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The role of prostaglandins during sexual maturation, ovulation and spermiation in the goldfish, Carassius… Bouffard, Maria Emilia Rachelle 1979

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THE EOIE OF PROSTAGLANDINS DURING SEXUAL MATURATION, OVULATION AND SPERMIATION IN THE GOLDFISH, CARASSIUS AURATUS BY MARIE EMILIA RACHELLE BOUFFARD B.Sc, University of Ottawa, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) No m 1 N o r _ T- - 9 f We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMEIA October, 1979 (c) Marie Emilia Rachelle Bouffard, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 ABSTRACT The objective of t h i s study was t c determine the involvement of prostaglandins i n the sexual development of both male and female goldfish. Carassius auratus . A chromatographic method was developed to separate the di f f e r e n t prostaglandin groups. To standardize the procedure, extraction and separation recoveries were measured using tritiated-prostaglandins. Radioimmunoassay was used to measure the PGB1, PGE1 and PGF2* in the plasma and gonad. I n i t i a l l y , a seasonal study was undertaken to assess the importance of prostaglandins during sexual maturation. Samples of plasma and gonad were assayed monthly for prostaglandins (from December to March) from two groups of f i s h , one held under natural photoperiod and the other under long photoperiod (16L:8D). Although monthly variations occurred i n a l l three prostaglandins examined, these changes did not correlate with changes i n gonadal maturationi Prostaglandins were then.measured in s e r i a l plasma samples of non-gravid and ovulating female goldfish. Ovulation was induced i n gravid f i s h by increasing the water temperature from 14° C to 20° C and by i n j e c t i n g human chorionic gonadotropin (HCG) . I t was found that: 1) PGF2 increased over 14 f o l d , 12 hours a f t e r the onset of ovulation (from pre-injection levels of 300 pg/ml to more than 4,000 pg/ml); however, t h i s increase appeared to commence immediately after ovulation. There was no change i n plasma P1GF20< levels in non-gravid control f i s h . The concentration of PGF2^in the ovarian f l u i d was over 9,000 pg/ml. 2) PGE1 decreased almost three-fold between the time of HCG in j e c t i o n (an average of 10 hours before ovulation) and 24 hours la;ter. The plasma PGE 1 l e v e l s in the non-gravid females were up to 20 times less than the gravid ovulating females. The concentration of PGE1 in the ovarian f l u i d was 630 pg/ml. 3) PGB1 l e v e l s decreased in the plasma of non-gravid and ovulating goldfish, following HCG i n j e c t i o n . The ovarian f l u i d contained 300 pg/ml of PGB1. P a r a l l e l experiments were performed on male goldfish that were spermiating. There were no s i g n i f i c a n t changes in plasma PGF20C within 24 hours of HCG i n j e c t i o n , whereas PGB1 decreased s l i g h t l y (as for the females), and PGE1 increased s i g n i f i c a n t l y 10 hours after HCG i n j e c t i o n . The findings of t h i s study suggest that PGF2 and PGE1 i n the ovarian f l u i d are the agents c o n t r o l l i n g ovulation in the female goldfish and that corresponding l e v e l s i n the blood contribute to other events associated with ovulation. The experiments on males indicate a possible r o l e for PGE1 during spermiation; however, i t i s d i f f i c u l t to ascertain i t s precise involvement at present. i v TABLE OF CONTENTS ABSTRACT - i i TABLE OF CONTENTS . i v LIST OF FIGURES ...................... . . . . . . . . . . . . . . . . . X ACKNOWLEDGEMENTS x i 1 GENERAL INTRODUCTION 1 1.1 HISTORICAL BACKGROUND 1 1.2 NOMENCLATURE AND STRUCTURE 2 1.3 OCCURRENCE AND RELEASE .5 1.4 BIOSYNTHESIS 6 1.5 METABOLISM ......... 7 1.6 MODE OF ACTION . . . . . . . . . . . . . . . . .............. 8 1.7 INVOLVEMENT OF PROSTAGLANDINS IN DIFFERENT AREAS 8 1.7.1 Circulation -.9 1.7.2 Nervous tissue 10 1.7.3 Inflamation . . . 1 0 1.7.4 prostaglandins in mammalian reproduction .11 1 . 7 . 4 . 1 Prostaglandins i n female reproduction . . . . 1 2 A) ovulation 13 B) maintenance of the corpus luteum . . . 1 3 C) mo t i l i t y of the reproductive t r a c t 14 1 . 7 . 4 . 2 Prostaglandins i n males 15 1.7.5 Prostaglandins' in f i s h 16 1.8 STATEMENT OF THE PROBLEM ..18 2 GENERAL TECHNIQUES . 20 2.1 FISH MAINTENANCE . 20 2.2 REAGENTS AND GLASSWARE ..... 20 2.3 COLLECTION AND EXTRACTION OF PROSTAGLANDINS ....21 2.3.1 E x t r a c t i o n from plasma .......21 2.3.2 E x t r a c t i o n from the gonad - 22 2.4 HISTOLOGY 23 2.5 COLUMN CHROMATOGRAPHY .............................. 23 2.5.1 P r e p a r a t i o n of the column 24 2.5.2 Gradient e l u t i o n apparatus 24 2.6 RADIOIMMUNOASSAY . 28 2.6.1 Sample p r e p a r a t i o n 28 2.6.1.1 PGF ... ...28 2.6.1.2 PGB 29 2.6.1.3 PGE ............. ..29 2.6.2 Radioimmunoassay procedure 29 2.6.3 C a l c u l a t i o n s 31 3 PRELIMINARY WORK .... 33 3.1 INTRODUCTION 33 3.2 REVIEW OF METHODOLOGY ............................... 33 3.2.1 E x t r a c t i o n 34 3.2.2 Separation 35 3.2.3 Assay methods .36 3.2.3.1 Bioassay 36 3.2.3.2 Chemical assays - 38 3.2.3.3 General b a s i s of radioimmunoassay ........38 A) radioimmunoassay of p r o s t a g l a n d i n s 39 3.3 VERIFICATIONS AND MODIFICATIONS OF PG ANALYTICAL TECHNIQUES -.41 3.3.1 Extraction e f f i c i e n c y .- 41 3.3.1.1 D i l u t i o n of labelled prostaglandins 41 3.3.1.2 Extraction recovery .....42 3.4 COLUMN CALIBRATION ....43 3.4.1 Preliminary chromatography methods and results 43 3.4.1.1 Gradient elution 47 A) apparatus 48 E) re s u l t s of gradient e l u t i c n cf prostaglandins 51 3.4.2 V e r i f i c a t i o n of methods on f i s h tissue 57 3.4.2.1 Radioimmunoassay of prostaglandins i n f i s h tissues 61 4 PROSTAGLANDINS DURING SEXUAL MATURATION; SEASONNAL CHANGES . 63 4.1 INTRODUCTION ................63 4.2 BACKGROUND REVIEW OF GONADAL DEVELOEMENT . .......63 4.2.1 Changes in gonadal histology with development .64 4.2.1.1 The female system 64 4.2.1.2 The male system 65 4.2.2 Endocrinological changes during gonadal development 65 4.2.2.1 The female system 66 4.2.2.2 The male system 66 4.2.3 Environmental influences on gonadal development 66 4.3 METHODS 67 4.3.1 Fish maintenance and sampling ..67 4.3.2 S t a t i s t i c s ......68 4.4 RESULTS FROM HISTOLOGY ...68 4.4.1 H i s t o l o g i c a l o b s e r v a t i o n s i n the female 68 4.4.2 H i s t o l o g i c a l o b s e r v a t i o n s i n the male 69 4.5 RESULTS OF THE PROSTAGLANDIN MEASUREMENT DURING DEVELOPMENT IN THE FEMALE 69 4.5.1 PGF2« l e v e l s i n the ovary ...... ...... ....70 4.5.2 PGF2^ l e v e l s i n the plasma 72 4.5.3 PGE1 i n the ovary 73 4.5.4 PGE1 l e v e l s i n the plasma 73 4.5.5 PGB1 l e v e l s i n the ovary 74 4.5.6 PGB1 l e v e l s i n the plasma 74 4.5.7 Comparison of the p r o s t a g l a n d i n l e v e l s i n t h e plasma and the ovary 75 4.6 PROSTAGLANDIN MEASUREMENT IN THE PLASMA AND TESTIS DURING DEVELOPMENT IN THE MALE 75 4.6.1 P G F ^ i n the t e s t i s ^ ....' ....... ..76 4.6.2 PGF2^ l e v e l s i n the plasma 78 4.6.3 PGE1 l e v e l s i n t e s t i s 79 4.6.4 PGE1 l e v e l s i n the plasma ...................... 79 4.6.5 PGB 1 l e v e l s i n the t e s t i s 80 4.6.6 PGB1 l e v e l s i n the plasma 80 4.6.7 Comparison of the p r o s t a g l a n d i n l e v e l s i n the plasma and the t e s t i s 81 4.7 DISCUSSION OF PROSTAGLANDINS IN THE FEMALE .81 4.8 DISCUSSION OF PROSTAGLANDINS IN THE MALE 83 4.9 CONCLUSION .... ..84 PROSTAGLANDINS DURING OVULATION IN THE GCIDFISH 86 5.1 INTRODUCTION 86 5.2 REVIEW OE THE MECHANISMS OE OVULATION .86 5.2.1 H i s t o l o g i c a l changes of the oocyte ..86 5.2.2 Endocrinological control of ovulation ..88 5.3 METHODS 90 5.3.1 f i s h maintenance 90 5.3.2 Females -.90 5.3.3 Controls ...93 5.3.4 S t a t i s t i c s . - i 93 5.4 RESULTS OF OVULATION EXPERIMENT 93 5.4.1 Prostaglandin l e v e l s in the ovarian f l u i d .....94 5.4.2 Prostaglandin levels i n the plasma of ovulating and non-gravid females ..94 5.4.2.1 PGF20( l e v e l s in Group I f i s h 94 5.4.2.2 PGF2o(> l e v e l s i n Group II f i s h .95 5.4.2.3 PGF20< l e v e l s i n Group III f i s h .95 5.4.2.4 PGF2 <levels in Group IV f i s h .96 5.4.2.5 PGE1 level s in Group I and Group II f i s h .98 5.4.2.6 PGE1 l e v e l s in Group III f i s h 98 5.4.2.7 PGE 1 levels in Group IV f i s h ...99 5.4.2.8 PGB1 level s in Group I f i s h 99 5.4.2.9 PGB 1 levels i n Group II f i s h 99 5.4.2.10 PGB1 levels i n Group III f i s h 100 5.4.2.11 PGB1 levels i n Group IV f i s h 100 5.5 DISCUSSION OF OVULATION . 100 5.5.1 PGF20( during ovulation 10 1 5.5.2 PGE1 during ovulation .........................103 5.5.3 PGB1 l e v e l s during ovulation 104 SPERMIATION IN THE GOLDFISH 106 i x 6.1 INTRODUCTION 106 6.1.1 T e s t i c u l a r changes d u r i n g spermation 106 6.1.2 Endocrine c o n t r o l o f s p e r m i a t i o n .107 6.1.2.1 Methods and r e s u l t s - .109 6.1.3 PGF^, PGE 1 and PGB1 l e v e l s i n the male p r i o r t o , and a f t e r , HCG i n j e c t i o n ....110 6.2 DISCUSSION 113 7 GENERAL DISCUSSION 114 BI BLIOGRAPHY -.135 APPENDIX . 154 X LIST OF FIGURES FIGURE 1: Molecular structure of various prostaglandins. . 4 FIGURE 2: Chromatographic apparatus 27 FIGURE 3: I n i t i a l elution pattern cf radioactive prostaglandins 46 FIGURE 4: Elution pattern of radioactive prostaglandins using a continual gradient technique. 51 FIGURE 5: I n i t i a l "on-off" gradient elution pattern of radioactive prostaglandins. 54 FIGURE 6: Successfull "on-off" gradient elution pattern of radioactive prostaglandins 56 FIGURE 7: Gradient elution pattern cf radioactive prostaglandins added to ovary homogenates 59 FIGURE 8: Seasonal variations i n prostaglandin lev e l s i n the female goldfish 72 FIGURE 9: Seasonal variations i n prostaglandin levels i n the male goldfish 78 FIGURE 10: Variations i n prostaglandin l e v e l s during HCG-induced ovulation in the goldfish 98 FIGURE 11: Variations in prostaglandin l e v e l s following HHG in j e c t i o n i n the male goldfish 112 FIGURE 12: Schematic representation of the proposed model of the role of prostaglandins in ovulation i n the goldfish 130 FIGURE 13: Schematic representation of the proposed model of the role cf prostaglandins i n spermiation in the g o l d f i s h . . 133 x i ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e t o my s u p e r v i s o r , Dr.A.M. P e r k s , f o r h i s g e n e r o s i t y and h i s p a t i e n c e d u r i n g my s t u d y , and f o r l e t t i n g me pursue my i n t e r e s t i n a " f i s h y " s u b j e c t . I would a l s o l i k e t o e x p r e s s my a p p r e c i a t i o n t o the members of my committee, Dr..W.S. Hoar and Dr. . J.E. P h i l l i p s , f o r t h e i r encouragement and g u i d a n c e , and f o r t h e i r h e l p i n t h e f i n a l s t a g e s of t h i s m a n u s c r i p t . I would a l s o l i k e t o thank the f o l l o w i n g people f o r t h e i r a s s i s t a n c e : Ms. S u z i n Crosby f o r d o i n g t h e h i s t o l o g y , Mr. C o l i n P a r k i n s o n f o r s o l v i n g many t e c h n i c a l problems, Norman S t a c e y , C h r i s K r e i s and B i l l H u l b e r t f o r many f r u i t f u l d i s c u s s i o n s and t e c h n i c a l s u g g e s t i o n s . I w i l l never f o r g e t Pamela Mace, M a r i a Weston, A d r i e n n e Peacock, Mary H u r l b u r t , Sue P l a t h , John B a i l e y and many o t h e r of t h e g r a d u a t e s t u d e n t s i n Z o o l o g y , f o r s h a r i n g the ups and downs o f grad u a t e s t u d e n t l i f e . F i n a l l y , I would l i k e t o d e d i c a t e my t h e s i s t o my p a r e n t s , Mary and E r n e s t B o u f f a r d , and I would l i k e t o thank them f o r t h e i r u n t i r i n g s u p p o r t and encouragement t h r o u g h o u t my s t u d i e s . 1 1 GENERAL INTRODUCTION 1_-_J[ HISTORICAL BACKGROUND In 1930, K u r z r o k and L i e b f i r s t d e s c r i b e d the p h y s i o l o g i c a l a c t i o n s of p r o s t a g l a n d i n s (PG). They had observed t h a t human s e m i n a l f l u i d had an e f f e c t on i s o l a t e d human u t e r i n e s t r i p s . R e l a x a t i o n o c c u r r e d i n t h e u t e r i from f e r t i l e women, but s t i m u l a t i o n o c c u r r e d i n t h e u t e r i n e s t r i p s from women w i t h complete or l o n g - s t a n d i n g i n f e r t i l i t y . G o l d b l a t t (1933) and Von E u l e r (1934, 1935, 1936, 1938, 1939) i n d e p e n d e n t l y demonstrated t h e p r e s e n c e of an a c t i v e p r i n c i p l e i n s e m i n a l plasma which s t i m u l a t e d smooth muscle and lowered b l o o d p r e s s u r e i n r a t s . T h i s substance r e a c t e d d i f f e r e n t l y from o t h e r n a t u r a l l y o c c u r r i n g s u b s t a n c e s a l r e a d y known. T h i s new compound was c a l l e d p r o s t a g l a n d i n , because o f i t s o c c u r r e n c e i n e x t r a c t s from t h e p r o s t r a t e and v e s i c u l a r g l a n d . However, E l i a s s o n (1959) l a t e r found t h a t t h e s e m i n a l v e s i c l e was the main s i t e o f PG p r o d u c t i o n , and net the p r o s t a t e . In 1934, Von E u l e r found t h a t the a c t i v e p r i n c i p l e i n the s e m i n a l v e s i c l e f l u i d c o u l d be e x t r a c t e d f r c m an a c i d s o l u t i o n w i t h l i p i d s o l v e n t s ; however, i t was a l s o s o l u b l e i n an a l k a l i n e medium. T h i s s i m p l e p u r i f i c a t i o n s t e p suggested t h a t the p r i n c i p l e was o f an a c i d n a t u r e , and had p r o p e r t i e s s i m i l a r t o those o f the f a t t y a c i d s . At Von E u l e r ' s s u g g e s t i o n , Bergstrom (1949) c o n f i r m e d t h e f a t t y a c i d n a t u r e of t h e p r o s t a g l a n d i n , and r e a l i z e d t h a t more than one u n s a t u r a t e d h y d r o x y - a c i d was p r e s e n t 2 i n the sheep seminal f l u i d . In 1957, prostaglandins were f i n a l l y i s o l a t e d in a c r y s t a l i z e d form by Bergstromm and S j o v a l l . These substances were called PGF and PGE. Bergstromm's group went on to d i f f e r e n t i a t e and i s o l a t e 13 other substances, a l l of which were based on the parent molecule, prostanoic acid. Subsequently large scale biosynthesis was successful, and prostaglandin research advanced rapidly into many areas of physiological, medical, pharmacological and b i o l o g i c a l inquiry. J L 2 NOMENCLATOEE AND STRUCTURE The term prostaglandin has remained i n the l i t e r a t u r e although i t i s a misnomer. Variations on the term prostaglandin were introduced and include both natural compounds and synthetic analogues. The l e t t e r s E and F were added to d i f f e r e n t i a t e between the two compounds which separated out when an extraction was peformed with an ether phosphate buffer solution. The E substance was found in the ether f r a c t i o n and the F i n the phosphate buffer ("fosfat" in Swedish). The A and B designate the respective derivatives of an acid or basic treatment of PGE. The stucture of the main prostaglandins and of the parent molecule, prostanoic acid, i s give i n Figure 1. The various prostaglandins d i f f e r only s l i g h t y in t h e i r structure, by modifications i n two areas of the molecule - the side chains and the cyclopentane r i n g . The l e t t e r s A, B, C, E or F indicate the changes i n the cyclopentane ri n g , whereas the numeral subscript denotes the number and position of double bonds i n the side 3 FIGUEE 1: Molecular structure of various prostaglandins and t h e i r parent molecule, prostanoic acid. 5 chain. The configuration of the naturally occurring prostaglandins i s r i g i d . The A or B are only used when re f e r i n g to PGF, and they designate the configuration at C 9; however, only the alpha series occurs naturally. There exists a number of derivatives and s t r u c t u r a l variants of prostaglandins, and these may be PG metabolites or synthetic compounds. In these cases, they are usually named afte r t h e i r closest s t r u c t u r a l r e l a t i v e , either the PG precursors, prostanoic acid, or the prostaglandins. Several of the PG precursors, such as the endoperoxides, the thromboxanes, and prostacyclin, i t s e l f , are potent yet unstable compounds. However, t h e i r structure and nomenclature w i l l not be outlined here. I i i OCCURRENCE AND RELEASE Host of the work concerning prostaglandin synthesis has been done on mammals, and almost a l l tissues analysed have been shown to produce PGs. The f i r s t account of PGs i n invertebrate tissues was i n 1969, when Weinheimer and Spraggins reported two isomers of PGA2 in the gorgonian c o r a l , Plexaura homonalla. Later, Light and Samuelsson (1972) demonstrated two more isomers of PGE2 i n the same animal. The f i r s t i n d i c a t i o n of PG production i n non-mammalian vertebrates was demonstrated by Christ and Van Dorp (1972); these workers found PG synthetase a c t i v i t y in many animal tissues, including those of birds, f i s h and amphibians..In 1972, a general survey was performed by Bito who studied the uptake of l a b e l l e d PGs i n both invertebrate and vertebrate tissues. He 6 found that marine bivalves could accumulate tr i t i a t e d - P G in most of their tissues and that other marine invertebrates could do so to a lesser extent. In addition, he showed that two species of elasmobranchs, and f i v e marine teleosts could concentrate tritiated-PG in the choroid plexus and the l i v e r . However, t h i s study did not demonstrate that the tissues produced PG, but only that they could incoporate them into t h e i r systems. ' There i s much variation in the occurrence and concentration of a s p e c i f i c prostaglandin i n different tissues. As a rule, the concentration i s usually i n the ng to pg range for each gram of tissue, or m i l l i l i t e r of f l u i d . One exception i s seminal f l u i d , where the greatest variety of prostaglandins are found, and i n the greatest concentration (approximatly 134ug/ml: Cenedella 1975) . l i i i BIOSYNTHESIS The chemical resemblance of prostaglandins to arachidonic acid suggested that i t could be a precursor of the prostaglandins. The f i r s t evidence that t h i s was true came when VanDorp and Bergstromm (1964) found that homogenates of sheep vesicular gland incubated with arachidonic acid could generate PGE2. The enzyme complex concerned was c a l l e d prostaglandin synthetase and appeared to be membrane-bound in the micrcsomal fr a c t i o n of the c e l l . The synthetic pathway involved several steps and was controlled by a feedback mechanism. Prostaglandin biosynthesis has several i n h i b i t o r s notably acetyl s a l y c i c acid (asperin) and indomethacin. These i n h i b i t o r s 7 appear to block the formation of c y c l i c p r o s t a g l a n d i n d e r i v a t i v e s at the enzyme l e v e l , r a t h e r than i n t e r f e r i n g with the a v a i l a b i l i t y of p r e c u r s o r a c i d s . T o t a l chemical s y n t h e s i s of PGs has been acheived f o r a l l the n a t u r a l l y o c c u r r i n g PG groups, and v a r i o u s " u n n a t u r a l " s t e r i o i s o m e r s have a l s o been produced. Although b i o s y n t h e s i s has been used s u c c e s s f u l l y t o produce PGs, chemical s y n t h e s i s i s more e f f i c i e n t and a l s o enables the p r e p a r a t i o n of PG analogues which have not been found i n nature. i i 5 . METABOLISM The main s i t e s of PG breakdown i n the mammal are the lungs, l i v e r , p l a c e n t a , kidney, spleen and adipose t i s s u e . Marazzy and Anderson (1974) d e s c r i b e d a 90 - 100% decrease cf PGE and PGF a f t e r passage through the lung. Although many enzymes are i n v o l v e d i n PG i n a c t i v a t i o n , the main one i s 15-hydroxy-PG-dehydrogenase. T h i s enzyme has been found i n the lung, as w e l l as i n most of the t i s s u e s mentioned above ( C u r t i s - P r i o r 1976). As the name of the enzyme suggests, i t c a t a l y s e s the dehydrogenation of the h y d r o x y l group at the C-15 p o s i t i o n thus g r e a t l y reducing the potency of the PG molecule. A f t e r t h i s , s e v e r a l other enzymes continue the process of PG degradation. 