ro l Treatments on B lood C a l c i u m . 132 30 4 25 Days o f Incubat ion Appendix F igure 18. The E f f e c t s o f 0.25M St ron t ium C h l o r i d e t ± J , 0.50M St ron t ium C h l o r i d e H , and Cont ro l Treatments on A l l a n t o i c F l u i d Ca l c i um. 133 8.1 11 12 13 14 15 16 Days o f Incubat ion pendix F igure 19 . The E f f e c t s o f 0.25M S t ron t ium C h l o r i d e 0.50M St ron t ium C h l o r i d e IS , and Cont ro l Q Treatments on Blood pH. 134 8 .5 8.01 H 12 13 14 15 16 Days o f Incuba t ion Appendix F igure 20 . The E f f e c t s o f 0.25M St ron t ium C h l o r i d e I 0.50M S t ron t ium C h l o r i d e H , and Cont ro l Treatments on A l l a n t o i c F l u i d pH. 135 50 QJ +•> O u o. cn E 40 > t> 30 QJ l/l >> c c o XI i -20 J 10 - • +-- -+• - H 1 i i : 4 4 K~4 4 4 - 4 4-- 4 4-- 4 4-- 4 - H 4-- •( 4-- ^ 4-- H 4-- H 4-- H Days o f Incubat ion Appendix F igure 21. The E f f e c t s o f 0.25M S t ron t ium C h l o r i d e 0.50M St ron t ium C h l o r i d e H , and Cont ro l E3 Treatments on Carbonic Anhydrase A c t i v i t y o f the C h o r i o a l l a n t o i c Membrane 136 137 22-21 20-_ 19 Days o f Incubat ion Appendix F igure :23. The E f f e c t s o f A c e t o - A c i d tiiiil , Aceto-Base H9, Aceto B, and Contro l 0 Treatments on A l l a n t o i c F l u i d Ca l c i um. 138 \ 139 9.04 11 12 13 14 15 Days of Incubation Appendix Figure 25. The Effects of Aceto-Acid t±I), Aceto-Base Aceto B , and Control ED Treatments on Allantoic Fluid pH. 140 36 J 31-o a. 26 5 21" w c «c u c o x> u to o o 16-11-11 12 13 14 Days of Incubation Appendix Figure 26. The Effects of Aceto-Acid Aceto El , and Control 15 Aceto-Base 16 Treatments on Carbonic Anhydrase Activity of the Chorioallantoic Membrane. 

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