8 l-_6 MODE OF ACTION Despite much research in t h i s area, the precise mechanism of prostaglandin action i s s t i l l far from clear. However, i t i s becoming more evident that PGs act at the molecular l e v e l via changes in the cyclonucleotides. Some of the evidence that both c y c l i c AMP and c y c l i c GMP are involved in the action of PGs w i l l be outlined b r i e f l y . Kuehl et a l (1972) discovered that the PGEs seemed to be the most e f f e c t i v e of the PGs i n increasing c y c l i c AMP l e v e l s i n the intact mouse ovary. This led to the discovery of membrane bound receptors i n lipocytes which had a p r e f e r e n t i a l a f f i n i t y for PGEs (Kuehl and Humes 1972). The PGFs did not seem to stimulate c y c l i c AMP production to the same extent, and their a f f i n i t y for the PGE receptors on the lipocytes was very weak. These findings led some investigators to look at the p o t e n t i a l relationship between PGF and c y c l i c GMP. Powell et a l (1974) discovered a PGF-sensitive receptor in ovine and human corpora lutea. Moreover, Dunham (1973) demonstrated an increase in the c y c l i c GMP/cyclic AMP r a t i o following application of PGF2^ to ovine and bovine veins, and a decrease following application of PGE2. These findings t i e i n well with observations of opposing actions of PGE and PGF on smooth muscle. ls.2 IIVOJ.VEMENT OF PROSTAGLANDINS IN DIFFERENT AREAS As stated previously, prostaglandins are found i n most tissues, and t h e i r physiological actions are manifested i n many areas. An overview of some of the research i n the major areas 9 w i l l be o u t l i n e d here, and some aspects w i l l be d i s c u s s e d i n more d e t a i l . 1.7.1 C i r c u l a t i o n P r o s t a g l a n d i n s seem to be i n v o l v e d i n many aspects of c i r c u l a t o r y homeostasis such as blood pressure, p e r i p h e r a l c i r c u l a t i o n and h e a r t r a t e . G e n e r a l l y the PGEs decrease blood pressure i n mammals; PGE1 i s the most potent f o l l o w e d by PGE2 and PGE3. This decrease i s brought about by p e r i p h e r a l v a s o d i l a t i o n . However, PGE1 i n c r e a s e s c a r d i a c output, and r a i s e s coronary flow; t h i s normally c o i n c i d e s with a f a l l i n blood pressure. The PGEs have l i t t l e or no e f f e c t on the r a t e and f o r c e of the i s o l a t e d h e a r t . However, they do seem to have a hyp e r t e n s i v e e f f e c t i n the kidney, and M c G r i f f e t a l (19 74) has suggested that PGE2, i n p a r t i c u l a r , c o u l d r e g u l a t e r e n a l blood flow. The PGFs are mainly concerned with non-vascular smooth muscle, although they do have some e f f e c t on the blood v e s s e l s . In g e n a r a l , when they do i n f l u e n c e v a s c u l a r muscle, they are v a s o c o n s t i c t o r s , but there i s much s p e c i e s v a r i a t i o n i n these s t u d i e s . O v e r a l l , the PGFs do not appear t o play a major r c l e i n c i r c u l a t o r y p h y s i o l o g y . 10 1.7.2 Nervous tissue Prostaglandins are present i n the central nervous system, as well as i n the cerebrospinal f l u i d , and PGF seems to predominate i n both (Coceani and Wolf 1965) . Many studies suggest that prostaglandins are implicated i n both the central and peripheral nervous systems. The PGEs appear to be implicated in sympathetic neurotransmission (Hedgvist 1970) as well as i n the mechanisms of thermoregulation and fever (Milton-and Wendant 1970). Administration of PGEs to rats w i l l increase body temperature very rapidly, and i t i s believed that the PGEs act as mediators of pyrogen-induced hyperthermia. Both PGE 1 and PGF20< seem to a f f e c t the brain stem cardioregulatory centers (Kaplan et a l , 1969), and they may also be involved with the regulation of the release of p i t u i t a r y hormones (LeMaire et a l 1974) . PGE2 and PGF2<< are released peripherally following stimulation of both sympathetic and parasympathetic nerves, and the release appears to be post-synaptic (Hedgvist 1970). 1.7.3 Inflamation Inflamation i s a l o c a l tissue response to foreign s t i m u l i , such as bateria or viruses, various chemicals or u l t r a v i o l e t and infrared l i g h t . I t may also be a component of the autoimmune reaction. Many factors are involved i n inflamation and i t s associated symptoms, such as pain, oedema, erythema and heat. Histamine, bradykinin, serotonin, certain plasma globulins and PGs seem to play a role in the inflamatory process. Exactly how 11 they interact has not yet been elucidated. There have been many instances were PGs have been i d e n t i f i e d after induced inflamation ( W i l l i s 1969; Piper and Vane 1969; Eakins et a l 1972). PGE1 and PGF.2«< have been i d e n t i f i e d in the inflamatory exudate during carrageenin-induced inflamation ( W i l l i s 1969) . In addition, PGE1 and PGE2 have been associated with the pain and inflamation of a r t h r i t i s . For t h i s reason, a s p i r i n and indomethacin, (PG i n h i b i t o r s ) , are e f f e c t i v e i n r e l i e v i n g the symptoms of a r t h r i t i c inflamation and pain. The exact role of PGs during inflamation i s not known. However, i t has been postulated that they may potentiate vascular permeability by inducing leakage at the c o l l e c t i n g venules (Kaley and Weiner 1971). 1.7.4 Prostaglandins i n mammalian reproduction The female reproductive cycle in mammals consists of an i n t r i c a t e series of endocrine steps leading from maturation of the oocyte (s) to ovulation^ This cycle repeats i t s e l f i f the egg (s) i s not f e r t i l i z e d . However, i f f e r t i l i z a t i o n occurs and implantation i s successful, another series of hormonal events takes place. The main organs involved in t h i s cycle are the hypothalamus, p i t u i t a r y , ovary and, to some extent, the adrenals. During pregnancy, the placenta takes over producton of most of the pregnancy-supporting hormones. The p r i n c i p a l reproductive hormones are l u t e i n i z i n g hormone (LH) and f o l l i c l e stimulating hormone (FSH) from the p i t u i t a r y , their respective releasing factors from the hypothalamus, and the steroids from 12 the ovary and the adrenals. When implantation occurs, chorionic gonadotropin i s produced from the placenta and i t maintains steroid production in the corpus luteum of the ovary. The discovery of the involvement of prostaglandins i n female reproduction has added a new dimension to the understanding of t h i s system. The importance of prostaglandins has been well documented in mammals, yet many questions remain unanswered, p a r t i c u l a r l y i n regard to t h e i r s p e c i f i c roles i n normal reproductive physiology. Prostaglandins have been i d e n t i f i e d i n the reproductive organs and f l u i d of both male and female mammals. In the female, PGs have been found in the ovary, uterus, menstrual f l u i d and amniotic f l u i d . They seem to be involved i n ovulation, l u t e o l y s i s , menstruation, p a r t u r i t i o n and spontaneous abortion. They are present in the t e s t i s and accessory g e n i t a l glands of male mammals, and human seminal f l u i d i s the richest source of PGs yet known. They appear to be implicated i n the processes of erection, ejaculation^ sperm mo t i l i t y and morphology. The following discussion w i l l attempt to cover some of the facets of PGs involvement i n these major areas of male and female reproductive physiology. 1.7.4.1 Prostaglandins in female reproduction 13 A) ovulation As the ovarian f o l l i c l e matures, the PG lev e l s within i t increase (LeMaire et a l 1975a). However, the exact relationship between PGs and oocyte maturation i s not yet clear. Studies have shown that in the ovary, i n the presence of PG i n h i b i t o r s , both the growth and maturation of the f o l l i c l e proceed normally, but that the f o l l i c l e s f a i l to rupture (Linder et a l 1974). This suggested that PGs were not e s s e n t i a l for oocyte maturation but may be important i n the release of the ovum. The necessity for PGs i n the expulsion of the ovum was futher supported by the following findings: 1) PGEs and PGFs both increase a f t e r HCG i n j e c t i o n i n rabbits (LeMaire et a l 1973), 2) indomethacin could block HCG induced ovulation (Yang 1973) , 3) exogenous administration of PGF2 C <could overcome the indomethacin block (Diaz-Infante et a l 1974) . B) maintenance of the corpus luteum Prostaglandins appear to play an important role in the control of the corpus luteum. However, the exact r o l e i s unclear. In v i t r o work indicates that PGs promote steroidogenesis i n most cases. However, in vivo, they are l u t e o l y t i c in most species. PGF20<appears to be the most potent, althought PGE2 i s also e f f e c t i v e in promoting l u t e o l y s i s . Goldberg and Eamwell (1975) have discussed three di f f e r e n t mechanisms for the l u t e o l y t i c effects of the PGs. Their f i r s t proposal i s that PGs i n t e r f e r e with the hormonal support of the corpus luteum. The second i s that PGs could have a dire c t l y t i c 14 e f f e c t on the s t e r o i d p r o d u c i n g c e l l s i n t h e ovary- T h i r d l y , t h e y suggest t h a t PGF2 o t c o u l d i n d u c e h y p o x i a i n the o v a r y by c o n s t r i c t i n g t h e o v a r i a n b l o o d v e s s e l s . The s t u d i e s so f a r have not d e l i n e a t e d w h ich, i f any, of the above e x p l a n a t i o n s i s c l o s e s t t o r e a l i t y . I t i s q u i t e p o s s i b l e t h a t the e f f e c t s o f PGs on the c o r p u s luteum are s p e c i e s dependant, making i t v e r y d i f f i c u l t t o a s c e r t a i n i f i n d e e d any one mechanism a p p l i e s . C) m o t i l i t y of the r e p r o d u c t i v e t r a c t The smooth muscle s t i m u l a t i n g p r o p e r t i e s of PGs would suggest t h a t they c o u l d i n f l u e n c e the m o t i l i t y o f the r e p r o d u c t i v e t r a c t . Indeed , i t i s w e l l e s t a b l i s h e d t h a t the PGEs i n h i b i t spontaneous c o n t r a c t i o n s of the f a l l o p i a n t u b e , whereas PGFs are s t i m u l a t o r y . PGs c o u l d c o n t r o l t u b a l c o n t r a c t i l i t y and ova t r a n s p o r t ; T h i s i s v e r y i n t e r e s t i n g , i n l i g h t o f t h e h i g h c o n c e n t r a t i o n o f PGs i n t h e semen. PGs have a l s o been shown t o a f f e c t u t e r i n e m o t i l i t y . The e x t e n t of s t i m u l a t i o n i s dependant on t h r e e f a c t o r s : 1) the mode of a d m i n i s t r a t i o n , 2) the r e p r o d u c t i v e s t a t u s o f t h e p e r s o n , and 3) which PG i s g i v e n ; I n f a c t , PGs have been p o s t u l a t e d t o be i n v o l v e d i n p a r t u r i t i o n , and have been used c l i n i c a l l y t o i n d u c e p a r t u r i t i o n and a b o r t i o n . 1 5 1.7.4.2 Prostaglandins i n males Depite the abundance of PGs i n the male reproductive system, r e l a t i v e l y l i t t l e research has been done in t h i s area. A comprehensive review on PGs i n male reproductive physiology has been written by Cenedella (1975). PGs are present i n r e l a t i v e l y high concentration in the semen and i n lower leve l s i n the accessory reproductive tissues. In the seminal plasma of man, PGEs are predominant over the other prostaglandins, with an average concentration of 50 ug/ml. The PGF levels are lower at approximatly 7 ug/ml. The PGA and PGB are, in combination, about the same concentration as the PGEs. Bygdeman et a l (1970) noted that seminal PGE lev e l s were lower in men who were unaccountably i n f e r t i l e . I t has been postulated that PGs could act i n t e r n a l l y or externally. Internally, PGs could influence sperm physiology, steroidogenesis, or the smooth muscle of the accessory sex glands. Externally, they could act upon the female reproductive t r a c t , as was mentionned previously. Unfortunately, very few studies have dealt with these questions. In males, PGs could play a r o l e i n regulating gonadotropin release in the p i t u i t a r y . Linder et a l (1974) discussed the rapid increase in LH following the administration of PGF20( to the male r a t . PGs may also influence steroidogenesis i n the male. Bartke et a l (1973) demonstrated a s i g n i f i c a n t decrease i n testosterone lev e l s following administration of PGs to mice. PGE2 was also e f f e c t i v e i n i n h i b i t i t i n g testosterone biosynthesis (Sakena et a l 1973) . As suggested i n the female, the action of PGs may be due to a decrease in blood flow to the 16 t e s t i s . The scarcity of information on the involvement of prostaglandins in the male reproductive system makes i t very d i f f i c u l t to asses their potential importance at t h i s time. 1.7.5 P r o s t a g l a n d i n s i n f i s h L i t t l e r e s e a r c h has been done on p r o s t a g l a n d i n s i n non-mammalian a n i m a l s . I n view of t h e demonstrated i m p o r t a n c e o f these s u b s t a n c e s i n so many f a c e t s o f p h y s i o l o g y , i t i s l i k e l y t h a t t h e r e i s some p h y l o g e n e t i c o v e r l a p i n t h e i r f u n c t i o n i n mammalian and non-mammalian systems. However, some r e c e n t r e s e a r c h on p r o s t a g l a n d i n s i n f i s h has y i e l d e d v e r y i n t e r e s t i n g r e s u l t s . Abramowitz and C h a v i n (1973) demonstrated t h a t some of the PGs were v e r y e f f e c t i v e i n e l i c i t i n g melanocyte d i s p e r s i o n i n t h e dermal melanophcres of t h e g o l d f i s h , C a r a s s i u s a u r a t u s L. They found t h a t PGB1 and PGB2 produced t h e g r e a t e s t r e s p o n s e , f o l l o w e d by PGE1 and PGE2; however, PGFI^ and PGF2 C < were o n l y s l i g h t l y e f f e c t i v e . They suggested t h a t the PGs were a c t i n g v i a the c y c l i c AMP system. I n t h a t same y e a r , Nomura, Ogata and I t o (1973), u s i n g v a r i o u s c h r o m a t o g r a p h i c t e c h n i q u e s , were a b l e t o i d e n t i f y 3 p r o s t a g l a n d i n s * PGFV PGE1, and PGE2 i n t h e t e s t e s of t h r e e s p e c i e s o f f i s h : t h e f l o u n d e r , P a r a l i c h t h y s o l i v a c e u s , the b l u e f i n t u n a , Thunnus thynnus and the chum salmon, Oncorhynchus k e t a . Two of t h e s e a u t h o r s l a t e r r e p o r t e d the p r e s ence of PGE2 i n t h e i n t e s t i n e of t h e s h a r k , T r i a k i s s c y l l i a (Ogata and Nomura 1975). Peyraud-Waitzenegger e t a l (1975) a l s o observed t h a t PGE2 17 had both cardiovascular and ventilatory e f f e c t s i n the carp, Cyprinus carpio L., which they attributed to the a c t i v a t i o n of adenyl cyclase by PGE2. The f i r s t research on reproduction concerning prostaglandins in f i s h was done be Stacey and Pandey (1975). They reported that indomethacin could block HCG-induced ovulation and spawning behaviour i n the female goldfish, Carassius auratus . In addition* they found that exogenous administration of PGF2*, PGE 1 or PGE2 could overcome the indomethacin block and induce ovulation and subsequent spawning. Their r e s u l t s suggested that prostaglandins could play a key r o l e in ovulation i n the goldfish.. This work was supported by the findings of Jalabert and Szollose (1975) who demonstrated that PGF2^ could induce in v i t r o ovulation of mature trout oocytes. However, they found that PGE2 had no v i s i b l e effect on the system. In a review on in v i t r o oocyte maturation and ovulation i n teleost f i s h , Jalabert (1975) proposed two s i t e s for the action of PGF20(on the oocyte: 1) the smooth muscle-like c e l l s located in the theca and 2) non-muscle c e l l s containing a c t i n - l i k e filaments. This proposition was made on the basis of observations by Szollose and Jalabert (unpublished data), that: 1) the response to PGF20(,was in h i b i t e d when calcium was absent i n the incubation medium or when calcium i n f l u x was i n h i b i t e d , and 2) that p a r t i a l contraction occurred with PGF2^ after i n h i b i t i o n of smooth muscle. Recently Goetz and Theofan (1979) have completed a s i m i l a r study using the perch, Perca flavesceus . However* they reported that EGE1 and PGE2, as well as PGF2* could stimulate i n v i t r o ovulation of mature 18 oocytes, and that PGE2 was the most potent of the three. The e f f e c t of prostaglandins on p i t u i t a r y gonadotropin (GTH) release was studied by Peter and B i l l a r d (1976) . They observed that in j e c t i o n s of PGF2^and PGE2 into t h i r d v e n t r i c l e of the goldfish suppressed serum GTH secretion and that PGE1 had no effect- If indeed prostaglandins play a role i n the i n i t i a t i o n of ovulation, these findings would suggest that they would not act by influencing gonadotropin secretion prior to ovulation. Singh and Singh (1977) reported that prostaglandins may affect thyroid a c t i v i t y in f i s h . They observed that administration of PGE 1 and PGF2oCto the c a t f i s h , Heteropneust.es f o s s i l i s , resulted i n a reduction of thyroid a c t i v i t y which was coincedent with a decrease i n TSH i n the p i t u i t a r y and an increase i n TSH le v e l s i n the blood. Kuo and Watanabe (1978) have investigated i n v i t r o changes in c y c l i c AMP levels following the administration of PGE2 and PGF2<< to mature oocytes from the mullet, Muqil cephalus , both prostaglandins increased the l e v e l of c y c l i c AMP i n the occyte, however, PGE2 was the most potent i n t h i s respect. This evidence suggests that the mode of action of the prostaglandins in f i s h i s similar to that observed in mammals. 1^8 STATEMENT OF THE PROBLEM Increasing information on prostaglandins i n f i s h suggests that they are implicated in the process of gonadal development and/or f i n a l maturation. In an attempt to c l a r i f y t h i s 19 r e l a t i o n s h i p , the changes i n t h r e e p r o s t a g l a n d i n s were measured i n male and female g o l d f i s h . C a r a s s i u s a u r a t u s , d u r i n g t h r e e gonadal e v e n t s : m a t u r a t i o n , o v u l a t i o n and s p e r m i a t i o n . P r i o r t o t h e p r e s e n t s t u d y , t h e r e was no e v i d e n c e f o r the p r e s ence o f p r o s t a g l a n d i n s i n g o l d f i s h t i s s u e s . P r o s t a g l a n d i n s have, however, been d e t e c t e d i n the t e s t i s of some f i s h (Nomura Ogata and I t o 1973); a l t h o u g h t h e r e has not even been an a t tempt t o measure them i n the o v a r i e s . 20 2 GENERAL TECHNIQUES 2..J FISH MAINTENANCE Goldfish, Carassius auratus, of the common comet variety were purchased from Grassy Porks Fisheries Company, Ma r t i n s v i l l e , Indiana. The f i s h were kept i n outdoor aquaria, i n flowing dechlorinated water, at 12-15°C and fed trout p e l l e t s two or three times a week ad libitum. Dead f i s h were removed immediately from the stock. Shipments of f i s h were kept for at least one month before experimentation. 2^2 REAGENTS AND GLASSWARE Only spectroguality or r e d i s t i l l e d reagents were used for extraction, chromatography and radioimmunoassay (RIA). Reagents and glassware were precooled to 5° C and were kept on ice throughout the experiment. To avoid adherance of PGs to the glass, a l l glassware exposed for long periods to the tissues was s i l i c o n i z e d ( S i l i c l a d , Clay Adams, N.Y.). Pipettes and micropipettes were not s i l i c o n i z e d , as no difference was found in r a d i o a c t i v i t y after measuring 3H-PGF20t and 3H-PGE2 with both s i l i c o n i z e d and non-siliconized micropipettes. A l l glassware was presoaked in chromic acid or i n a bacterioside-fungicide detergent (Mucosit, Mrez Co. Ltd.) before noriral washing. The formulae for the various experimental solutions are given i n the Appendix. The phosphate buffer and the extraction solution were prepared just p r i o r to use. The phosphate buffer 21 contained an a n t i o x i d a n t , p - a m i n o s a l i c y c l i c a c i d , to reduce the breakdown of PGs. • 2^3 COLLECTION AND EXT1ACTI0N OF PROSTAGLANDINS F i s h were a n e s t h e t i z e d i n a 0.02% s o l u t i o n of t r i c a n e methane sulphonate (MS 222, Sandoz) and weighed p r i o r to sampling. The f i s h were handled i n a moist paper towel to prevent e x c e s s i v e d r y i n g . 2.3.1 E x t r a c t i o n from plasma Blood was taken v i a the caudal v e s s e l using a 3.0 ml d i s p o s a b l e p l a s t i c s y r i n g e with a 23 or 25 gauge needle, depending on the s i z e of the f i s h . The s y r i n g e contained 0.1 ml of a sodium heparin s o l u t i o n (heparin, sodium s a l t , Sigma #H-3125, Sigma, S t - L o u i s , Mo) made i n phosphate b u f f e r s a l i n e (2 IO/ml) with p - a m i n o s a l i c y c l i c a c i d (PBS-A) . The blood was put i n t o a s i l i c o n i z e d t e s t tube and c e n t r i f u g e d a t 2500g, a t 4° C f o r 10 minutes (Refrigerated-Automatic c e n t r i f u g e , S e r v a l l Co.). The plasma was removed and measured with a d i s p o s a b l e p i p e t t e , and placed i n a 15 ml c o n i c a l c e n t r i f u g e tube c o n t a i n i n g 3.0 ml of petroleum e t h e r ; t h i s removed the n e u t r a l l i p i d s . A f t e r removal of the ether phase, 3.0 ml of an e x t r a c t i o n s o l u t i o n ( e t h y l a c e t a t e : isopropanol:0.2N HCl, 3:3:1) was added to the plasma and mixed thoroughly. To t h i s mixture, 3.0 ml of d i s t i l l e d water and 2.0 ml of e t h y l acetate were added and mixed; the mixture was then c e n t r i f u g e d at the medium speed 22 setting (in an International Model HN IEC: International Eguipment Company, Needham Heights, Mass.), at 4 0 C for 10 minutes . The organic layer was coll e c t e d i n a la b e l l e d test tube and kept on i c e . An additionnal 3.0 ml of ethyl acetate was added to the centrifuge tube, mixed, and centrifuged as before. This second organic layer was combined with the f i r s t and evaporated under a stream of nitrogen at 40°C. The residue was covered with Parafilm (Parafilm, American Can Co., Dixie/Marathon, Greenwich, CT 06830), and kept at -30° C u n t i l the chromatography step. 2.3.2 Extraction from the gonad The gonad was removed and weighed i n cold PBS-A. A sample of approximatly one gram was dissected out quickly, and homogenized over ice in 1.0 ml PBS-A and 3.0 ml of the extraction solution given above (Thomas glass homogenising tube, #A30677, Philadelphia, Pa. and mechanized using a Cole-Parker motor, model E2E64, Cole-Parker Istruments and Equipment Co., Chicago, I I . ) . The homogenate was then added to 2.0 ml ethyl acetate and 3.0 ml d i s t i l l e d water i n a 15 ml conical centrifuge tube, and processed i n the same fashion as was described for plasma. Some of the remaining tissue was used for histology. 23 2^4 HISTOLOGY Samples of the ovary and the t e s t i s were taken from each f i s h and fixed in either Bouin's solution, or i n a solution of 2% gluteraldehyde with 1% formalin i n PBS. Following f i x a t i o n , the tissues were dehydrated and embedded in pa r a f f i n ; and sectioned at 7um. The sections were stained with Mallory's T r i p l e Stain or Hematoxylin-Eosin, for determination of sexual maturity. 2^5 COLUMN CHROMATOGRAPHY The extracted samples contained a mixture of prostaglandins. S i l i c i c acid chromatography was performed cn a l l the samples in order to separate out the di f f e r e n t PG groups from each sample. The basis for t h i s separation on s i l i c i c acid i s that the PGs have different p o l a r i t i e s , and by changing the po l a r i t y of the eluting solvents i t i s possible to separate the three major PG groups. A modification of the Jaffe and Eehrman (1974) technique was used, and w i l l be described here. The ef f i c i e n c y of the extraction procedure, and of the column chromatography elution w i l l be outlined i n the section on Preliminary Work. The following modifications of the Jaffe and Behrman's method were used throughout these studies. 24 2.5.1 Preparation of the column S i l i c i c acid (Sil-A-200, Mesh 60-200, lot*115C-0185, Sigma, St-Louis, Mo.) was f i r s t suspended in d i s t i l l e d water and decanted several times to remove the f i n e s . I t was then dried at 115*C and maintained at t h i s temperature u n t i l use. A 0.5 g weight of s i l i c i c acid was placed i n a round pouring device which consisted of a 20ml capacity glass vessel with a 1 cm hole on top and a 1.5 cm long spout at a 130 angle (see Figure 2b). The column consisted of a 9 cm s i l i c o n i z e d Pasteur pipette plugged with glass wool. A few drops of benzene:ethyl acetate in a 6:4 volume r a t i o (refered to as E:EA) were added to the s i l i c i c acid to make a slurry. The spout f i t t e d the top of the column and by adding B:EA dropwise to the top of the pouring device, the contents were e a s i l y transferred to the column; The column was then f i t t e d with a piece of teflon tubing, making i t possible to control the flow with a clamp. The column was then washed with approximately f i v e m i l l i l i t r e s of B:EA. The s i l i c i c acid i s a white powder which turns translucent blue when B:EA i s added. It becomes more opaque when water or methanol are present. If the column was not blue after the f i r s t wash, then additionnal B:EA would be applied u n t i l the column became translucent. 2.5.2 Gradient elution apparatus Two 100 ml graduated cylinders were adapted with spouts at their bases (Figure 2a). Cylinder B had two spouts: the f i r s t spout connected with cylinder A by a small section of teflon 25 tubing; the second spout was the outlet for the eluting solvent. Clamps were used to control the flow between the two cylinders and the outflow. Cylinder B contained 31 ml of B:EA, and cylinder A held a mixture of 15 ml methanol (M) and 15 ml B: EA. Both had magnetic s t i r r i n g bars, and were held on a magnetic s t i r r e r . The sample was thawed on i c e and then s c l u b i l i z e d f i r s t i n 0.2 ml of benzene:ethyl acetate:methanol (6:4:1), and a further 0.8 ml of B:EA was added to the sample and vortexed. The one ml of f i n a l extract was added to the top of the column and l e f t to s e t t l e for a few seconds. The sides of the column were tapped gently to ensure an even interface. The sample was run onto the column and stopped quickly so as to prevent the column from running dry. 1.0 ml of B:EA was used to rinse the tube containing the sample. This 1.0 ml rinse was then gently placed on the column and the gradient e l u t i o n started. The gradient e l u t i o n protocol was as fellows: with the valve between the cylinders closed, 2.0 ml B:EA were delivered to the column. The valve between the cylinders was opened, and 3.0 ml of a mixture were delivered to the column, while the contents of the cylinders were allowed to mix. Whenever the valve was open, the magnetic s t i r r e r was switched on to insure proper mixing of the two solutions. The valve was then closed and a further 7.0 ml were delivered to the column. The valve was opened for the f i n a l 6 ml of eluent. With t h i s "on-off" gradient elution technique i t was possible to control the methanol content i n the outflow to the column. Eluates were collected with an automatic f r a c t i o n c o l l e c t o r 26 FIGURE 2: Chromatographic a p p a r a t u s . a) g r a d i e n t e l u t i o n a p p a r a r u s . b) g l a s s p o u r i n g d e v i c e f o r p r e p a r a t i o n of s i l i c i c a c i d columns. 27 a ) M a g n e t i c S t i r r e r B o 4K D r o p C o u n t e r CUD F r a c t i o n C o l l e c t o r b ) 28 (LKB, Ultrovac, Ltd.). The fractions were collected and measured by volume using a drop counter. The f i r s t four m i l l i l i t r e s contained the PGB and PGA groups and the next eight m i l l i l i t r e s contained the PGEs, whereas the f i n a l six m i l l i l i t r e s eluted the PGF f r a c t i o n . The f r a c t i o n s were evaporated tc dryness at 40 C under a stream of nitrogen, and then covered with Parafilm, and kept at -30° C u n t i l the assay. 2^6 RADIOIMMUNOASSAY RIA k i t s for the measurement of prostaglandins F£, E1 and E1 were obtained from C l i n i c a l Assays Inc., Cambridge, Mass. A l i s t of the reagents included i n the k i t i s given i n the Appendix. The assay was performed i n disposable polypropylene tubes (Falcon #2053, 12x75 mm). Duplicates were carr i e d but for both the standard curve and the unknown samples and a standard curve was done for each assay. The basic protocol outlined i n the k i t instructions was followed, except f o r a few steps which w i l l be noted below. 2.6.1 Sample preparation 2.6.1.1 PGF The samples were removed from the freezer and thawed i n an ice bath. -Then 1.4 ml of isogel t r i s buffer (ITB) were added to each tube by means of an egual volume dispenser (Repipet, Fisher 29 S c i e n t i f i c ) and vortexed several times to ensure that the sample was completely dissolved in the buffer. 2.6.1.2 PGB The same proceedure was followed as with PGF^, except that only 1.0 ml of ITB was added to each tube. 2.6.1.3 PGE An antibody toward PGE was not available. However, i t was possible to convert PGE to PGB, and then measure PGE by using the PGB antibody. The conversion was performed as follows: 1.0 ml of ITB was added to a l l the tubes and and mixed with 0.1 ml of 1N NaOH. The tubes were placed i n a b o i l i n g water bath for fi v e minutes, removed, and then cooled in an ice bath. The pH was measured by a microelectrode (Fisher S c i e n t i f i c Ltd.) and adjusted to approximatly pH 7 using 0.1 ml of 1N g l a c i a l acetic acid. Because of the small volume involved, i t was d i f f i c u l t to adjust the pH precisely, so a range of 6.8 to 7.8 was accepted. 2.6.2 Radioimmunoassay procedure The reagents were thawed on ice and the antibody , tracer, standards, rabbit normal serum (RNS) and goat anti - r a b b i t serum (GARS) were reconstituted with ITB and kept on ice. Working standards were prepared by making a series of 3-fold d i l u t i o n s s t a r t i n g with the stock standard. The f i n a l six solutions ranged 30 between 9.2 and 2400 PG per 100 u l for PGF 2^  and 16.4 and 4000 PG per ul for PGB1. The reagents were added to duplicate tubes following the protocol on page 178 i n the Appendix for PGF2^and on page 164 for PGE1 and PGB1 (the ITB was added by Repipet; the standards by disposable micropipettes; the unknowns by disposable glass pipettes; the tracer and antiserum by one or two ml glass pipettes). The tube was mixed gently a f t e r each addition (Vortex, Co Ltd). The tubes were then incubated i n a water bath at 37° C for 1.5 hours, then 0.1 ml of RNS and 0.1 ml of GARS were added immediatly to each tube and vortexed. The tubes were covered with Saran wrap (Dow Chemicals of Canada Limited, Toronto, Ont.) and put in a p l a s t i c bag and incubated for 20-24 hours (this step precipitates the antibody-antigen complex). After the incubation, the tubes were centrifuged at 4000g, at 4' C for 50 minutes. The supernatant from tubes #1 and #2 (see Appendix) were decanted in t o separate s c i n t i l l a t i o n v i a l s ; the r a d i o a c t i v i t y in these tubes i s the Total Counts (TC) . After decanting the supernatants from a l l the remaining tubes, into a waste container, the tubes were placed upside down in a rack, and lined with absorbant paper with a p l a s t i c backing. Each tube was car e f u l l y wiped with absorbant tissue twisted on the end of an applicator s t i c k . The precipitate was disolved i n 0.5 ml of 0.1N NaOH, then neutralized with 0.5 ml cf 0.1N HCl. This solution was decanted into a corresponding s c i n t i l l a t i o n v i a l , and the tube was shaken gently to insure maximal transfer. A t i l t i n g dispenser was used to transfer 10 ml of s c i n t i l l a t i o n f l u i d to each (LSC C o c k t a i l , #3-4986, J.T. Baker chemicals. 31 P h i l l i s b u r g , N.J., or. NEF-948 Riafluor, New England Nuclear, Lachine, Quebec.) v i a l . The v i a l s were l e f t to e q u i l i b r a t e for one hour and then counted on the ISOCAP/300 l i q u i d s c i n t i l l a t i o n counter (Nuclear Chicago) for 10 minutes. Two problems which can be encountered using BIA are the deterioration of reagents and the adsorption of proteins and small molecules to glassware and p l a s t i c s . The k i t reagents were lyophylized, kept at -30* C and were used only once, immediately after reconstitution. Adsorption problems were avoided by adding gelatin to the buffer. This non-binding protein not only prevents adsorption but also seems to enhance antibody uptake (Murphy and Marvin, in press). 2.6.3 Calculations The background count was the average counts per minute (cpm) , l e f t i n tubes #1 and #2 after removal cf the supernatant. This value represented the trapping of r a d i o a c t i v i t y i n the precipitate and was subtracted from the t o t a l cpm i n each v i a l . Quench correction was performed by the Channels Ratio Method. This consisted of running a series of standard solutions with a known amount of r a d i o a c t i v i t y and various amounts of quenching (radioactivity i n decomposition per minute or dpm). The e f f i c i e n c y of counting for the unknowns can be determined by the guench curve given as the A/B r a t i o (cpm i n channel A / cpm i n channel B) against the absolute e f f i c i e n c y of counting (total cpm/absolute dpm i n the standard). The f i n a l radioactive value i s i n dpm, and i s calculated by f i r s t subtracting the background 32 from each sample, and then d i v i d i n g t h i s v a l u e by i t s e f f i c i e n c y of counting. Fol l o w i n g the quench c o r r e c t i o n f o r each samrle, the b i n d i n g r a t i o can be c a l c u l a t e d by d i v i d i n g the dpms f o r the unknown sample by the average dpm i n the t o t a l bound (TB) tubes, (#3 and #4). The standard curve i s obtained by p l o t t i n g the b i n d i n g r a t i o s f o r each c o n c e n t r a t i o n of standard a g a i n s t the l o g a r i t h m of the c o r r e s p o n d i n g c o n c e n t r a t i o n . The b i n d i n g r a t i o s of the unknowns were then c a l c u l a t e d and the p r o s t a g l a n d i n l e v e l f o r each sample de f i n e d f o r each tube, averaged f o r the d u p l i c a t e s , and c o r r e c t e d f o r d i l u t i o n . The f i n a l v a l u e s are given i n pg per ml plasma or per g t i s s u e . The mean, standard d e v i a t i o n , standard e r r o r and the c o e f f i c i e n t of v a r i a r t i o n were c a l c u l a t e d f o r the d i f f e r e n t groups of animals and t i s s u e s by the use of an IBM 370 computer. To assess the q u a l i t y of the k i t ' s reagents, the b i n d i n g c a p a c i t y was estimated by c a l c u l a t i n g the r a t i o of T o t a l b i n d i n g (tubes #3 & #4) over T o t a l Counts (tubes #1 S #2). Any k i t with a r a t i o under 0.3 was not used. The normal range f o r the r a t i o was 0.4 to 0.5. 33 3 PRELIMINARY WORK INTRODUCTION The development of an accurate and reproducible method for the analysis and i d e n t i f i c a t i o n of prostaglandins i n f i s h tissue was c r u c i a l for t h i s study. The established techniques for PG assay had been developed on mammalian tissue; therefore, i t was e s s e n t i a l to v e r i f y these procedures using f i s h tissue. A selection of the most suitable separation and assay procedures was made following an analysis of the available methods. Therefore, prostaglandin methodology w i l l be reviewed b r i e f l y . The v e r i f i c a t i o n s and modifications made on these procedures w i l l be described in d e t a i l . 3^2 REVIEW OF METHODOLOGY Two major preparatory steps are usually performed before prostaglandins can be measured i n tissue. The f i r s t p u r i f i c a t i o n process i s an extraction with organic solvents. Salmon and Karim (1976) outlined the three main reasons for organic extraction: the process increases the s p e c i f i c i t y of the analysis, concentrates the PGs (thereby increasing assay s e n s i t i v i t y ) , and removes substances such as protein, and l i p i d s , which could i n t e r f e r e with the assay procedure. The second step i s the separation of PGs i n t o the three major groups: 1)PGA & PGE, 2) PGE and 3) PGF. Group separation w i l l also increase the s p e c i f i c i t y of the analysis and further remove substances which 34 could i n t e r f e r e with the assay. Another advantage of s e p a r a t i o n , i s t h a t i t enables the a n a l y s i s of more than one PG frcm a s i n g l e sample. However, these steps w i l l undoubtably be a source cf some l o s s of PGs. I t i s t h e r e f o r e necessary to determine the extent of e r r o r by monitoring the recovery of l a b e l l e d p r o s t a g l a n d i n s f o l l o w i n g e x t r a c t i o n and s e p a r a t i o n . T h i s i s e s p e c i a l l y t r u e i n t h i s study, as the recommended p u r i f i c a t i o n steps had been t e s t e d i n mammals, r a t h e r than f i s h . 3 . 2.1 E x t r a c t i o n Various e x t r a c t i o n methods have been o u t l i n e d i n the l i t e r a t u r e . They are a l l based on the dual nature of the p r o s t a g l a n d i n molecule, whereby PGs are s o l u b l e i n both p o l a r and o r g a n i c s o l v e n t s . The PG molecule i s p o l a r ; t h e r e f o r e a n e u t r a l s o l v e n t system would not give a good y i e l d and a b a s i c s o l v e n t would i o n i z e the c a r b o x y l i c group on the p r o s t a g l a n d i n . E i t h e r of these f a c t o r s would decrease the e x t r a c t i o n p o t e n t i a l . To ensure a good recovery , i t i s necessary to a c i d i f y the aqueous phase of the e x t r a c t o n s o l u t i o n (pH 3 to 4 ) . Once the PGs have been e x t r a c t e d i n an a c i d i c s o l u t i o n , they can e a s i l y be removed i n an o r g a n i c s o l v e n t and removed by e v a p o r a t i o n p r i o r to f u r t h e r p u r i f i c a t i o n . 35 3.2.2 S e p a r a t i o n Due t o the r e l a t i v e p o l a r i t i e s of t h e d i f f e r e n t p r o s t a g l a n d i n groups, i t i s p o s s i b l e t o s e p a r a t e them by c o n t r o l l i n g the p o l a r i t y o f t h e d e v e l o p i n g s o l v e n t s . Chromatographic t e c h n i q u e s a r e most f r e q u e n t l y used f o r s e p a r a t i n g PGs. Column chromatography i s used m a i n l y f o r s e p a r a t i o n of PG groups w h i l e t h i n l a y e r chromatography and r e v e r s e phase chromatography a r e used f o r both group and i n d i v i d u a l s e p a r a t i o n of PGs. Although i t would have been d e s i r a b l e t o have i n d i v i d u a l s e p a r a t i o n of PGs, the pr o c e d u r e s i n v o l v e d a r e t e d i o u s and r e q u i r e a g r e a t e r p u r i f i c a t i o n of t h e sample, and a r e t h e r e f o r e not p r a c t i c a l on a l a r g e s c a l e . Column chromatography w i t h s i l i c i c a c i d i s t h e most w i d e l y used t e c h n i q u e f o r p r o s t a g l a n d i n s e p a r a t i o n . Salmon and Karim (1976) have p o i n t e d out some of i t s advantages: " i t ( s i l i c i c a c i d chromatography) can be used t o s e p a r a t e out s m a l l amounts of EGs, g i v e s low " b l a n k s " i n most assay systems and i s r e l a t i v e l y s i m p l e " . I t i s a l s o i n e x p e n s i v e and r e a d i l y a v a i l a b l e . The one problem sometimes e n c o u n t e r e d d u r i n g s i l i c i c a c i d chromatography, i s poor r e p r o d u c i b i l i t y . However, t h i s can be minimized by u s i n g t h e same b a t c h o f s i l i c i c a c i d (Kibby, Bronn and Minton, 1977), and by b e i n g c o n s i s t e n t t h r o u g h o u t the ex p e r i m e n t s . 36 3-2-3 Assay methods Assay methods for prostaglandins have been the major setback i n PG research; U n t i l the l a s t decade, the only r e l i a b l e method for measuring PGs was the bioassay. With the increasing knowledge and inte r e s t i n PG research, new assay technigues, such as radioimmunoassay (RIA) have been developed. In addition, other chemical assays have been adapted, and bioassays have been improved. Each assay technique has some advantages over the other. It i s therefore e s s e n t i a l to weigh out the research problem against the l i m i t a t i o n s of the assay. During the description cf the di f f e r e n t assay techniques, the reasons f o r choosing RIA for this study w i l l be discussed. 3.2.3.1 Bioassay Numerous bioassay systems have been developed based on the smooth muscle stimulating a c t i v i t y of the PGs, and i s o l a t e d smooth muscle preparations are the most common bioassay techniques. Salmon and Karim (1976) reviewed the most frequently used methods, which included: g e r b i l colon, rat and hamster stomach and fundus, rabbit duodenum and jejunun, guinea pig ileum, chick rectum and the rat mesenteric vascular bed preparation. More exotic smooth muscle preparations, such as smooth muscle from the uterus and i n t e s t i n e s cf the f r u i t bat and the goldfish, are outlined i n Berstrom, Carlson and Weeks (1968) . In 2iY_2 preparations, such as blood pressure and blood flow 3 7 a s s a y s , a r e sometimes used f o r measurement of PGs. They are e s p e c i a l l y s e n s i t i v e t o PGE and PGA; however, they are not as s e n s i t i v e as t h e smooth muscle p r e p a r a t i o n s . P a r a l l e l b i o a s s a y s , u s i n g s e v e r a l t i s s u e s w i t h v a r y i n g s e n s i t i v i t i e s t c the d i f f e r e n t PGs, are used t o i d e n t i f y v a r i o u s PGs from the same sample. Of the p a r a l l e l b i o a s s a y s , the b l o o d bath t e c h n i q u e i s p r o b a b l y t h e most s o p h i s t i c a t e d (Vane 1969). I t c o n s i s t s of s u p e r f u s i n g h e p a r i n i z e d b l o o d c o n t i n u o u s l y over i s o l a t e d assay organs and t h e n r e t u r n i n g t h e b l o o d to the a n i m a l . T h i s t e c h n i q u e o f f e r s i n f o r m a t i o n not o n l y on t h e b e h a v i o u r c f PGs over t i m e , but a l s o on the i n t e r a c t i o n o f PGs w i t h o t h e r s u b s t a n c e s . The i o n i c c o m p o s i t i o n of t h e medium, the p r e s e n c e o f i n t e r f e r i n g s u b s t a n c e s i n t h e sample, and t h e p o t e n t i a l s e n s i t i z a t i o n of PGs to t h e m s e l v e s can a l l a f f e c t t h e a c t i o n o f PGs i n the smooth muscle p r e p a r a t i o n . P u r i f i c a t i o n of the sample must preceed PG b i o a s s a y to i n s u r e maximum s p e c i f i c i t y . Some problems can be overcome by t h e use o f s e l e c t i v e a n t a g o n i s t s t o i n t e r f e r i n g s u b s t a n c e s , such as smooth muscle a c t i v a t o r s . Futhermore, the a d d i t i o n o f a PG a n t a g o n i s t would i n h i b i t spontaneous g e n e r a t i o n of PGs d u r i n g t h e a s s a y . O v e r a l l , PG b i o a s s a y s are a s e n s i t i v e and r e l i a b l e method and i n some c a s e s , t h e r e s u l t s from b i o a s s a y s have been c o n f i r m e d by s p e c i f i c c h e m i c a l a n a l y s i s . However, b i o a s s a y s on a l a r g e s c a l e a r e not p r a c t i c a l , even though many of the problems a s s o c i a t e d w i t h them can be reduced t o a minimum. 38 3-2.3.2 Chemical assays S e v e r a l chemical assays have been adapted f o r measuring PGs and have been reviewed by Salmon and Karim (1976). Enzyme a n a l y s i s (Anggard 1971) and u l t r a - v i o l e t spectrophotometry (Shaw and Ramwell 1969) are both s p e c i f i c but have low q u a n t i t a t i v e ab i l i t i e s . There are three g a s - l i q u i d chromatographic methods f o r measuring PGs: F l a m e - I o n i z a t i o n Detector, E l e c t r o n Capture Detector and Mass Spectrophotometer. These techniques are a l l very s e n s i t i v e , but r e q u i r e extensive sample p r e p a r a t i o n . Moreover, the i n s t r u m e n t a t i o n r e q u i r e d i s very expensive. 3.2.3.3 General b a s i s of radioimmunoassay Radioimmunoassay (RIA) i s a c o m p e t i t i v e b i n d i n g assay which r e l i e s on the s t e r e o s p e c i f i c b i n d i n g p r o p e r t i e s " of a p r o t e i n antibody. Enzymes, e x t r a c e l l u l a r p r o t e i n s ( t r a n s i n s ) and t i s s u e r e c e p t o r s may a l s o f u n c t i o n as b i n d i n g p r o t e i n s i n c o m p e t i t i v e b i n d i n g assays. The f i r s t RIA was developed f o r i n s u l i n by Yalow and Berson i n 1960. Since then many RIA's have been developed f o r metabolic substances i n very low c o n c e n t r a t i o n . C l i n i c a l l y , they have been important i n measuring lew c o n c e n t r a t i o n s of p r o t e i n and s t e r o i d hormones, vit a m i n s , metabolic substances,blood p r o t e i n s and some drugs. The t h r e e subtances i n v o l v e d i n the RIA r e a c t i o n are the a n t i g e n , the l a b e l l e d antigen and the s p e c i f i c antibody. Both the l a b e l l e d and u n l a b e l l e d a n t i g e n compete f o r b i n d i n g s i t e s on the antibody. The t o t a l amount of the two a n t i g e n s should exceed 39 the number of binding s i t e s on the antibody as the reaction equilibrium i s reached when a l l the binding s i t e s are f i l l e d . Following a suitable incubation period, the bound and unbound fractions are seperated and the percent binding determined. If the concentration of the l a b e l l e d antigen and the antibody are kept constant, the antibody-antigen complex of these two w i l l be inversely proportional to the amount of unlabelled antigen. A standand curve i s obtained by adding known amounts of the unlabelled antigen to a constant mixture of the antibody and antigen, and then p l o t t i n g the percent bound against the concentration of unlabelled antigen added (100% being antibody and la b e l l e d antigen only). The concentraticn of antigen in an unknown sample can be read from the standard curve by calculating the percent binding i n the sample obtained under the same conditions. A) radioimmunoassay of prostaglandins Antibodies towards PGs were f i r s t described by Levine and Van Vunakis (1970). Eecause of the small size of the PG molecule, i t i s necessary to conjugate the PG with a protein in order to generate antibodies. The l a b e l l e d markers are usually t r i t i a t e d PGs. These markers are r e l a t i v e l y stable and have a good a f f i n i t y for the corresponding antibody. Some reseachers have been sucessful i n iodonating a tyrosine methyl ester of PGF2<< (Levine and Van Vunakis 1970; and Ohki et a l 1974) . Although the iodinated PG has more s p e c i f i c a c t i v i t y than the t r i t i a t e d form, i t does not seem to possess the same antigenic 40 q u a l i t i e s and f o r t h i s reason i t i s not commonly used. RIA f o r PGs o f f e r many advantages over o t h e r methods f o r t h e i r measurement. One u s e f u l f a c t o r i s the h i g h s e n s i t i v i t y . F u r t h e r m o r e , t h e p r e c i s i o n o f t h e assay i s i n s u r e d by c a r e f u l l y m o n i t o r i n g the p i p e t t i n g and the c o u n t i n g of r a d i o a c t i v i t y . They a r e a l s o r e l a t i v e l y r a p i d a s s a ys; once the experiment i s s e t up, many samples can be p r o c e s s e d s i m u l t a n e o u s l y . The major problems of RIA a r e c r o s s - r e a c t i v i t y between t h e d i f f e r e n t PG groups and i n t e r f e r i n g s u b s t a n c e s . While o r g a n i c e x t r a c t i o n and s e p a r a t i o n does m i n i m i z e t h e s e problems, c r o s s -r e a c t i v i t y s t i l l d e c r e a s e s t h e s p e c i f i c i t y o f the assay . D e s p i t e group s e p a r a t i o n of PGs, c r o s s - r e a c t i v i t y w i t h i n a PG group s t i l l o c c u r s . I n d i v i d u a l PGs cannot be measured p r e c i s e l y u n l e s s t h i n l a y e r chromatography i s performed as w e l l . However, t h i s i s not done r o u t i n e l y , as i t i s much t o o t e d i o u s f o r a l a r g e number of samples, and i n consequence, r e s u l t s a re o f t e n quoted f o r PG groups, r a t h e r than i n d i v i d u a l PGs. R e c e n t l y , i t has been r e p o r t e d t h a t s i l i c i c a c i d chomatography removes most of the non e s t e r i f i e d f a t t y a c i d s which were one of the main i n t e r f e r i n g s u b s t a n c e s i n the as s a y (Gold and Edgar 1978). RIA k i t s f o r t h e i d e n t i f i c a t i o n of PGs are a v a i l a b l e from C l i n i c a l Assays I n c . D e t a i l s on the c o n t e n t s and c r o s s -r e a c t i v e t y of the a n t i b o d i e s are g i v e n i n t h e Appendix. RIA was chosen as the assay method f o r t h i s s t u d y because of i t s h i g h s e n s i t i v i t y , and i t s a d a p t a b i l i t y f o r the r a p i d a n a l y s i s of a l a r g e number of samples. 41 i i i IIEIFICATIONS AND MODIFICATIONS OF PG ANALYTICAL TECHNIQUES The following discussion outlines a l l the steps taken to v e r i f y the extraction , separation and assay of PGs in f i s h tissue- Methods and r e s u l t s are described together for each of the above procedures. This description follows the approximate order i n which the techniques were performed, and includes the reasons for modifications. 3-3.1 Extraction e f f i c i e n c y 3.3.1.1 D i l u t i o n of labelled prostaglandins Tritium l a b e l l e d PGs were purchased from New England Nuclear* Boston, Mass. The following PGs were s o l u b i l i z e d in 70% ethanol and shipped on dry i c e : #482 PGE2 85,6,8, 11,12,14,15- 3H(N) ) l o t * 932-108, s p e c i f i c a c t i v i t y 117 Ci/mmol, concentration of 0.025 mCi; 0.000075 in 0.25 ml. #345 PGF20( (9-3H (N) ) lot# 787-250, s p e c i f i c a c t i v i t y 9.2 Ci/mmol* concentration of 0.05 mCi; 0.0019mg in 0-5 ml. The v i a l s were stored at -70°C u n t i l use. A one hundred f o l d d i l u t i o n was performed on the standards as follows: the PG standards were removed from the freezer and thawed on i c e . Using a 100 ul Hamilton syringe, 200 u l of 70% ethanol was placed i n a glass ampoule which had been previously s i l i c o n i z e d and autoclaved- To t h i s , 10 u l of H-PG was added with a Hamilton syringe, and t h i s was followed by another 750 u l of 70% ethanol. 42 (50 u l a l i g u o t s ) - F i n a l l y , a f u r t h e r 40 uml c f 70% e t h a n c l were added t o make up a t o t a l o f 1.0 ml per ampoule. The ampoules and s t o c k s o l u t i o n s were then p l a c e d at -70° C ( H a r r i s deep f r e e z e ) . The f i n a l c o n c e n t r a t i o n s of 3H-PGs were: 3H-PGE2; 3,000pg/ml ( a p p r o x i m a t l y 24, 000 dpm/ 10 ul) 3H-PGF2j -38,000 pg/ml ( a p p r o x i m a t l y 20,000 dpm/10 u l ) . 1 I t was known t h a t PGE2 r e a d i l y a d sorbes t o g l a s s s u r f a c e s ; t h e r e f o r e , the e x t e n t o f t h i s a d s o r p t i o n was t e s t e d by measuring d u p l i c a t e 10 u l volumes of bo t h 3H-PGE2 and 3H-PGF2^ u s i n g s i l i c o n i z e d and n o n - s i l i c o n i z e d m i c r o p i p e t t e s and a 50 u l Ha m i l t o n s y r i n g e . The f i n a l dpms i n each a l i g u o t were not s i g i f i c a n t l y d i f f e r e n t from each o t h e r , f o r both PGs t e s t e d . 3.3.1.2 E x t r a c t i o n r e c o v e r y A 20 u l volume o f e i t h e r H-PGF or H-PGE was added t o the sample a t the homogenate s t a g e , o r d i r e c t l y t o the plasma. T h i s s t e p was f o l l o w e d by the e x t r a c t i o n p r o c e d u r e s o u t l i n e d i n the g e n e r a l m e t h o d s ( J a f f e and Behrman, 1974). The r a d i o a c t i v i t y was measured i n an a l i g u o t of the f i n a l o r g a n i c phase c o n t a i n i n g t h e e x t r a c t e d p r o s t a g l a n d i n s . The a l i g u o t was p l a c e d i n a s c i n t i l l a t i o n v i a l , f o l l o w e d by 10 ml o f s c i n t i l l a t i o n f l u i d , then counted f o r 10 minutes i n t h e ISOCAP. The p e r c e n t r e c o v e r y was c a l c u l a t e d by d i v i d i n g t h e dpms i n the a l i g u o t by the t o t a l dpms added, t h e n m u l t i p l y i n g by the r a t i o o f t h e volume i n t h e sample over t h e volume i n t h e a l i g u o t , and f i n a l l y m u l t i p l y i n g t h i s v a l u e by 100. The average % y i e l d +SEM (# of samples) f o r 3H-PGE2 , when added t o the ovary homogenate was 86.5% ±2.9 (3) 43 and, for ^-PGF^, 85.3% ±1.8 (4). When both tracers were added together, an average of 85.6% ±0.9 (5) of the r a d i o a c t i v i t y was recovered in the ovary homogenates, and 84.5% +4.6 (3) in the plasma samples. 3^4 COLUMN CALIBRATION 3.4.1 Preliminary chromatography methods and results S i l i c i c acid (Sigma, SIL-A-200 Mesh 60-200 l o t #115C-0185) was suspended in d i s t i l l e d water, l e f t to s e t t l e for a few minutes, and then decanted. This was repeated several times to remove the fine s . Removal of the fines ensures a better flow rate as well as decreasing the r i s k of blocking the column. The wet s i l i c i c acid was dried at 115 C for at least 24 hours, and kept at t h i s temperature u n t i l use. This a c t i v a t i o n process i s important since removing the free water from the s i l i c i c acid improves the adsorptive strength of the gel (Trueblocd and Malmberg 1949). To further increase the adsorptive c h a r a c t e r i s t i c s , a prewash using B:EA was performed, prior to loading the column. If no water was present, the column would be a translucent blue. However* i f the column was even p a r t i a l l y opaque, more B:EA would be used to wash the cclunm, u n t i l the blue color was achieved. The column consisted of a Pasteur pipette which had been s i l i c o n i z e d ( S i l i c l a d , Clay Adams, NY) and plugged with glass wool. 0.5 g of s i l i c i c acid was mixed into two ml cf B:I A. The 44 slurry was poured into the pipette, while adding more B:EA to the flask to ensure complete recovery. (Subsequently, a pouring device was made to f a c i l i t a t e this task.) The column was attached to a drop counter over the f r a c t i o n - c o l l e c t o r with teflon tubing. The flow from the column could be controlled by means of a clamp. The column was washed with fiv e ml cf B:EA and clamped o f f . The sample was prepared by measuring either 10 or 20 ul of tr i t i a t e d - P G i n a test tube and blowing over i t with N gas u n t i l i t was evaporated. It was then suspended in 0.2 ml of a B: EA and methanol solution (6:4:1), and a further 0.8 ml of B:EA was added. An equivalent amount of the labelled PG was put into a s c i n t i l l a t i o n v i a l i n order to determine t o t a l dpms. The sample was loaded onto the column, which was clamped off after a l l the sample had run into the g e l . Each dif f e r e n t PG group was eluted off by adding the following solutions, according to the "methods outlined in Ja f f e and Berhman (1974): PGA and PGB f r a c t i o n : 6 ml of benzene:ethyl acetate, 60:40 (B: EA) . - PGE f r a c t i o n : 12 ml of benzene:ethyl acetate:methanol, 60:40:2. PGF f r a c t i o n : 4 ml of benzene:ethyl acetate:methanol, 60:40:10. 1 the column was clamped off after each solvent had run through, and i t was then opened for the next solvent. One m i l l i l i t r e samples were collected and transferred to a s c i n t i l l a t i o n v i a l to be counted. The percent recoveries were calculated by dividing the dpms i n the sample by the t o t a l dpms added x 100. The r e s u l t s of these experiments are given i n Figure 3. In 45 FIGUBE 3: i n i t i a l e l u t i o n p a t t e r n o f r a d i o a c t i v e p r o s t a g l a n d i n s . E l u t i o n p a t t e r n o b t a i n e d u s i n g the method of J a f f e and Berhgman (1974) . (note: each d i v i s i o n on the a b c i s s a r e p r e s e n t s one 3.0 ml f r a c t i o n ) Hb 47 some c a s e s , t h e r e s u l t s a r e s a t i s f a c t o r y , but i n o t h e r s , t h e y a r e not. I n two columns, t h e r e s o l u t i o n of PGE was not good, as t h e r a d i o a c t i v i t y was spread over a v e r y wide a r e a . Some t e c h n i c a l problems were p r e s e n t i n the system; th e s e c o u l d have had a d i r e c t e f f e c t on the r e s o l u t i o n o f t h e p r o s t a g l a n d i n s . The c l a m p i n g o f the column (when ch a n g i n g s o l u t i o n s ) c o u l d e a s i l y have d i s t o r t e d t h e bands by the p r o d u c t i o n of d i f f e r e n t i a l f l o w a t t h e i n t e r f a c e o f the g l a s s . A l s o , i t was d i f f i c u l t , when changing s o l u t i o n s , not t o d i s t u r b t h e s u r f a c e of the column. F i n a l l y , i t i s i m p o s s i b l e , u s i n g t h i s system, t o m a i n t a i n an even s o l v e n t f l o w , as the p r e s s u r e head d e c r e a s e s d u r i n g t h e column run; 3.4.1.1 G r a d i e n t e l u t i o n I n an attempt t o a l l e v i a t e some of the above problems, and f a c i l i t a t e h a n d l i n g of the e l u t i o n s o l v e n t s , a g r a d i e n t e l u t i o n system was s e t up. G r a d i e n t e l u t i o n i s r o u t i n e l y used t o s e p a r a t e l i p i d mixures ( H i r s h and Ahrens 1958). B a s i c a l l y , g r a d i e n t e l u t i o n c o n s i s t s o f g r a d u a l l y i n c r e a s i n g t h e c o n c e n t r a t i o n of a s o l v e n t i n an e l u t i o n medium, t h e r e b y c r e a t i n g a c o n c e n t r a t i o n g r a d i e n t which e n a b l e s a group of compounds w i t h s l i g h t y d i f f e r e n t s o l u b i l i t i e s t o be s e p a r a t e d . Bygdeman and Samuelsson (1966) d e s c r i b e d a c o n t i n u o u s g r a d i e n t method f o r s e p a r a t i n g the PG groups on a s i l i c i c a c i d column by i n c r e a s i n g the c o n c e n t r a t i o n o f e t h y l a c e t a t e i n benzene. In t h e i r s t u d y , the r e c o v e r y and s e p a r a t i o n were v e r y good; however, the number of t r i a l s was low: t h r e e f o r PGF and t h r e e 48 for PGE. Furthermore, t h e i r set-up was not p r a c t i c a l for processing a large number of samples. To overcome the processing d i f f i c u l t i e s , and to maintain a high degree of e f f i c i e n c y , a smaller version of t h e i r system was designed. The elution pattern was modelled on Jaffe and Behrman's (1977) method of increasing the methanol concentration i n B:EA. This procedure, which w i l l be described, made i t simple to determine the best elution seguence for the optimum resolution and separation of the PG groups. A) apparatus S i l i c i c acid was prepared as described i n the General Techniques. A diagram of the gradient e l u t i o n apparatus i s shown in Figure 2 . Two 100 ml glass graduated cylinders were adapted with spouts at t h e i r bases. Cylinder A had one outlet, and cylinder B had two outlets, each at 180 degrees to the other. A short section of s i l i c o n tubing (ID 1.35 mm, OD 3.35 mm, LKB #2030-962) joined the two cylinders. A 13-14 cm section of the same tubing, f i t t e d with a 10 u l disposable pipette, was attached to the second outlet of cylinder B, and led d i r e c t l y to the column. The cylinders were held on fixed p l a s t i c bases, resting on a magnetic s t i r r e r (Fllexa-Mix, Fisher S c i e n t i f i c ) . The s t i r r e r was mounted on a support stand by means of a clamp. Solvent flow rates could be altered to maintain a constant pressure head on the column, by r a i s i n g or lowering the s t i r r e r . Magnetic s t i r r i n g bars in each cylinder ensured complete mixing of the solvents. The column was held to a stand by means of a 49 t h r e e - p r o n g clamp over the f r a c t i o n c o l l e c t o r . A 5 u l d i s p o s a b l e p i p e t t e was h e l d between two p i e c e s of s i l i c o n t u b i n g , and was j o i n e d t o the drop c o u n t e r on t h e f r a c t i o n c o l l e c t o r . The s m a l l d i a m e t e r of the p i p e t t e reduced the s o l v e n t o u t f l o w , t h e r e b y i n c r e a s i n g t h e r e s o l u t i o n by i n c r e a s i n g t h e time t h e sample had to t r a v e l over t h e g e l . B) r e s u l t s of g r a d i e n t e l u t i o n o f p r o s t a g l a n d i n s V a r i o u s s o l v e n t p a t t e r n s were attempted w i t h t h e g r a d i e n t e l u t i o n a p p a r a t u s . Except f o r a few n o t e d c a s e s , the column bedding c o n s i s t e d o f 0.5 g of s i l i c i c a c i d which had been prewashed w i t h 5 ml o f B:EA. 20 u l o f one of the t r i t i a t e d - P G S was evaporated under N suspended i n 0.2 ml of B:EA and methanol (6:4:1) . 0.8 ml of B: EA was t h e n added, and the whole m i x t u r e was l o a d e d on the column. The r e s u l t s o f the f i r s t g r a d i e n t e l u t i o n t r i a l s a re g i v e n i n F i g u r e 4. In t h e s e e x p e r i m e n t s c y l i n d e r A c o n t a i n e d 15 ml of methanol and 15 ml o f B:EA, w h i l e c y l i n d e r B had 30 ml of B:EA. There i s an o b v i o u s o v e r l a p between PGE and PGF i n t h e s e columns. The PGFs came o f f much e a r l i e r t h a n they d i d w i t h J a f f e and Behrman's method. T h i s c o u l d be due t o the s o l v e n t becoming p o l a r t o o r a p i d l y , and e l u t i n g o f f the mere p o l a r PGFs a l o n g w i t h t h e PGEs. However, when t h e methanol c o n c e n t r a t i o n was d ecreased t o 9.0 ml i n 21.0 ml o f B:EA, the PGF peak was not s h i f t e d s i g n i f i c a n t l y , as can be seen i n t h e column on t h e f a r r i g h t i n F i g u r e 4b.. I n an attempt t o improve r e s o l u t i o n by i n c r e a s i n g column l e n g t h , 0.6 g o f s i l i c i c a c i d was used i n s t e a d 50 FIGOEE 4: e l u t i o n p a t t e r n of r a d i o a c t i v e p r o s t a g l a n d i n s using a c o n t i n u a l g r a d i e n t e l u t i o n technique a) with 0-5 g of s i l i c i c a c i d and 30-0 ml (B: EA) : (methanol) i n t o 30.0 ml B:EA b) s o l i d and s l a s h e d l i n e s : with 0.6 g s i l i c i c a c i d and 30.0 ml 1:1 (E: EA) : (methanol) i n t o 30.0 ml B: EA c) dotted l i n e s : with 0.5 g of s i l i c i c a c i d and 30.0 ml 9:21 (E:EA) : (methanol) i n t o 30 ml B: EA. (note: each tube contained 1.0 ml of e l u a t e . The broken l i n e below represents the time during which the g r a d i e n t was on.) a ) Tube Number 52 of 0.5 g. (three experiments). A continuous gradient was started with methanol:B:EA 1:1 in cylinder A and 30 ml B:EA i n cylinder B: the results from these experiments are shown i n Figure 4b. These results were not s a t i s f a c t o r y , as the FGF and PGE peaks s t i l l overlapped. Therefore 0.5 g of s i l i c i c acid was used i n a l l subsequent columns. Some promising results were obtained in the elution pattern of two columns (Figure 5a). The elution of these two columns was started with 2.0 ml of B:EA, after which a continuous gradient was resumed with 30 ml of methanol:B:EA (1:1) i n A, and 30 ml of B:EA in B: t h i s s l i g h t change in elution shifted the PGF peak from the 8-9 position to the 11-12 position. A few "on-off" gradients were attempted (Figure 5b). The cylinders contained the same solution as described previously. The "on-off" was accomplished by clamping the tubing between the cylinders with a microhemostat. The "on-off" settings are shown under the elution pattern for each column. The results from the three columns in Figure 5b were not s a t i s f a c t o r y , as the PGF peak was s t i l l in the 8-9 position, d i r e c t l y over the PGE peak in every case. However, the PGF peak shown i n Figure 5c had shifted to the 13-14 position, with very l i t t l e overlap with PGE peak. A sat i s f a c t o r y version of the elu t i o n pattern was f i n a l l y found (Figure 6a). The cylinders contained the following: A had 30 ml of the methanol: B:EA (1:1) mixture and B had 32 ml of B:EA. The elution was started with 2.0 ml of E:EA, and then the gradient was turned on for the next 3.0 ml, turned off for 6.0 ml, and on again for the f i n a l 6.0 ml. The peaks for PGE f e l l i n 53 FIGURE 5: I n i t i a l "on-off" gradient elution pattern radioactive prostaglandins. A l l columns had 0.5g of s i l i c i c acid and the gradient elution consisted of 30.0 of 1:1 (B : EA) : (methanol) into 30.0 ml of B:EA. a) gradient elu t i o n started a f t e r eluting the f i r s t 2.0 ml with E:EA B) gradient was on for the f i r s t 7.0 ml, stopped for 5.0 ml, then started again for the f i n a l 4 ml. C) gradient o f f for the f i r s t 2.0 ml, on for 4.0 ml, stopped for 4.0 ml, then on for the f i n a l 7.0 ml. % R A D I O A C T I V I T Y % R A D I O A C T I V I T Y S R A D I O A C T I V I T Y ro oo —» ro _i T 1 y 1 1 1 1 J1 1 1 1 1 1- I 1 1 1 1 1^  1 1 1 1 1 1 1 ' 1- I 1 1 1 1 1 1 \ 1 1 1 1 1 r — i r-55 FIGURE 6: Successfull "on-off" gradient elution pattern of radioactive prostaglandins. In a l l columns the gradient was off for the f i r s t 2.0 ml, on for the next 4.0 ml, o f f for 5.0 ml, then on for the f i n a l 6.0 ml. a) with i n d i v i d u a l prostaglandins on a column. b) with two prostaglandins together on a column. a) -¥ 1 1 1 1 1 1 1 7 T 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 T u b e N u m b e r 57 the 8 to 10 position, and the peaks for PGF had shifted over to the 14 to 16 position. Under these same conditions, a combination of the two tracers was used on the columns shown in Figure 6b. The elution pattern of these columns correlate well with the pattern obtained with the single tracer experiments shown in Figure 6a. The bands of the double tracer experiments do not overlap, as the r a d i o a c t i v i t y i n the 12 and 13 position i s r e l a t i v e l y low and the peaks are sharp. The percent recoveries are acceptable: 78.4% ±1.1 for PGE2, and 56.5% ±9.8 for PGF2^ Although t h i s elution pattern appeared to be the most suitable for separating out the PGE and PGF groups, i t was necessary to test i t s performance using f i s h t i s s u e . During these experiments, i t was also possible to measure the effectiveness of recovery from the extraction procedure. 3.4.2 V e r i f i c a t i o n of methods on f i s h tissue Following the extraction recovery experiments with f i s h tissue, there was a s u f f i c i e n t amount of extract, containing t r i t i a t e d PGs, to measure the column e f f i c i e n c y . A measured volume of t h i s f l u i d was evaporated under N at 40" C and the residue was resuspended in 0.2 ml of benzene;ethyl acetate: methanol (6:4:1), and run on the column. To t e s t the r e p r o d u c i b i l i t y of the separation pattern using f i s h tissue, the "on-off" gradient e l u t i o n method was performed using the f i s h extracts. The e l u t i o n pattern was followed exactly as described for columns #18 to #28; i . e . , with 30 ml of methanol: B:EA (1:1) 58 i n c y l i n d e r A and 32 ml of B:EA i n c y l i n d e r E. The " o n - o f f " g r a d i e n t p a t t e r n used was as f o l l o w s : a) s t a r t i n g w i t h 2.0 ml of B:EA from B w i t h t h e g r a d i e n t o f f b) 3.0 ml w i t h the g r a d i e n t on c) 6.0 ml w i t h t h e g r a d i e n t o f f d) f i n i s h i n g w i t h the f i n a l 6.0 ml w i t h the g r a d i e n t on. The f r a c t i o n s were c o l l e c t e d i n one m i l l i l i t r e volumes. The r e s u l t s from t h e s e e x p e r i m e n t s a r e shown i n F i g u r e 7. The r a d i o a c t i v e d i s t r i b u t i o n c o r r e l a t e s w e l l w i t h the p a t t e r n found when u s i n g t r a c e r a l o n e ; however, t h e pe r c e n t r e c o v e r y was somewhat l o w e r . There were some problems which o c c u r r e d i n i t i a l l y , and c o u l d have decreased the e f f i c i e n c y of the column. In some i n s t a n c e s , the column would p l u g a f t e r a p p l i c a t i o n of t h e sample. In t h e s e c a s e s , t h e sample e x t r a c t was u s u a l l y f l o c c u l e n t and very d e e p l y c o l o u r e d . I n an attempt t o overcome t h i s , a p i n c h of s i l i c i c a c i d was added t o the sample p r i o r t o e v a p o r a t i o n and p e r m i t t e d t o s e t t l e f o r a few seconds. The l i q u i d was removed, and p l a c e d i n a n o t h e r t e s t tube. The re m a i n i n g s i l i c i c a c i d was washed once w i t h B:EA, and t h i s r i n s e was a l s o added t o the t e s t tube; The f i n a l e x t r a c t was e v a p o r a t e d t o dryn e s s and resuspended w i t h 0.2 ml of the l e a d i n g s o l u t i o n s . Presumably, the b a t c h e x t r a c t i o n w i t h s i l i c i c a c i d c o u l d account f o r some of t h e l o s s o f r a d i o a c t i v i t y . F o r t u n a t e l y , t h i s c l o u d i n e s s o n l y o c c u r r e d d u r i n g these p r e l i m i n a r y e x p e r i m e n t s , and i t was t h e r e f o r e not n e c e s s a r y t o re p e a t t h i s s t e p f o r any o f t h e o t h e r samples. 59 FIGUEE 7: g r a d i e n t e l u t i o n p a t t e r n o f r a d i o a c t i v e p r o s t a g l a n d i n s added t o ovary homogenates. 40r-• 3 H P G E 0 & 3 H - PGR H- P G E I Z T T Z H - P G F 2 30r-20 U < o Q < ON? 10 1 2 3 4 5 6 7 8 9 10 11 12 Tube Number 16 17 61 To f u r t h e r maximize the t r a n s f e r o f PGs i n the e x t r a c t from the t e s t tube t o the column* t h e t e s t t u b e was washed w i t h 1.0 ml of B:EA a f t e r l o a d i n g the e x t r a c t on t h e column. T h i s r i n s e was added to the column t o s t a r t the e l u t i o n ( c o n s e q u e n t l y c y l i n d e r B c o n t a i n e d o n l y 31 ml o f B: EA) . T h i s p r o t o c o l was performed f o r a l l subsequent samples. There was some concern about u s i n g an a c i d i c e x t r a c t i o n s o l u t i o n f o r the i n i t i a l e x t r a c t i o n of PGs, as t h e r e was a p o s s i b i l i t y o f some l o s s of PGE by c o n v e r s i o n t c PGB under a c i d i c c o n d i t i o n s . However, o n l y a v e r y s m a l l amount o f r a d i o a c t i v i t y was found i n t h e PGA and PGB f r a c t i o n f o l l o w i n g e x t r a c t i o n w i t h H-PGE2, and t h i s l o s s was c o n s i d e r e d t o be n e g l i g i b l e . 3.4.2.1 Eadioimmunoassay of p r o s t a g l a n d i n s i n f i s h t i s s u e s To a s s e s s the amount of t i s s u e n e c e s s a r y f o r t h e measurement of p r o s t a g l a n d i n s i n f i s h t i s s u e , s e v e r a l samples of o v a r y , t e s t i s and plasma were e x t r a c t e d and the PG f r a c t i o n s were s e p a r a t e d on t h e column. These samples were th e n assayed f o r t h e t h r e e PGs by RIA. A p p r o x i m a t e l y one gram or one m i l l i l i t e r of t i s s u e was used, and except f o r the PGB, t h i s q u a n t i t y was s u f f i c i e n t f o r the d e t e c t i o n of PGs. The mean PG v a l u e s measured were as f o l l o w s : PGF2 I < -plasma ( f o u r s a m p l e s ) : 1165.4 pg/ml - t e s t i s (two s a m p l e s ) : 109.4 pg/g -ovary ( t h r e e s a m p l e s ) : 226. 4 pg/g 62 PGE1 -plasma (four samples): 112.5 pg/ml - t e s t i s (two samples): 90.0 pg/g -ovary (three samples): 0.0 pg/g PGB1 -below threshold i n a l l (nine) samples From these findings, i t was decided that a minimun cf one gram of ovary or t e s t i s would be used for PG measurement, and that plasma from two f i s h would be pooled, since cne ml did not contain s u f f i c i e n t PGB1 for detection; In the course of the succeeding experiments, two other f i s h tissues, muscle and kidney, were assayed f o r PGs. The findings were as follows: PGF2: 44. 4 pg/g PGE 1: 1370. 0 pg/g PGB 1: 72. 8 pg/g : PGF2w: 1481.1 pg/g PGE 1: 5217.6 pg/g PGB1: 8.2 pg/g This i s the f i r s t record of prostaglandins i n these tissues in Carassius auratus. 63 4 PROSTAGLANDINS DURING SEXUAL MATURATION! SEASONNAL CHANGES i i i l INTRODUCTION In mammals, prostaglandins have been shown to influence gonadotropin release (Ratner et a l 1974; Carlson et a l 1973; Sato et a l 1974). Labhestwar (1972a, b) has suggested that prostaglandins affect steroid synthesis, as he found that PGF^, and to a lesser extent PGE2, are l u t e o l y t i c agents. Furthermore, PGF^^was found to increase e s t r a d i o l concentration i n the ovary, whereas progesterone secretion dropped (Labhestwar, 1974) . Because these hormones are ess e n t i a l for gonadal maturation i n a l l animals, i t seemed l i k e l y that PGs could function as a control of gonadal maturation. The following experiments were performed to observe any possible changes in PG l e v e l s i n the gonad and plasma during maturation of male and female goldfish between late F a l l and early Spring. Two experimental groups of male and female goldfish were set up: to accelerate the maturation process, one group was subjected to a long photoperiod regime (16L:8D), and the other was under natural photoperiod. Plasma and gonad samples were taken for PG measurement over t h i s time period from both groups. !L.2 BACKGROUND REVIEW OF GONADAL DEVELOPMENT In the female, the process of ovarian maturation has been extensively studied (Lam et a l 1978) and the h i s t o l o g i c a l development has been well documented (Yamamato and Yamazaki 64 1961). Recently, there has been more insight into endocrinological involvement and physical influences, such as temperature, photoperiod, or n u t r i t i o n * which may be associated with the functionning of gonadal maturation. The male reproductive system i s not as well known Although the h i s t o l o g i c a l changes which occur during maturation have been outlined (Hoar 1969), the h i s t o l o g i c a l and endocrinological events have not been as well researched as they have i n the female. Some background on the h i s t o l o g i c a l and endocrinological changes i n both females and males w i l l be described below. 4.2.1 Changes in gonadal histology with development 4.2.1.1 The female system In the female, oogonial p r o l i f e r a t i o n i s followed by a meiotic prophase in which oogonia are transformed i n t o primary oocytes. The oocytes then go through two growth phases; the i n i t i a l growth of the oocyte, pre-vitellogenesis, i s follewed by vi t e l l o g e n e s i s , which involves the deposition of two kinds of yolk, yolk granules and yolk vesicles, into the oocyte. Yamamato and Yamazaki (1966) outlined the h i s t o l o g i c a l changes of the developing ovary in the goldfish, Carassius auratus, and they divided the pre - v i t e l l o g e n i c phase into three stages: chromatin nuclear stage (1) , and early and la t e perinucleolar stages (2 S 3). They also divided the vit e l l o g e n e s i s phase into a further 65 seven stages: yolk v e s i c l e (4), primary, secondary and t e r t i a r y yolky oocytes (5,6 & 7) , migratory nucleus stage (8) prematuration (9) and ripe (10) stages. A t r e t i c f o l l i c l e s are found during a l l stages of development but are predominant a f t e r the spawning season. The rhythm of oocyte development i n the goldfish i s asynchronous. This type of development i s distinguished by having oocytes at various stages i n the ovary, and i n the a b i l i t y to spawn several times during a season. As the ovary matures, more of the l a t e r stages of oocytes are present; however, there are always some of the l a t e r stages present, even at f u l l maturity. 4.2.1.2 The male system In the male, spermatogenesis follows a similar but simpler pattern of development. The primary c e l l s , or spermatogonia, go through mitotic p r o l i f e r a t i o n to y i e l d primary spermatocytes, followed by a meiotic d i v i s i o n to give secondary spermatocytes, which further divide to give the spermatids, which, in turn, metamorphose into the spermatocytes, or motile sperm. 4.2.2 Endocrinological changes during gonadal development 66 4.2.2.1 The female system Lam et a l (1978) reviewed the endocrine c o n t r o l of r e p r o d u c t i o n i n the female g o l d f i s h . In t h e i r summary they s t a t e t h a t the p i t u i t a r y appears to p l a y a c e n t r a l r o l e i n most, i f not a l l , of the stages and processes of oogenesis. Both the p i t u i t a r y hormones and estrogens seem to be necessary during v i t e l l o g e n e s i s . There are i n d i c a t i o n s t h a t e s t r a d i o l mediates h e p a t i c v i t e l l o g e n i n p r o d u c t i o n , whereas the p i t u i t a r y c o n t r o l s i t s i n c o p o r a t i o n i n t o the oocytes ( J a l a b e r t 1967). H u r l b u r t (1977) showed that t h y r o i d hormones i n f l u e n c e v i t e l l o g e n e s i s but only i n c o n j u n c t i o n with gonadotropin (GTH). 4.2.2.2 The male system The e n d o c r i n o l o g i c a l c o n t r o l of spermatogenesis has not been as e x t e n s i v e l y s t u d i e d i n the g o l d f i s h . However, the p i t u i t a r y does play an e s s e n t i a l r o l e i n the growth and f u n c t i o n of the t e s t i s . Yamazaki and Donaldson (1968) showed t h a t hypophysectomy i n h i b i t e d t e s t i s growth and induced r e g r e s s i o n i n the g o l d f i s h ; however, i n j e c t i o n s of p u r i f i e d salmon p i t u i t a r y gonadotropin c o u l d completely r e s t o r e spermatogenesis i n these f i s h . 4.2.3 Environmental i n f l u e n c e s on gonadal development Environmental f a c t o r s are a l s o important i n the r e p r o d u c t i v e processes of the g o l d f i s h ; photoperiod and temperature have been i n v e s t i g a t e d with r e s p e c t t o t h e i r e f f e c t s 6 7 on oogenesis and on gonadotropin levels ( G i l l e t et al 1 9 7 8 ) . Their studies indicate that high temperatures stimulate GTH secretion. However, the increase in GTH was not always correlated with an increase i n gonadal development since high temperatures induced gonadal regression. They also reported that long photoperiod stimulated oogenesis i n the winter. 4. 2 METHODS 4.3.1 Fish maintenance and sampling In mid-November, 1 9 7 6 , approximately 150 healthy f i s h , weighing between 60 and 100 g, were removed from the stock tank and divided i n t o three groups. Two indoor tanks were set up with fluorescent lamps on a timing device set at 16 hours l i g h t and eight hours dark. One tank held 50 females and the other 50 males (however, there was some mixing i n both tanks). A third tank under natural photoperiod held a mixture cf both males and females. The males were d i f f e r e n t i a t e d by the presence of small tubercules on their front pectoral f i n s ; these are absent i n the female. The f i s h were sampled in mid-December and then at focr-week in t e r v a l s i n January, February and March. The natural-photoperiod f i s h were sampled at the same time as the long-photoperiod f i s h . Blood and gonadal tissue was taken for PG measurement. The gonad was weighed for GSI and a sample was taken f o r histology. 68 The t i s s u e s were p r o c e s s e d f o r t h e radioimmunoassay of PGF, PGE and PGB as o u t l i n e d i n the G e n e r a l Techniques. 4.3.2 S t a t i s t i c s An a n a l y s i s of v a r i a n c e (ANOVA) was performed on the data from t h e s e e x p e r i m e n t s . A DBC program, the BMD 10V f o r unequal c e l l s i z e , was used f o r t h e c o m p u t a t i o n . The Newman-Kuels m u l t i p l e range t e s t (Zar 1974) was performed on the d i f f e r e n t groups a t a s i g n i f i c a n c e l e v e l of 0.05. 4_. 4 RESULTS FROM HISTOLOGY Alt h o u g h the gono-somatic f i s h , t h i s measurement was r e f l e c t i o n of s e x u a l m a t u r i t y i comparison between f i s h . i n d e x (GSI) was r e c o r d e d f o r each not found t c be a v e r y s e n s i t i v e nd was not used as a parameter o f 4.4.1 H i s t o l o g i c a l o b s e r v a t i o n s i n t h e female H i s t o l o g i c a l e x a m i n a t i o n o f t h e gonad was done on a l l 45 f e m a l e s from both p h o t o p e r i o d groups sampled between December and March. I n a l l the f e m a l e s , many s t a g e s of o o g e n e s i s were found i n the o v a r i a n s e c t i o n s , as would be expected w i t h an asynchronous development. None of the l a s t t h r e e s t a g e s of o o c y t e development ( m i g r a t o r y n u c l e u s , p r e m a t u r a t i o n and r i p e o o c y t e s ) were p r e s e n t i n any of the s e c t i o n s . The t e r t i a r y y o l k y oocyte s t a g e was t h e most advanced s t a g e observed i n any month, 69 b u t o n l y a p p r o x i m a t e l y one h a l f o f t h e o v a r i e s had d e v e l o p e d t o t h i s s t a g e . A l l t h e o t h e r s t a g e s were p r e s e n t i n v a r i a b l e r a t i o s . I n a d d i t i o n , a t r e t i c f o l l i c l e s w e r e o b s e r v e d i n 35- 4 5 % o f t h e f i s h t a k e n i n e v e r y month. T h e r e were no s i g n i f i c a n t h i s t o l o g i c a l d i f f e r e n c e s b e t w e e n t h e n a t u r a l - a n d t h e l o n g -p h o t o p e r i o d f e m a l e s , n o r were t h e r e any d i f f e r e n c e s i n s t a t e s o f m a t u r i t y b e t w e e n t h e months. 4.4.2 H i s t o l o g i c a l o b s e r v a t i o n s i n t h e male The 45 m a l e s s t u d i e d f r o m b o t h p h o t o p e r i o d g r o u p s were u n i f o r m l y m a t u r e , a s a l l t h e t e s t i c u l a r s e c t i o n s showed s e m i n i f e r o u s t u b u l e s w h i c h were v e r y l a r g e and d i s t e n d e d w i t h s p e r m a t o z o a . F u r t h e r m o r e , t h e male p o s s e s s e d s m a l l t r a n s l u c e n t t u b e r c u l e s ( p e a r l o r g a n s ) on t h e u p p e r s i d e s c f t h e i r p e c t o r a l f i n s ; t h e s e s e c o n d a r y s e x c h a r a c t e r i s t i c s a r e an e x t e r n a l i n d i c a t i o n t h a t t h e f i s h i s s e x u a l l y m a t u r e . 4-_5 RESULTS OF THE PROSTAGLANDIN MEASUREMENT DURING DEVELOPMENT IN THE FEMALE A t o t a l o f 45 f i s h were s a m p l e d b e t w e e n December and Ma r c h . E l e v e n f e m a l e s f r o m t h e l o n g p h o t o p e r i o d g r o u p a nd t h r e e f r o m t h e n a t u r a l p h o t o p e r i o d g r o u p were s a m p l e d e a c h month d u r i n g t h e f o u r month s t u d y p e r i o d ( e x c e p t f o r F e b r u a r y , where 12 f e m a l e s f r o m t h e l o n g p h o t o p e r i o d g r o u p were s a m p l e d ) . A f t e r s p i n n i n g t h e b l o o d f o r p l a s m a , t h e p l a s m a was p o o l e d b e t w e e n two f i s h . 70 4.5.1 PGF2 level s i n the ovary The mean +SEM ovarian levels of PGF2 1 <in both of the long photoperiod females are given i n Figure 8. The highest value (6 18.0 ±189.2 pg/ml) was found i n January, and the lowest l e v e l (256.0 ±66.6 pg/ml) was found i n February. No s i g n i f i c a n t difference was found between the monthly l e v e l s of PGF2,* . The mean ovarian lev e l s i n the natural photoperiod females are also given i n Figure 8 . There was a si m i l a r r i s e in the mean PGF2^ levels i n January (1001.4 ±556. 2 pg/ml) . The lowest mean was, however, in< March (277.8 ±74.3 pg/ml). Nevertheless, there was no s i g n i f i c a n t difference between the means from each month. Furthermore, there was no s i g n i f i c a n t difference between the PGF2^ le v e l s i n the natural- and long-photoperiod females. 4.5.2 PGF2 level s i n the plasma The mean ±SEM plasma PGF2„<. le v e l s i n the long and natural photoperiod females are given in Figure 8 for each month. The levels ranged from 976.4 ±375.7 pg/ml i n January, to 252.6 ±64.3 pg/ml i n February, i n the long-photoperiod f i s h . The natural-photoperiod group had a range of 438.9 pg/ml tc 1282.0 pg/ml i n February (note: there were only two samples i n thi s group, after pooling the plasma). The analysis of variance showed there was no s i g n i f i c a n t difference between the mean l e v e l of PGF2 in each month for both photoperiod groups. There was also no s i g n i f i c a n t difference between the two photoperiod regimes. 71 FIGURE 8: Seasonal v a r i a t i o n s i n p r o s t a g l a n d i n l e v e l s i n t h e female g o l d f i s h . The mean ±SEM l e v e l s of PGF2(<, PGE1 and PGE 1 i n t h e ovary ( l e f t hand s i d e ) and i n the plasma ( r i g h t hand s i d e ) of c o r r e s p o n d i n g f e m a l e s d u r i n g t h e f o u r month stu d y p e r i o d . S t a r r e d p o i n t s r e p r e s e n t means which 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 from each o t h e r (Newman-Kuels, p=0.05) . 72 ! 73 4.5.3 PGE 1 in the ovary Mean tSEM ovarian levels of PGE1 for the two photoperiod groups are given i n Figure 8. The peak value of PGE1 i n the long-photoperiod f i s h (834.0 ±155.5 pg/g) was found i n January, and the lowest value (404.0 ±.50.6 pg/g) was found i n March. These levels were not 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. The natural-photoperiod females had a peak l e v e l of PGE1 i n December (1027.0 ±67.7 pg/g), f a l l i n g to a low in March (171.0 +82-9 pg/g)- Although the decrease in mean level s i s quite sharp, i t i s obscured by the high standad deviation, owing to a low sample s i z e . There was no s i g n i f i c a n t difference between long- and natural-photoperiod f i s h , but when an analysis of variance was done on the pooled monthly values of PGE1, the decrease observed i n March was found to be s i g n i f i c a n t l y different (p= 0.05) from the other monthly values. 4.5.4 PGE1 level s i n the plasma Figure 8 gives the mean tSEM PGE1 levels i n plasma per month for the long- and the natural-photoperiod females. In the long photoperiod group, there was a s i g n i f i c a n t r i s e in PGE1 lev e l s , from 91.0 ±36.3 pg/ml in December to 1000.0 ±270.5 pg/ml in March. In the natural photoperiod animals, the lowest mean was i n January, at 102-9 pg/ml, and the peak was in February, at 1108.3 pg/ml. There was no s i g n i f i c a n t difference between the monthly levels of PGE1 in the natural photoperiod females. An analysis of variance indicated that the monthly means of the long- and natural-photoperiod animals were not s i g n i f i c a n t l y 74 different from each other. 4.5.5 PGB1 lev e l s i n the ovary The mean ±SEM ovarian levels of PGB1 are given in Figure 8 for the long-photoperiod f i s h . There i s a s i g n i f i c a n t decrease in PGB1 from 490.0 ±38.2 pg/g i n December, tc 20.0 ±7.2 pg/g i n March. The PGB1 l e v e l s i n the natural photoperiod f i s h , are also given i n Figure 8 and show a si m i l a r pattern; however, there i s no s i g n i f i c a n t difference between any of these values. There i s no s i g n i f i c a n t difference between the long-photoperiod and the natural-photoperiod animals. After peeling the values of these two groups, the analysis of variance indicated that a l l means except those i n January and February, 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. 4.5.6 PGB 1 levels i n the plasma Figure 8 gives the mean ±SEM plasma PGB1 levels for each month for long and natural photoperiod females. But the l e v e l s fluctuated in both groups, the peaks were found i n December and February and the lower values i n January and March. In the long-photoperiod group, the highest value was 387.9 ±73.6 pg/ml and the lowest was 74.7 pg/ml i n March. In the natural photoperiod group, the peak was 543.3 pg/ml i n December and 45.6 pg/ml i n January. The fluctuations in PGB1 i n either photoperiod group were not s i g n i f i c a n t l y different between each month. There was no s i g n i f i c a n t difference between the photoperiod groups. 7 5 However, when a l l the photoperiod values are pooled, the decreases between December and January were found to be s i g n i f i c a n t l y d i f f e r e n t (p=0.05). 4.5.7 Comparison of the prostaglandin l e v e l s i n the plasma and the ovary An analysis of variance did not show a s i g n i f i c a n t difference between monthly PGF2w.and PGB 1 le v e l s in the plasma and the gonad in both long- and natural-photoperiod f i s h , and in the pooled values. However, a s i g n i f i c a n t difference was found in PGE1 levels between plasma and ovary i n January, February and March, when the two photoperiod groups were pooled. 4^6 PROSTAGLANDIN HEASDBEMENT IN THE PLASMA AND TESTIS CORING DEVELOPMENT IN THE MALE A t o t a l of 45 males were sampled between December and March. Every month, eleven males were sampled from the long-photoperiod group and three from the natural-photoperiod group (except for December, when four males were sampled). The plasma from two samples was pooled i n these experiments, as for the females. 76 4.6.1 PGF2Uin the t e s t i s The mean ±SEM monthly l e v e l s of PGF2 o Cin the t e s t i s of the goldfish are shown i n Figure 9 for natural- and long-photoperiod males. There was no s i g n i f i c a n t difference between the natural and long-photoperiod f i s h . Both groups had a peak PGF20< l e v e l i n January of 2135.2 ±837.0 pg/ml for the long-photoperiod group, and 4003.8 ±1799.9 pg/ml fo r the natural-photperiod group. These level s decreased f i v e to 10 f o l d by March, to 418.6 ±174.0 pg/ml and 314.3 ±103.58 pg/ml, respectively. This increase i n mean PGF2 d C observed in January was not found to be s i g n i f i c a n t l y d ifferent from the other months, for both natural- and long-photoperiod f i s h . However, the mean pooled values of PGF2^in January were s i g n i f i c a n t l y d i f f e r e n t from the other months. 4.6.2 PGF2^ levels i n the plasma The mean +SEM plasma levels of PGF2 o Cin the males are given in Figure 9. These values did not show the large increase which was observed i n the gonad. The range i n plasma EGF2<<in the long-photoperiod f i s h was from 806.8 ±229.2 pg/ml in December, f a l l i n g to 550.7 +179.3 pg/ml i n March. In the natural-photoperiod group, the highest l e v e l was i n March, at 758. 1 ±120.3 pg/ml, and the lowest was in December, at 112.9 pg/ml. There was no s i g n i f i c a n t difference between the monthly values in both natural- and long-photoperiod, nor was there any difference between the two photoperiod groups. Futhermore, no. s i g n i f i c a n t differences were found after pooling the values. 77 FIGURE 9: Seasonal v a r i a t i o n s i n p r o s t a g l a n d i n l e v e l s i n t h e male g o l d f i s h . The mean +SEM l e v e l s of PGF2^, PGE 1 and PGB 1 i n the t e s t i s ( l e f t hand s i d e ) and i n t h e plasma ( r i g h t hand s i d e ) of c o r r e s p o n d i n g males d u r i n g t h e f o u r month stu d y p e r i o d . Double s t a r r e d p o i n t s a r e means which were s i g n i f i c a n t l y d i f f e r e n - t front s i n g l e s t a r r e d p o i n t s (Newman-Kuels, p=C. 05) . 18 79 4.6.3 PGE1 level s i n t e s t i s Figure 9 gives the mean ±SEM t e s t i c u l a r levels of PGE1 i n males for each month. An increase i n the mean le v e l s of PGE1 occurs in March in the natural-photoperiod f i s h , and in February in the long-photoperiod f i s h (1161.8 ±484.6 pg/g and 970.0 +646.0 pg/ml, respectively). However, these increases were 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 other months due tc the large standard deviation of the mean. There was no s i g n i f i c a n t difference between the two photoperiod groups. The pooled monthly values of PGE1 observed i n the t e s t i s were not 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. 4.6.4 PGE 1 level s in the plasma Figure 9 shows the the mean ±SEM monthly PGE1 le v e l s found in the male plasma. The levels f o r the long-photoperiod animals ranged from 160.2 ±37. 1 pg/ml i n December to 674. 5 ±37.3 pg/ml in March. In the natural-photoperiod group, the highest mean was 1010.4 pg/ml i n December, and the lowest was 275.8 pg/ml in February. There was no s i g n i f i c a n t difference between the monthly values in either photoperiod groups The two photoperiod groups were not found to be 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, and no s i g n i f i c a n t difference was found between the monthly means of the pooled values. 80 4.6.5 PGB 1 l e v e l s i n the t e s t i s F i g u r e 9 shows the mean ±SEM t e s t i c u l a r PGB1 l e v e l s f o r each month. The PGB 1 l e v e l s i n both the n a t u r a l - and long-photoperiod f i s h decreased s i g n i f i c a n t l y between December and January, and again between February and March. Furthermore, the i n c r e a s e observed between January and February was a l s o s i g n i f i c a n t . The h i g h e s t mean l e v e l s of PGB1 were 641.5 ±66.0 pg/ml and 594.9 ±50.5 pg/ml i n the n a t u r a l - and long-photoperiod groups, r e s p e c t i v e l y . The lowest mean l e v e l s were 39.6 pg/ml i n the n a t u r a l - , and 62.4 ±18.0 pg/ml i n the long-photcperiod groups. There was no s i g n i f i c a n t d i f f e r e n c e i n the monthly l e v e l s between the two photoperiod groups. When these values were pooled, the outcome was the same, i n that a l l months were d i f f e r e n t from one another except January and March. 4.6.6 PGB1 l e v e l s i n t h e plasma The mean +SEM plasma PGB1 l e v e l s i n the males are given i n Figure 9. The p a t t e r n of monthly changes p a r a l l e l e d those i n the gonad. A s i g n i f i c a n t decrease occurred between December and January i n long-photoperiod and i n n a t u r a l - p h o t o p e r i o d f i s h . The highest l e v e l s were found i n December and the means were 515.0 +53.5 pg/ml i n the l o n g - and 745.1 pg/ml i n the n a t u r a l -photoperiod f i s h . The lowest l e v e l s o ccurred i n January i n both groups, and the l e v e l s were 62.4 ±10.9 pg/ml and 34.3 pg/ml i n the l o n g - and n a t u r a l - p h o t p e r i o d groups, r e s p e c t i v e l y . There was no s i g n i f i c a n t d i f f e r e n c e between the two photoperiod groups. When the values were pooled, the i n c r e a s e i n February was again 8 1 found t o be s i g n i f i c a n t l y d i f f e r e n t from J a n u a r y and March. 4 . 6 . 7 Comparison of t h e p r o s t a g l a n d i n l e v e l s i n t h e plasma and the t e s t i s There was no s i g n i f i c a n t d i f f e r e n c e i n the monthly v a l u e s of PGE1 and PGB1 between the gonad and plasma i n the male. However, a f t e r p o o l i n g t h e v a l u e s o f t h e two p h o t o p e r i o d groups, the J a nuary mean PGF2„<. l e v e l i n the gonad was found t o be s i g n i f i c a n t l y h i g h e r t h a n t h a t o f the plasma. JK7 DISCUSSION OF PROSTAGLANDINS The r e PGs d u r i n g changes i n gonadal ma e f f e c t on a Due t o and t h e v e r s t u d y , i t o c c u r r i n g i a n t i c i p a t e d m a t u r i t y i n s t a t e s wou was not t h e m a t u r i t y , f a c t , t h a t IN THE FEMALE not c l e a r l y d e f i n e t h e r o l e o f he g o l d f i s h . At t h i s p o i n t , the o t c o r r e l a t e w i t h changes i n o p e r i o d appear t o have a d i r e c t s u i t s o f t h i s s t u d y do s e x u a l m a t u r a t i o n of t monthly PG l e v e l s do n t u r i t y , nor does phot ny of t h e PG l e v e l s . t h e asynchronous n a t u r e o f o o g e n e s i s i n the g o l d f i s h y narrow range of o v a r i a n s t a t e s observed i n t h i s i s d i f f i c u l t t o u n d e r s t a n d the changes which are n PG l e v e l s over the p e r i o d of s t u d y . I t had been t h a t t h e l o n g p h o t o p e r i o d would a c c e l e r a t e gonadal the f e m a l e , and t h a t a d e f i n i t e p o l a r i t y c f o v a r i a n I d r e s u l t between December and March. However, t h i s c a s e , as t h e r e was l i t t l e d i f f e r e n c e i n o v a r i a n based on h i s t o l o g y , between any of t h e months. In t h e r e was o n l y a low r a t i o o f t e r t i a r y o o c y t e s i n the 82 l a s t month of s a m p l i n g . F u r t h e r m o r e , p h o t o p e r i o d d i d not seem t o a f f e c t PG l e v e l s , as t h e r e was no d i f f e r e n c e between t h e n a t u r a l - and t h e l o n g - p h o t o p e r i o d groups. The PG l e v e l s of i n d i v i d u a l f i s h were i n v e s t i g a t e d i n o r d e r t o see whether any of the extreme v a l u e s belonged t o f i s h which had s i m i l a r o v a r i a n c h a r a c t e r i s t i c s . The PG c o n c e n t r a t i o n i n f i s h which had a g r e a t e r preponderance of a t r e t i c f o l l i c l e s was 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 mean v a l u e o f t h a t month. O v a r i a n l e v e l s of PGF2^were found t o have t h e g r e a t e s t v a r i a t i o n w i t h i n a month. A f t e r l o o k i n g at o v a r i a n PGF2^ v a l u e s i n i n d i v i d u a l f i s h , i t was observed t h a t a l l the v e r y h i g h v a l u e s of PGF2o(. were measured i n females whose o v a r i e s had o o c y t e s i n the t e r t i a r y y o l k s t a g e . However, not a l l o v a r i e s h a v i n g t e r t i a r y o o c y t e s had a h i g h P G F 2 * c o n c e n t r a t i c n . T h i s o b s e r v a t i o n suggested t h a t the more mature o v a r y has a g r e a t e r c a p a c i t y f o r pr o d u c i n g PGF2^upon h o m o g e n i z a t i o n than t h e p r e - v i t e l l o g e n i c o r e a r l i e r s t a g e s o f v i t e l l o g e n e s i s . I f t h i s i s so, the wide v a r i a t i o n i n P G F 2 ^ c o n c e n t r a t i o n which was observed c o u l d be due to the r e l a t i v e q u a n t i t y o f t e r t i a r y o o c y t e s homogenized i n the sample. The plasma PGF2 c Cand PGB1 c o n c e n t r a t i o n s c o r r e l a t e d w e l l w i t h t h e i r c o r r e s p o n d i n g v a l u e s i n the o v a r y , as no s i g n i f i c a n t d i f f e r e n c e was found between the two t i s s u e s . However, between December and March t h e r e was a s i g n i f i c a n t i n c r e a s e i n mean plasma PGE1 i n the fe m a l e , with a co n c o m i t a n t decrease i n mean o v a r i a n PGE1. T h i s i s d i f f i c u l t t o e x p l a i n , as the go n a d a l s t a t e o f the f i s h does n ot seem t o c o r r e l a t e i n any way w i t h t h e PGE1 l e v e l s i n t h e plasma o r i n t h e gonad. A l t h o u g h PGE1 does not 83 appear to have a direct role i n reproduction, i t may be acting i n d i r e c t l y by influencing the c i r c u l a t i o n of the ovary due to i t s vasodilatory capacity. This has, i n f a c t , been suggested to be the case in some mammals ( Smith et a l 1967). The pooled ovarian PGE1 and PGB1 levels showed a s i g n i f i c a n t decrease in March, and the PGF2<< l e v e l s also decreased at t h i s time, but not s i g n i f i c a n t l y . If, indeed, oogenesis i s proceeding toward larger yolk-laden oocytes, i t i s possible that at t h i s point the l i p i d pool i n the ovary i s being diverted toward oocyte production, rather than toward PG production. 4^8 DISCUSSION OF PROSTAGLANDINS IN THE MALE In the males, the PG levels i n the t e s t i s and in the plasma fluctuate over the sampling period. However, as in the females, these changes cannot be corrolated with sexual maturity, as no h i s t o l o g i c a l changes were observed in the t e s t i s over t h i s period. A l l the male goldfish sampled were i n a mature , prespawning reproductive state and, although there was a range in the GSI values of 2.26 to 5.46, there was no in d i c a t i o n of a corre l a t i o n between the GSI and the PG l e v e l . Furthermore, photoperiod did not seem to have an effect on PG l e v e l s in the male. There was a dramatic increase i n the mean PGF2^ levels i n the t e s t i s during January (Figure 9) . A very large standard error of the mean i s associated with these l e v e l s : the mean of the pooled values i s 2644 pg/ml; yet half of the values are ever 84 3000 pg/ml, and the other half are well below 1C00pg/ml. There i s no h i s t o l o g i c a l clue to explain what might be influencing PG synthesis i n these f i s h . Clemens and Grant (1964) reported seasonal variation i n the hydration of the t e s t i s cf the goldfish. It i s possible that changes in hydration may somehow be influencing PG l e v e l s , and that t h i s may account for some of the v a r i a b i l i t y in PG concentration i n the t e s t i s . However, there are two arguments against t h i s : f i r s t , that GSI, which i s an i n d i r e c t measure of gonadal water content, does not appear to r e f l e c t PG l e v e l s , and secondly, that only one of the prostaglandins, PGF2, shows a high degree of v a r i a b i l i t y . It i s also possible that prostaglandins control c i r c u l a t o r y changes that, in turn, are responsible for hydratuicn. However* there are no data to support this hypothesis at present. The mean PG l e v e l s in the t e s t i s and i n the plasma p a r a l l e l each other, except for PGF2^which i s very high in the gonad i n January and February, but stable i n the plasma throughout the four month period. Nevertheless, these results suggest that plasma PGE1 and PGB1 could be used as a r e f l e c t i o n of t h e i r concentration i n the gonad; however* th i s would not be true for PGF2=(, and i t would be inadvisable to use plasma PGF2^ concentrations to assess the condition of the gonad. i t i J CONCLUSION The r e s u l t s of t h i s study are inconclusive i n regards to PG involvement i n gonadal maturation. However, t h i s i s the f i r s t account of the presence of PG i n the ovary, t e s t i s and plasma of 8 5 the g o l d f i s h , and c e r t a i n o b s e r v a t i o n s suggest t h a t the ovary i s c a p a b l e of p r o d u c i n g a h i g h c o n c e n t r a t i o n of PGF2(. I t seems p l a u s i b l e t h a t PGs do p l a y a r o l e i n t h e s e x u a l m a t u r a t i o n o f the g o l d f i s h , a l t h o u g h t h e s e e x p e r i m e n t s > d i d net demonstrate t h e i r p r e c i s e i n v o l v e m e n t . 86 5 PROSTAGLANDINS DURING OVULATION IN THE GOLDFISH 5 i J INTRODUCTION P r o s t a g l a n d i n s have been suggested t o be i n v o l v e d i n o v u l a t i o n i n mammals ( C l a r k et a l 1978; L e M a i r e e t al,1973) , and in d o m e t h a c i n has been shown t o b l o c k o v u l a t i o n i n g o l d f i s h (Stacey and Pandey 1975). The i n d o m e t h a c i n b l o c k can be overcome i n the g o l d f i s h by t r e a t m e n t w i t h PGF2 £ <,PGE1 and PGE2 (Stacey and Pandey 1975). F u r t h e r m o r e , J a l a b e r t and S z o l l o z i (1975) r e p o r t e d t h a t PGF2 o tcould s t i m u l a t e i n v i t r o o v u l a t i o n of t r o u t o o c y t e s . The e x p e r i m e n t s i n p r e v i o u s s e c t i o n s c f t h e p r e s e n t s t u d y have demonstrated t h e presence of p r o s t a g l a n d i n s i n t h e plasma and gonads of male and female g o l d f i s h ; however, they d i d not a l l o w d e t e r m i n a t i o n of the r o l e o f PGs i n gonadal m a t u r a t i o n . T h e r e f o r e , t h e e x p e r i m e n t s o u t l i n e d i n t h i s s e c t i o n were undertaken t o study the p o s s i b l e i n f l u e n c e of PGs i n t h e f i n a l s t a g e s of oocyte m a t u r a t i o n , and i n o v u l a t i o n i n the fema l e . 5^2 REVIEW OF THE MECHANISMS OF OVULATION 5.2.1 H i s t o l o g i c a l changes o f t h e o o c y t e I n t h e g o l d f i s h , o o g e n e s i s proceeds u n t i l t h e t e r t i a r y o o c yte s t a g e and t h e o v a r y , p r i o r t o o v u l a t i o n , c o n t a i n s p r i m a r l y o o c y t e s a t t h i s stage of development. The maintenance 87 of these v i t e l o g e n i c o o c y t e s appears t o be under t h e c o n t r o l of the p i t u i t a r y (Yamazaki 1965; S t a c e y 1977; H u r l h u r t 1977; Lam et a l 1978). Yamamato and Yamazaki (1967) o u t l i n e d the f o l l o w i n g events which o c c u r i n t h e f i n a l m a t u r a t i o n and o v u l a t i o n c f the g o l d f i s h o o c y t e s : 1) There i s an i n c r e a s e d v a s o d i l a t i o n i n the f o l l i c u l a r l a y e r . 2) G e r m i n a l v e s i c l e m i g r a t i o n (GVM) t a k e s p l a c e a p p r o x i m a t e l y f i v e hours p r i o r t o o v u l a t i o n . T h i s c o n s i s t s of t h e m i g r a t i o n of the n u c l e u s t o the a n i m a l p o l e o f t h e o o c y t e . 3) G e r m i n a l v e s c i c l e breakdown (GVBD) , or t h e d e g e n e r a t i o n of t h e membrane s u r r o u n d i n g t h e n u c l e u s , o c c u r s . 4) A decrease i n the number and s i z e of t h e lys c s o m e s p r e s e n t i n the g r a n u l o s a c e l l s . (The lysosomes c o n t a i n both p r o t e a s e s and ca r b o h y d r a s e s . ) 5) A d e g e n e r a t i o n of the m i c r o v i l l i which connect t h e ooc y t e w i t h t h e g r a n u l o s a (perhaps due t o p r o t e o l y t i c enzymes ( J a l a b e r t 1976)). 6) An a c c u m u l a t i o n of f l u i d i n the i n t r a c e l l u l a r spaces between the t h e c a e x t e r n a and t h e c a i n t e r n a (due, perhaps, t o t h e p r o d u c t i o n of o s m o t i c a l l y a c t i v e s u b s t a n c e s by t h e ca r b o h y d r a s e enzymes). 7) These s t e p s l e a d t o f o l l i c l e r u p t u r e and t h e a c t i v e e x p u l s i o n of t h e o o c y t e i n t o the o v a r i a n c a v i t y ( o v u l a t i o n ) . T h i s s e r i e s of e v e n t s a l s o o c c u r s i n o t h e r t e l e o s t f i s h ; 88 Jalabert (1975) has described s i m i l a r ( i n v i t r o ) changes i n the mature trout oocyte. 5.2.2 Endocrinological control of ovulation The endocrine control of the f i n a l maturation and ovulation in teleosts has been studied by many authors (see reviews by Yamamato and Yamasaki 1967; Jalabert 1976; Lam et a l 1978), and a brief description of some of the major c o n t r o l l i n g agents w i l l be given here. Although the p i t u i t a r y plays a ce n t r a l role in coordinating the ovulatory process, other hormones such as the ovarian steroids, corticosteroids and prostaglandins have alsc been shown to be involved in the process. Several studies have demonstrated that substances which can stimulate gonadotropin (GTH) production, such as clomiphene c i t r a t e and certain l u t e i n i z i n g hormone-releasing hormones (IH-EH), can induce ovulation i n i n t a c t goldfish (Pandey and Hoar 1972; Lam et a l 1975; Lam et a l 1976). Furthermore, administration of p a r t i a l l y p u r i f i e d salmon gonadotropin (SG-G 100) restores ovulation i n hypophysectomized goldfish (Yamasaki 1962; Yamasaki and Donaldson 1968). A gonadotropin surge has been demonstrated during ovulation in the goldfish (Breton et a l 1972) and recently, Stacey et a l (1979) were able to define the precise timing of t h i s surge. They reported that GTH starts to increase approximately 10-12 hours prior to ovulation and remains elevated u n t i l ovulation, then decreases sharply. The p i t u i t a r y appears to be controlled by environmental factors such as l i g h t 89 and temperature (Hontela and Peter 1978; B i l l a r d et a l 1S78). Steroid hormones appear to play an important role at the time of ovulation i n f i s h . Khoo (1974) claimed that progesterone was capable of inducing in vivo ovulation in goldfish at both 12 and 20 C. Jalabert (1976) reported that 17-*-hydroxy-20-/6-dihydroprogesterone (17-<<-OH20-/3-P) was the most e f f e c t i v e in promoting in v i t r o oocyte maturation i n gol d f i s h , rainbow trout and northern pike. However, t h i s s t e r o i d could not induce ovulation in the goldfish at 12*C (Pandey, unpublished observations). However, i t appears that 17-X-OH-20-A-P stimulates GVBD only after GVM has occurred and that GVM i s under p i t u i t a r y control (Jalabert et a l 1977) . The corticosteroids seem to play an i n d i r e c t role in ovulation i n some f i s h . Certain c o r t i c o s t e r o i d s , p a r t i c u l a r l y , 11-deoxycorticosterone and 11-deoxycortisol, are e f f e c t i v e i n inducing i n v i t r o ovulation i n the goldfish (Jalabert et a l 1973; Jalabert 1976). The c o r t i c o s t e r o i d pathway could offer another control mechanism in ovulation which could be important in stress-related ovulation. Research on the involvement of prostaglandins during ovulation in f i s h was described in the Introduction. However, a b r i e f mention w i l l be made here of some of these findings. Stacey and Pandey (1975) found that indomethacin, a prostaglandin i n h i b i t o r , could block HCG-induced ovulation i n the goldfish, and that PGF2ot, PGE 1 and PGE2 could restore the response. Jalabert and S z o l l o z i (1976) demonstrated that PGF2 was very e f f e c t i v e in stimulating ovulation i n mature oocytes of the rainbow trout. Jalabert (unpublished r e s u l t s , 1976) also 90 reported that the PGF 2^  ovulatory response occurred with mature oocytes i n the goldfish and northern pike. Recently, Goetz and Theofan (1979) have observed that PGE2, and not PGF2* or PGE1, could induce i n v i t r o ovulation of perch oocytes. To further elucidate the role of prostaglandins during ovulation in f i s h , PGF2, PGE1 and PGB1 were measured in the plasma of the goldfish during ovulation. The results of t h i s experiment are reported below. 5-_3 METHODS 5.3.1 Fish maintenance A stock of approximately 150 f i s h weighing between 40 and 80 g each was purchased in A p r i l and kept in outdoor aguaria at ambient temperature (about 14° C) . Feeding was increased to at least once a day to promote growth and development of the f i s h . Frozen brine shrimp was included i n the d i e t , along with the trout p e l l e t s . Around mid-June i t was evident that some of the females were becoming gravid, indicated by the distended apperance of the abdomen and an enlarged gonadopore. 5.3.2 Females Ovulation was induced in gravid females by increasing the water temperature to 20°C, and i n j e c t i n g f i s h with human chorionic gonadotropin (HCG) as described by Stacey and Pandey 91 (1S75) . The advantage of t h i s method i s that ovulation i s almost inev i t a b l e , as well as easily predicted. Fish w i l l usually ovulate eight to 12 hours following HCG i n j e c t i o n . Selected f i s h were removed from the stock tank and groups of four to f i v e were placed i n 20 gallon tanks. The temperature of the water was approximately 1H" C and imitation vegetation was made using strands of black p l a s t i c , to cover about 30% of the tank. In l a t e afternoon, (1600-1800 h) on the day prior to sampling, the water was turned o f f and a glass water heater was turned on, to gradually increase the temperature to 20"C. S e r i a l blood samples were taken from each f i s h beginning the following morning. Between 0800 and 0900 h, a f i s h was anesthetized i n MS222 (0.02% i n water), weighed, and a blood sample of between 1.0 and 1.5 ml was taken. The f i s h was then injected intraperitonealy with a solution of HCG (Human Chorionic Gonadotropin, ,1000 IU/ml PBS; #CG-2, Sigma, St-Louis, Mo.) at 10 10 per g weight, and then returned to the tank. This procedure was repeated for the remaining f i s h . Individual f i s h were recognized by coloration or by f i n c l i p s . The blood was then processed as described previously. Starting at seven hours after HCG i n j e c t i o n , the f i s h were checked every hour for ovulation. Ovulation was indicated by the occurrence cf a stream of oocytes running f r e e l y from the ovipore when gentle pressure was applied to the abdomen. At ovulation, and on the morning a f t e r , the f i s h were stripped of as many eggs as possible. They were collected i n a polystyrene test tube and kept on i c e . After a few minutes of 92 s e t t l i n g , some of t h e o v a r i a n f l u i d was c o l l e c t e d and p r o c e s s e d i n t he same f a s h i o n as the gonadal t i s s u e s f o r the PG a s s a y . O v u l a t i o n e x p e r i m e n t s were performed on t h r e e s e t s o f f i s h . The p r e l i m i n a r y work was done on Group I which c o n s i s t e d o f s i x g r a v i d f e m a l e s . Two or t h r e e b l o o d samples were t a k e n from each of t h e s e f i s h , the f i r s t p r i o r t o HCG i n j e c t i o n and the second a t o v u l a t i o n . A t h i r d sample was t a k e n from two o f t h e s i x f i s h , on the morning f o l l o w i n g o v u l a t i o n . Group I I was a s m a l l s e t of t h r e e f e m a l e s which had o v u l a t e d s p o n t a n e o u s l y by t h e morning, f o l l o w i n g the i n c r e a s e i n water t e m p e r a t u r e . These f i s h were sampled t w i c e , t h e f i r s t b l o o d sample was t a k e n i m m e d i a t e l y t h a t morning, and the .second 24 hours l a t e r , on the f o l l o w i n g morning. U n f o r t u n a t e l y , i t was i m p o s s i b l e t o a s s e s s t h e exact t i m e o f o v u l a t i o n i n t h e s e t h r e e f i s h ; however, spontaneous o v u l a t i o n u s u a l l y o c c u r r e d a t 0400 0500 h, f o l l o w i n g an i n c r e a s e i n water temperature ( S t a c e y , p e r s o n n a l communication). The t h i r d s e t of f i s h . Group I I I , c o n s i s t e d o f 20 g r a v i d female f i s h . Four b l o o d samples were c o l l e c t e d from these f i s h . As w i t h Group I , t h e f i r s t sample was t a k e n p r i o r t o HCG a d m i n i s t r a t i o n , t h e second sample was t a k e n 6 hours l a t e r , and the t h i r d a t o v u l a t i o n . The f o u r t h and f i n a l sample was ta k e n on t h e morning f o l l o w i n g o v u l a t i o n (24 hours a f t e r HCG i n j e c t i o n ) . Two f i s h from t h i s s e t had not o v u l a t e d w i t h i n the 24 hour p e r i o d f o l l o w i n g HCG i n j e c t i o n . Samples were a l s o t a k e n from t h e s e f i s h , a t the d e s c r i b e d t i m e s . I n t h e s e cases the t h i r d sample was u s u a l l y t a k e n about 15 t o 16 hours a f t e r HCG a d m i n i s t r a t i o n . The mean time of o v u l a t i o n o f the r e m a i n i n g f i s h was 10 hours after HCG i n j e c t i o n . 5. 3. 3 Controls An experiment was set up to check whether s e r i a l sampling affected PG l e v e l s . Blood samples were taken from f i v e non-gravid females held i n cold (14° C) running water. The time i n t e r v a l s were the same as for the ovulating f i s h ; however, a 0.6% saline solution (0.01 ml/g) was injected instead of HCG. Thereafter, blood samples were taken i n the same time seguence, and processed i n the prescribed fashion. 5.3.4 S t a t i s t i c s An analysis of variance was performed cn a l l the data using the UBC ANOVAR program. The Newman-Keul multiple range test was performed on the different groups with a l e v e l of significance of 0.05. The paired t - t e s t was used on - the prelimenary data, where only two samples were taken from each f i s h ; again a signi f i c a n c e l e v e l of 0.05 was used. The s t a t i s t i c s were computed on the IBM 370 at UBC. 5.4 RESULTS OF OVULATION EXPERIMENT 94 5.4.1 Prostaglandin lev e l s i n the ovarian f l u i d The mean ±SEM leve l s of PGF2^in the ovarian f l u i d was 9088 ±2306.9 pg/ml, that of PGE1 was 636.0 ±137.4 pg/ml and PGB1 was 303.1 +64.2 pg/ml. It should be noted that i n four of the eight samples , the le v e l of PGF2^ .was so high that i t s binding r a t i o was off the standard curve and that the highest value on the curve (2400 pg) was given to these samples. This was probably an underestimate of the true value of the PGF20<: concentration i n these samples. 5.4.2 Prostaglandin lev e l s i n the plasma of ovulating and non-gravid females 5.4.2.1 PGF2^ level s i n Group I f i s h In the preparatory experiment, Group I, two blood samples were taken from each of six gravid females. The f i r s t was taken prior to HCG administration* and the second at ovulation. The mean ±SEM PGF2^ l e v e l before HCG i n j e c t i o n was 532.6 ± 49.3 pg/ml, and had increased to 4322.1 + 1221.7 pg/ml at ovulation. However, t h i s increase was not shown to be s i g n i f i c a n t (paired t - t e s t ) , as the high mean was due to two very high values cut of the six samples. The samples taken from the two f i s h 24 hours after HCG inj e c t i o n had a mean PGF2*level of 4778.0 pg/ml. 95 5-4.2.2 PGF2 o tlevels i n Group II f i s h In Group II f i s h , where three females had ovulated spontaneously by early morning, the PGF2*levels in the plasma were very high- The mean values of PGF2«;in the f i r s t sample was 7754 pg/ml; however, t h i s dropped to 885.2 pg/ml 24 hcurs l a t e r , a f t e r stripping the f i s h of eggs. 5-4.2.3 PGF2*levels i n Group III f i s h The PGF2oC prof i l e during ovulation i s shown i n Figure 10 for the f i s h from Group I I I , where four blood samples were taken. The mean ±SEM PGF2ct l e v e l s i n the plasma taken in the morning following the increase in water temperature and prior to HCG i n j e c t i o n was 317.4 ±30.3 pg/ml. There i s nc s i g n i f i c a n t change in the six hours following HCG administration; the mean plasma PGF2 0 <level at t h i s time was 292. 8 ±46.3 pg/ml. However, the mean PGF2^ did increase to 979- 3 ±323. 3 pg/ml at ovulation, when oocytes could be squeezed from the f i s h . This increase was 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 f i r s t two readings. The PGF^ levels jumped to 4093.9 ±558.8 pg/ml 12 hours after ovulation. At t h i s time, more eggs could be squeezed from the female, and generally there was a considerable volume of eggs. Two HCG-injected f i s h did not ovulate within 24 hours. The PGF2^in these individ u a l s remained under 300 pg/ml in a l l plasma samples. 96 5.4.2.4 PGF2o(.levels i n Group IV f i s h The mean ±SEM l e v e l s of PGF2,< in the non-gravid saline injected females, Group IV, are given in Figure 10 . There was a s l i g h t decrease i n PGF2oCover 24 hours i n these f i s h . The mean + SEM i n the f i r s t sample was 245.3 +94.3 pg/ml and dropped to 94.6 ±41.9 pg/ml i n the fourth sample. This decrease was not found to be s i g n i f i c a n t . 5.4.2.5 PGE1 lev e l s i n Group I and Group II f i s h The six females i n Group I had a PGE1 l e v e l of 7324.8 +903.4 pg/ml prior to ovulation. This value decreased two f o l d , to 4402.2 +1103.0 pg/ml at ovulation. The two females sampled at 24 hours after HCG i n j e c t i o n had a mean PGE1 l e v e l of 2 139.4 pg/ml. A three f o l d decrease in PGE1 lev e l s was found in the spontaneously ovulating f i s h , Group I I , from 7187.6 pg/ml to 2100.3 pg/ml between the f i r s t sample taken after ovulation, and the sample taken 24 hours l a t e r . 5.4.2.6 PGE1 levels i n Group III f i s h The changes in plasma PGE1 are shown i n Figure 10 . An overall decrease (two fold) was observed i n PGE1 i n the 24 hour period between HCG i n j e c t i o n and 12 hours after ovulation i n t h i s group of f i s h . The highest lev e l s were found prior to HCG administration , 260 1. 3 +396. 5 pg/ml, and decreased s l i g h t l y to 1471.7 ±283.0 i n the subsequent six hours. PGE1 leveled at 97 FIGURE 10: v a r i a t i o n s i n p r o s t a g l a n d i n l e v e l s d u r i n g HCG-induced o v u l a t i o n i n the g o l d f i s h . PGF2^ PGE 1 and PGB 1 i n the female g o l d f i s h . The t h i c k l i n e r e p r e s e n t s changes which o c c u r r e d i n g r a v i d females b e f o r e and a f t e r HCG i n j e c t i o n (10 IU//ml) and a t o v u l a t i o n (mean time 10 hours a f t e r HCG i n j e c t i o n ) . The t h i n l i n e r e p r e s e n t s changes which o c u r r e d i n non-g r a v i d , s a l i n e - i n j e c t e d (0.6% s a l i n e , 0.01ml//g) females over t h e same t i m e p e r i o d , (note: means w i t h a f i v e p o i n t s t a r a r e not s i g n i f i c a n t l y d i f f e r e n t from means w i t h a s i x p o i n t s t a r . ) 99 1607.1 ±370.2 pg/ml at ovulation* then decreased further to 1076.5 ±419.7 pg/ml 12 hours after ovulation. The decrease observed between the f i r s t and l a s t samples was s i g n i f i c a n t at the p=0.05 l e v e l . 5.4.2.7 PGE1 levels i n Group IV f i s h The mean PGE1 plasma l e v e l s i n the non-gravid, saline-injected f i s h were very low i n comparison to the ovulating f i s h . These l e v e l s ranged between 30.0 and 120.0 pg/ml and were 20 times lower than the highest mean in Group I I I . Figure 10 gives the changes in plasma PGE1 over the 24 hour sampling period. There was no s i g n i f i c a n t change in PGE1 leve l s over t h i s time. 5.4.2.8 PGB1 l e v e l s i n Group I f i s h In the preliminary study, a s l i g h t but i n s i g n i f i c a n t decrease was found in the plasma PGB1 leve l s (800.0 ±96.5 to 550.4 ±89.8 pg/ml). Furthermore, there was no change i n PGB1 in the plasma of the two f i s h sampled 12 hours afte r ovulation (679.0 pg/ml). 5.4.2.9 PGB1 l e v e l s in Group II f i s h The PGB1 l e v e l s i n the spontaneously-ovulating f i s h did not change between the f i r s t sample after ovulation and the second 24 hours l a t e r (806.6 ±135.5 to 806.6 ±299.5 pg/ml). 100 5.4.2.10 PGB1 level s i n Group III f i s h The mean PGB1 plasma lev e l s i n the f i s h from Group I I I are shown i n Figure 10 . There was a s i g n i f i c a n t decrease i n PGB1 between the pre-injection sample and the second sample (470.5 +39.5 to 266.3 +45.8 pg/ml), and between the ovulatory sample and the l a s t sample (270. 1 +42.6 to 147.7 +34.4 pg/ml). 5.4.2.11 PGB1 le v e l s i n Group IV f i s h There was a s i g n i f i c a n t decrease between the f i r s t and f i n a l samples in the non-gravid, sali n e - i n j e c t e d females. The mean PGB1 level s i n the non-gravid sali n e - i n j e c t e d females are shown i n Figure 10 . There was a s i g n i f i c a n t decrease in PGB 1 between the f i r s t and f i n a l blood samples i n t h i s group. 5^5 DISCUSSION OF OVULATION The above experiments strongly suggest that PGs play an important role i n ovulation i n the gold f i s h . S i g n i f i c a n t changes were found i n PGF2^and in PGE1 level s i n the plasma. There was a s i g n i f i c a n t decrease i n PGE1 during the 24 hour sampling period. Furthermore, the level s of PGE1 i n the gravid, HCG-primed females were over 20 times greater than i n the non-gravid sal i n e - i n j e c t e d females. These findings would suggest that PGE1 may be involved in f i n a l oocyte maturation, prior to ovulation. In the l i t e r a t u r e , i t has often been shown that PGF2 c <is the most i n f l u e n t i a l of the prostaglandins at the time of ovulation i n mammals (LeMaire et a l 1975; Clark et a l 1978). In the present 10 1 s t u d y , a l t h o u g h t h e r e was an i n c r e a s e i n PGF2 o {at the time of o v u l a t i o n , i t does not seem l i k e l y t h a t plasma PGF2«_was d i r e c t l y r e s p o n s i b l e f o r t h e i n d u c t i o n of o v u l a t i o n , as t h i s r i s e o c c u r r e d o n l y a f t e r o v u l a t i o n . However, t h e PGF2 < < l e v e l i s v e r y h i g h i n t h e o v a r i a n f l u i d , which bathes the o o c y t e s , and might i n f l u e n c e o v u l a t i o n i n the g o l d f i s h . The r e s u l t s f r c m the s a l i n e - i n j e c t i o n experiment demonstrated t h a t r e p e t i t i v e s a m p l i n g had no s i g n i f i c a n t e f f e c t on e i t h e r plasma PGF2*or PGE1 i n t h e g o l d f i s h . However* i t does appear t o have some e f f e c t on the plasma l e v e l s o f PGB1 , as t h e r e was a s i g n i f i c a n t d e c r e a s e i n PGB1 l e v e l s d u r i n g t h e s e r i a l s a m p l i n g o f the n o n - g r a v i d , s a l i n e - i n j e c t e d f i s h (Group IV) . The r e l a t i o n s h i p between t h e f i n d i n g s i n the l i t e r a t u r e and t h o s e i n t h e p r e s e n t study w i l l be d i s c u s s e d i n more d e t a i l i n the G e n e r a l D i s c u s s i o n . 5.5.1 PGF2 d u r i n g o v u l a t i o n O v u l a t i o n was checked every hour by g e n t l y massaging the abdomen of t h e f i s h ; I n n i n e o u t of 17 females i n t h i s group, o n l y a few eggs had o v u l a t e d ( l e s s t h a n 0.5ml). In t h e s e i n s t a n c e s , the p r o c e s s o f o v u l a t i o n was c o n s i d e r e d t o be j u s t b e g i n n i n g . I n the r e m a i n i n g e i g h t f i s h , many eggs had o v u l a t e d when the b l o o d sample was t a k e n , and o v u l a t i o n was c o n s i d e r e d t o have t a k e n p l a c e s h o r t l y b e f o r e the check. The mean plasma PGF2^ c o n c e n t r a t i o n i n c r e a s e d at o v u l a t i o n , but t h i s i n c r e a s e was not s i g n i f i c a n t . The apparent i n c r e a s e was caused by a b i a s due t o a few v e r y h i g h l e v e l s of PGF2*. I t i s i n t e r e s t i n g t o note t h a t the h i g h e r l e v e l s o f PGF2^were found i n t h o s e f i s h which had 102 o v u l a t e d a g r e a t e r volume of eggs. The range of PGF2*values i n the nine " j u s t ^ o v u l a t i n g " f i s h i s 103 t o 850.0 pg/ml with a mean ±SEM of 395.5 + 72. 1 pg/ml. The range of the PGF2 o Clevels i n the other e i g h t f i s h which had ovulated a g r e a t e r volume of eggs i s 332 to 5600 pg/ml with a mean ±SEM of 1808.9 +638.5 pg/ml. Since blood samples were taken at o v u l a t i o n and 12 hours l a t e r , i t i s d i f f i c u l t t o know e x a c t l y when PGF2*. s t a r t e d to i n c r e a s e i n the blood and how r a p i d l y i t d i d so. I t seems p l a u s i b l e from the above r e s u l t s t h a t the i n c r e a s e i n PG i n the plasma i s r e l a t e d t o the volume of ovulated eggs i n the o v a r i a n c a v i t y and t h a t t h i s i n c r e a s e i n the plasma i s very r a p i d . Ovulated eggs were removed f o l l o w i n g blood sampling. T h i s was done at the f i r s t o b s e r v a t i o n of o v u l a t i o n , and a g a i n the f o l l o w i n g morning, at which time the volume of eggs had g r e a t l y i n c r e a s e d . On one occasion* many eggs had ovulated a t the f i r s t o v u l a t o r y sampling, and the plasma PGF2^1evels i n t h i s f i s h at t h i s time were 5600 pg/ml. The f o l l o w i n g morning, the plasma PGF2^had decreased to 119.9 pg/ml* with very few eggs remaining i n the o v a r i a n c a v i t y . A s i m i l a r decrease i n PGF2t<was observed f o l l o w i n g egg removal i n the t h r e e s p o n t a n e o u s l y - o v u l a t i n g f i s h . The mean o v u l a t o r y l e v e l i n these f i s h was 7753.3 pg/ml. These samples were taken s e v e r a l hours a f t e r o v u l a t i o n . Twelve hours l a t e r , the mean PGF2*level had decreased t o 885.2 pg/ml; a g a i n , t h i s was a f t e r removal of many of the eggs. I t would appear from these observations t h a t the removal of the ovulated eggs leads to a decrease i n PGF2*in the plasma. These f i n d i n g s coupled with the presence o f a very high c o n c e n t r a t i o n of PGF20< i n the o v a r i a n f l u i d , would suggest t h a t the ovary i s the source of the PGF2t< 103 found i n the plasma. PGF2o<. c o u l d be l e a k i n g from the o v a r i a n f l u i d t o t h e plasma a f t e r o v u l a t i o n . PGF^ c o u l d a l s o be produced by the o v i d u c t upon s t r e t c h i n g . However, not a l l t h e o o c y t e s are i n t h i s a r e a a f t e r o v u l a t i o n , as most of the o v u l a t e d eggs remain i n the o v a r i a n c a v i t y , p r i o r t o spawning ( S t a c e y , 1974). The i n c r e a s e i n PGF2 e <cannot be a t t r i b u t e d d i r e c t l y t o the HCG i n j e c t i o n , as plasma PGF2otlevels d i d not change i n the two H C G - i n j e c t e d f i s h which d i d not o v u l a t e . T h i s f u r t h e r s u p p o r t s the h y p o t h e s i s t h a t t h e s o u r c e o f PGF2^ c o u l d be the o v a r i a n f l u i d p r e s e n t i n the o v a r i a n c a v i t y , and/or i n t h e o v i d u c t f o l l o w i n g o v u l a t i o n . 5.5.2 PGE1 d u r i n g o v u l a t i o n The PGE1 l e v e l s decrease i n the plasma d u r i n g t h e o v u l a t i o n p e r i o d . T h i s d e c r e a s e was observed i n f i s h f r c m Groups I , I I and I I I , which c o n t a i n e d g r a v i d f i s h o n l y . The f i s h from Groups I and I I demonstrated a two t o t h r e e t i m e s g r e a t e r PGE 1 c o n c e n t a t i o n than t h e f i s h i n Group I I I . There i s no apparent reason f o r t h i s d i s c r e p a n c y , as r e p e t i t i v e sampling o f t h e s a l i n e - i n j e c t e d f i s h d i d not a f f e c t PGE1 l e v e l s . Moreover, of t h e two H C G - i n j e c t e d f i s h which d i d not o v u l a t e , one showed a decrease i n plasma PGE1, but the o t h e r showed no change. There i s a c l e a r d i f f e r e n c e i n t h e plasma PGE1 c o n c e n t r a t i o n between g r a v i d and n o n - g r a v i d females. The mean plasma PGE1 l e v e l s i n t h e g r a v i d f e males i n the H C G - i n j e c t i o n e x periment are 10 t o 30 t i m e s g r e a t e r than the l e v e l s found i n t h e n o n - g r a v i d f e males used i n t h e s a l i n e - i n j e c t i o n experiment. 104 The h i g h c o n c e n t r a t i o n of PGE1 i n the g r a v i d f i s h d i d not appear to be d i r e c t l y l i n k e d t o o v u l a t i o n , as the two g r a v i d f e m a l e s which d i d not o v u l a t e a l s o had h i g h e r PGE1 l e v e l s t h a n t h o s e i n the s a l i n e group. T h i s s u g g e s t s t h a t PGE1 may be a n e c e s s a r y component of the f i n a l phases o f the m a t u r a t i o n p r o c e s s , i m m e d i a t l y p r i o r t o o v u l a t i o n . The s o u r c e o f PGE1 i s not c l e a r ; presumably i t i s s y n t h e s i z e d i n the o v a r y , as was suggested f o r PGF2. However, PGE1 does not f o l l o w the same s e c r e t i o n p a t t e r n as PGF2 C <. P G F ^ i n c r e a s e s d u r i n g o v u l a t i o n w h i l e PGE1 d e c r e a s e s , and the r e l a t i v e c o n c e n t r a t i o n of PGF2 o Lin the o v a r i a n f l u i d i s over 10 t i m e s t h a t o f PGE1 . The decrease observed i n the plasma c o u l d be due t o a s h i f t i n the p r e c u r s o r l i p i d s toward t h e p r o d u c t i o n of PGF2„,. 5.5.3 PGB1 l e v e l s d u r i n g o v u l a t i o n The plasma PGB1 l e v e l s d e c r ease i n both the o v u l a t i n g f i s h and i n the s a l i n e - i n j e c t e d f i s h . T h i s s u g g e s t s t h a t r e p e t i t i v e b l o o d s a m p l i n g may d e p l e t e PGB1 i n the plasma. There i s l i t t l e d i f f e r e n c e between plasma PGB1 l e v e l s i n t h e g r a v i d f i s h i n Group I I I and t h e n o n - g r a v i d f e m a l e s from Group IV. However, f i s h i n t h e p r e p a t o r y e x p e r i m e n t s . Group I , and the s p o n t a n e o u s l y o v u l a t i n g f i s h , Group I I , e x h i b i t e d plasma PGE1 l e v e l s which were two t o t h r e e t i m e s h i g h e r t h a n i n f i s h from the o t h e r two groups. The c o n c e n t r a t i o n of PGB1 i n the o v a r i a n f l u i d was t h e l o w e s t of a l l the PGs measured i n t h e f e m a l e . A l t h o u g h PGB1 i s 105 found i n the plasma and the o v a r i a n f l u i d d u r i n g o v u l a t i o n , t h e r e i s no e v i d e n c e t h a t i t i s changing d u r i n g t h i s t i m e . T h i s would suggest t h a t PGB1 does not p l a y a s i g n i f i c a n t r o l e d u r i n g o v u l a t i o n . The o v e r a l l f i n d i n g s i n t h e s e e x p e r i m e n t s suggest t h a t both P G I ^ a n d PGE 1 a r e i m p o r t a n t i n t h e f i n a l m a t u r a t i o n p r o c e s s , and a t o v u l a t i o n , i n t h e g o l d f i s h . PGE1 may t o be n e c e s s a r y p r i o r t o o v u l a t i o n , as i t i s i n a very h i g h c o n c e n t r a t i o n i n t h e plasma at t h i s t i m e , whereas PGF^^seems t o be more i m p o r t a n t a t , or a f t e r , o v u l a t i o n . There i s no e v i d e n c e t h a t PGB 1 i n f l u e n c e s o v u l a t i o n or t h e f i n a l s t a g e s of oocyte m a t u r a t i o n . A l l t h r e e PGs are p r e s e n t i n o v a r i a n f l u i d , w i t h PGF2 i n t h e h i g h e s t c o n c e n t r a t i o n . T h i s suggests t h a t t h e ovary i s t h e main sou r c e o f PGs . The i m p l i c a t i o n s of t h e s e r e s u l t s , t o g e t h e r w i t h t h e f i n d i n g s o f o t h e r r e s e a r c h e r s , w i l l be c o n s i d e r e d i n t h e G e n e r a l D i s c u s s i o n . 1 0 6 6 SPEEMIATION IN THE GOLDFISH 6 X J INTRODUCTION 6.1.1 T e s t i c u l a r changes d u r i n g s p e r m a t i o n S p e r m i a t i o n i n f i s h i s d e f i n e d as t h e r e l e a s e of mature sperm i n t o the sperm d u c t s , and i s due to a t h i n n i n g of t h e semen (Clemens and Grant 1964). S p e r m i a t i o n u s u a l l y b e g i n s e a r l y i n the spawning season and the t e s t i s remains i n t h i s s t a t e d u r i n g the e n t i r e spawning p e r i o d . S e v e r a l a u t h o r s (Yamamato and Yamazaki 1967; Yamasaki and Donaldson 1968b) have d i s c u s s e d the p r o c e s s of s p e r m i a t i o n i n the g o l d f i s h . S p e r m i a t i o n s t a r t s s h o r t l y a f t e r the l a s t spawning season and i s completed r a p i d l y w i t h i n one t o two months. A t the end of s p e r m a t o g e n e s i s , the spermatozoa a r e found i n t h e lumen of the t e s t i c u l a r l o b u l e s , where they are f r e e from the S e r t o l i c e l l s , and remain i n t h i s s t a t e t h r o u g h o u t th e s p e r m i a t i o n p e r i o d . P r i o r t o s p e r m i a t i o n , none of the l o b u l e s a r e connected to the s e m i n a l t u b u l e . However, s h o r t l y b e f o r e s p e r m i a t i o n , l o n g t u b u l a r l o b u l e s a r e formed as a r e s u l t of breakage of many o f the w a l l s between t h e l o b u l e s ; t h e s e l o b u l e s a r e t h e n connected t o t h e s e m i n a l t u b u l e s . F u r t h e r m o r e , the e p i t h e l i a l c e l l s of t h e sperm d u c t s and t h e i n t e r s t i t i a l c e l l s become h y p e r t r o p h i e d a t t h i s t i m e . There i s a l s o a marked h y p e r t r o p h y o f t h e b l o o d c a p i l l a r i e s i n both t h e t e s t i c u l a r l o b u l e s and the s e m i n a l t u b u l e s . I n a d d i t i o n , gonadal h y d r a t i o n i n c r e a s e s c o n s i d e r a b l y , 107 and s u b s e q u e n t l y a f l u i d i t y o f t h e m i l t d e v e l o p s . Yamazaki and Donaldson (1968b) have l i n k e d t h e h y p e r t r o p h y observed i n t h e c e l l s of t h e sperm d u c t s w i t h t h e i n c r e a s e i n s e m i n a l f l u i d i t y , and have suggested t h a t t h e s e c e l l s may be s e c r e t i n g f l u i d i n t o the d u c t s . Yamamato and Yamazaki (1967), f o l l o w i n g h i s t o c h e m i c a 1 o b s e r v a t i o n s , have a t t r i b u t e d t h i s i n c r e a s e i n gonadal water c o n t e n t t o an i n c r e a s e i n t h e enz y m a t i c breakdown of c a r b o h y d r a t e , which would f a v o r water uptake by the t i s s u e s . T h e i r h i s t o c h e m i c a l and u l t r a s t r u c t u r a l o b s e r v a t i o n s i n d i c a t e d t h a t both t h e S e r t o l i c e l l s and the sperm du c t s a r e very a c t i v e a t t h i s t i m e . A l t h ough the male i s f u n c t i o n a l l y mature from the onset of s p e r m i a t i o n , Sanchez-Eodriguez e t a l (1978) have r e p o r t e d t h a t t h e r e a r e changes i n sperm volume and the number of sperm r e l e a s e d over t h e s p e r m i a t i o n p e r i o d i n t h e rainbow t r o u t , Salmo g a i r d n e r i . I t i s l i k e l y t h a t t h e s e changes a l s o c c c u r i n the g o l d f i s h , and t h a t f l u c t u a t i o n s i n t e s t i c u l a r h y d r a t i o n would a l s o o ccur over t h i s p e r i o d (Clemens and Grant 1965). 6.1.2 E n d o c r i n e c o n t r o l of s p e r m i a t i o n I t has been w e l l e s t a b l i s h e d t h a t t h e p i t u i t a r y p l a y s a major r o l e i n the c o n t r o l of s p e r m i a t i o n i n f i s h (Clemens and Grant 1964,1965; Yamamato and Yamazaki 1967; Yamazaki and Donaldson 1968a, b,; B i l l a r d 1978; B i l l a r d e t a l 1978; Shanchez-E o r i g u e z e t a l 1978). An i n c r e a s e i n t e s t i c u l a r h y d r a t i o n i n t h e ca r p and g o l d f i s h , and s e m i n a l t h i n n i n g i n rainbow t r o u t have been observed f o l l o w i n g i n j e c t i o n s o f p i t u i t a r y e x t r a c t s 1C8 (Clemens and Grant 1964, 1965) . Yamamato and Donaldson 1968a, b) have shown t h a t spontaneous s p e r m i a t i o n i n g o l d f i s h does not o c c u r f o l l o w i n g hypophysectomy, and t h a t p i t u i t a r y e x t r a c t s and HCG c o u l d induce s p e r m i a t i o n i n both i n t a c t and hypophysectomized f i s h w h i l e LH and FSH were not e f f e c t i v e . Crim §1 (1975) have demonstrated t h a t plasma g o n a d o t r o p i n l e v e l s , of s e v e r a l s p e c i e s of s a l m o n i d s , are much h i g h e r a t the end of s p e r m a t o g e n e s i s and d u r i n g the spawning season than a t any o t h e r time. Yamazaki and Donaldson (1969) have a l s o found h i s t o c h e m i c a l e v i d e n c e of 3 - ^ - h y d r o x y s t e r o i d dehydrogenase i n the i n t e r s t i t i a l c e l l s of the g o l d f i s h . They observed t h a t hypophysectomy reduced t h e a c t i v i t y of t h i s enzyme* w h i l e a d m i n i s t r a t i o n of salmon g o n a d o t r o p i n (GTH) r e s t o r e d t h i s a c t i v e t y . However, they found no change i n enzyme a c t i v i t y d u r i n g s p e r m i a t i o n , d e s p i t e f i n d i n g s t h a t salmon-GTH induced h y p e r t r o p h y of the i n t e r s t i t i a l c e l l s and s p e r m i a t i o n i n hypophysectomized f i s h . They concl u d e d t h a t , a l t h o u g h t h e r e appeared t o be no c o r r e l a t i o n between 3-/3-h y d r o x y s t e r i o d dehydrogenase and s p e r m i a t i o n , t h e r e was an i n d i c a t i o n t h a t GTH may i n f l u e n c e s t e r o i d p r o d u c t i o n w hich, i n t u r n , c o u l d i n i t i a t e s p e r m i a t i o n . S anchez-Eodriguez et a l (1.978) have l o o k e d a t weekly plasma g o n a d o t r o p i n and androgen l e v e l s i n t r o u t , over a 12 week p e r i o d f o l l o w i n g the f i r s t i n d i c a t i o a n o f s p e r m i a t i o n . They r e p o r t e d t h a t plasma GTH was h i g h at t h e onset of s p s e r m i a t i o n , decreased i n t h e f o l l o w i n g 6 weeks, t h e n s t a r t e d t o i n c r e a s e , r e a c h i n g maximum l e v e l s on t h e 12th week. Androgen l e v e l s f o l l o w e d a r e v e r s e p a t t e r n . From t h e s e r e s u l t s , t h e y p o s t u l a t e d 109 t h a t the androgens c o u l d have a n e g a t i v e feedback on g o n a d o t r o p i n s e c r e t i o n . T h i s h y p o t h e s i s was a l s o made f o l l o w i n g e x p e r i m e n t s by B i l l a r d , E i c h a r d and B r e t o n (1977) and by B i l l a r d (1978). T h e i r s t u d i e s showed t h a t a d m i n i s t r a t i o n o f v a r i o u s androgens suppessed the i n c r e a s e i n g o n a d o t r o p i n s e c r e a t i o n which o c c u r s i n t r o u t f o l l o w i n g c a s t r a t i o n , and t h a t these r e s p o n s e s were g r e a t e s t d u r i n g t h e spawning p e r i o d . O v e r a l l , the p i t u i t a r y seems t o c o n t r c l the p r o c e s s of s p e r m i a t i o n i n f i s h . However, s t e r o i d s a l s o appear t o be i n v o l v e d , and t h e e x a c t f u n c t i o n s of these hormones have yet t o be e l u c i d a t e d . 6.1.2.1 Methods and r e s u l t s I n an a ttempt t o observe the p o s s i b l e i n v o l v e m e n t o f p r o s t a g l a n d i n s i n s p e r m i a t i o n i n t h e g o l d f i s h , PGF2,, PGE1 and PGB1 l e v e l s were measured i n the plasma of s p e r m i a t i n g g o l d f i s h , over a 24 hour p e r i o d f o l l o w i n g HCG a d m i n i s t r a t i o n . Four b l o o d samples were taken from each o f 10 s p e r m i a t i n g males. The f o l l o w i n g procedure was p a t t e r n e d on the p r e c e e d i n g o v u l a t i o n e x p e r i m e n t : t h e water was warmed t o 20°C and b l o o d samples were t a k e n the f o l l o w i n g morning. A f t e r t h i s , HCG (Human C h o r i o n i c G o n a d o t r o p i n 1,000 ID/ml PBS; Sigma, S t - L o u i s ) was i n j e c t e d at 10 IO/g, and b l o o d samples were t a k e n a t s i x , 10 and 24 hours a f t e r HCG i n j e c t i o n . The 10 hour sampling time was chosen as i t was t h e mean ti m e o f o v u l a t i o n i n t h e f e m a l e s . Four b l o o d samples were t a k e n from each f i s h . B l o o d samples were pr o c e s s e d as d e s c r i b e d p r e v i o u s l y and assayed f o r each p r o s t a g l a n d i n . A l l 110 males were mature, as m i l t was produced when p r e s s u r e was a p p l i e d t o the abdomen. 6.1.3 PGF2^, PGE1 and PGB1 l e v e l s i n the male p r i o r t c , and a f t e r , HCG i n j e c t i o n F i g u r e 11 shows the p r o f i l e of PGF2^in t h e plasma of t h e male g o l d f i s h d u r i n g the 24 hour p e r i o d f o l l o w i n g HCG a d m i n i s t r a t i o n . The mean ±SEM of PGF2^ i n t h e plasma sample p r i o r t o i n j e c t i o n was 236. 1 ±42.4 pg/ml. T h i s l e v e l decreased s l i g h t l y i n t h e l a s t sample t o 172.9 ±63.1 pg/ml; t h e r e was, however, no s i g n i f i c a n t d i f f e r e n c e between any of t h e samples. The changes i n t h e mean ±SEM i n plasma PGE1 f o l l o w i n g HCG i n j e c t i o n a r e g i v e n i n F i g u r e 11. There i s a s i g n i f i c a n t i n c r e a s e i n the PGE1 l e v e l from t h e p r e - i n j e c t i o n sample (990.7 + 171.0 pg/ml) to the t h i r d sample (2351. 4 ±470. 0 p g / m l ) , t a k e n t e n hours l a t e r . The mean PGE1 l e v e l had decreased t o 1432. 1 ±117.1 pg/ml by the f o l l o w i n g morning; however, t h i s d e c r e ase was not s i g n i f i c a n t . F i g u r e 11 shows t h e changes observed i n t h e mean ±SEM i n plasma PGB1 i n the same f i s h . There i s a s i g n i f i c a n t d e c r e ase between the p r e - i n j e c t i o n sample (561.1 ±45.6 pg/ml) and t h e t h i r d sample (452.2 +52.9 pg/ml), t a k e n t e n hour s l a t e r . A f u r t h e r d e c r ease was observed i n the f o u r t h sample (298.0 164.5 pg/ml) the f o l l o w i n g morning, which i s s i g n i f i c a n t l y d i f f e r e n t from t h e o t h e r t h r e e samples. 111 FIGURE 11 v a r i a t i o n s i n p r o s t a g l a n d i n l e v e l s f o l l o w i n g HCG i n j e c t i o n i n male g o l d f i s h . The mean +SEM l e v e l s o f PGF2^ PGE 1 and PGB1 i n the mature male g o l d f i s h b e f o r e and a f t e r HCG i n j e c t i o n (10 IU//ml) . The s t a r r e d p o i n t s i n d i c a t e means 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 each o t h e r (Newman-K u e l s , p = 0. 05) . C o n c e n t r a t i o n (pgm. ml. / P lasma) ZH 113 6-_2 DISCUSSION In the male go l d f i s h , plasma leve l s of PGF2^ did not fluctuate in the 24-hour period following HCG administration. However, there were s i g n i f i c a n t changes i n PGE1 and PGB1 levels of the same f i s h . PGE1 increased more than two f o l d 10 hours after HCG i n j e c t i o n , while i n the same time period, PGB1 dropped s i g n i f i c a n t l y to approximately 20% of the pre-injection l e v e l . PGB1 had decreased a further 30% by the following morning- I t i s d i f f i c u l t , at t h i s time, to correlate the changes in plasma PGE1 and PGB 1 with changes in the reproductive status of the f i s h . The male goldfish used in t h i s study were a l l f u l l y mature and in a state of spermiation. For t h i s reason, the changes in the prostaglandins cannot be d i r e c t l y attributed to the onset, or the induction, of spermiation, but may be regarded as changes due to an acute increase i n gonadotropin i n the f i s h . These are the f i r s t findings which indicate that prostaglandins may be involved in the spermiation process in the goldfish. However, the function and mode of action of the prostaglandins in the reproductive systems of male f i s h s t i l l remains to be elucidated. The relationship between prostaglandins and other hormones involved i n the spermiation process w i l l be considered i n the General Discussion. 114 7 GENERAL DISCUSSION The e x p e r i m e n t s on s e a s o n a l v a r i a t i o n s i n p r o s t a g l a n d i n s i n the e a r l y p a r t o f t h i s t h e s i s demonstrated the p r e s ence o f p r o s t a g l a n d i n s i n t h e plasma and gonads c f male and female g o l d f i s h ; however, they d i d not e s t a b l i s h t h e i r p o s s i b l e i n v o l v e m e n t i n the e a r l y gonadal m a t u r a t i o n o f e i t h e r sex. N e v e r t h e l e s s , t h e r e was an i n d i c a t i o n t h a t o v a r i e s h a v i n g y o l k y o o c y t e s a r e c a p a b l e o f a g r e a t e r s y n t h e s i s of PGF2^than the s e i n an e a r l i e r s t a g e . The o v u l a t i o n s t u d y suggested t h a t PGE1 and PGF2 o t are p r o b a b l y a s s o c i a t e d w i t h the f i n a l m a t u r a t i o n and o v u l a t i o n of t h e g o l d f i s h o o c y t e . The h i g h c o n c e n t r a t i o n c f PGF\ found i n t h e o v a r i a n f l u i d s u g g e s t s t h a t PGF2 o Cis v e r y i m p o r t a n t a t o v u l a t i o n * o r i n t h e p r o c e s s e s i m m e d i a t e l y f o l l o w i n g o v u l a t i o n . I n the males, however, s i g n i f i c a n t changes were observed o n l y i n PGE1. PGB1 d i d not appear t c be concerned w i t h any o f the r e p r o d u c t i v e p r o c e s s e s i n v e s t i g a t e d i n e i t h e r males or f e m a l e s . The p r e s e n t d i s c u s s i o n w i l l r e l a t e t h e s e r e s u l t s t o t h e f i n d i n g s of o t h e r r e s e a r c h e r s . T h i s w i l l be f o l l o w e d by a model of the p o s s i b l e r o l e o f p r o s t a g l a n d i n s i n g c l d f i s h r e p r o d u c t i o n , and an o u t l i n e of some of the p o s s i b l e mechanisms of t h e i r a c t i o n . A l t h o u g h the s e a s o n a l s t u d y d i d not show any changes o c c u r r i n g i n p r o s t a g l a n d i n l e v e l s t h a t c o u l d be a t t r i b u t e d t o gonadal m a t u r a t i o n , t h i s does not n e c e s s a r i l y mean t h a t p r o s t a g l a n d i n s are u n i m p o r t a n t i n t h i s p r o c e s s . The f a c t t h a t p r o s t a g l a n d i n s are p r e s e n t i n the gonad and f l u c t u a t e d u r i n g m a t u r i t y s u g g e s t s t h a t they may be f u n c t i o n i n g i n the o v e r a l l maintenance of the gonad. I t i s p o s s i b l e t h a t p r o s t a g l a n d i n s are 115 i n t e r a c t i n g w i t h o t h e r hormones from t h e p i t u i t a r y and the ovary and t e s t i s , d u r i n g the development of t h e gonad of both male and female g o l d f i s h . However, i n mammals, the i n v o l v e m e n t of p r o s t a g l a n d i n s i n t h e r e p r o d u c t i v e system appears t o be i n t h e f i n a l s t a g e s of m a t u r i t y , p r i o r t o o v u l a t i o n . I t i s t h e r e f o r e not s u r p r i s i n g t o f i n d a s i m i l a r p a t t e r n o c c u r r i n g i n f i s h . I n both f i s h and mammals, the r o l e of p r o s t a g l a n d i n s i n s p e r m i a t i o n i s not c l e a r , but t h e r e i s a s t r o n g p o s s i b i l i t y t h a t PGF2 0 (and PGE1 a r e v e r y i m p o r t a n t i n o v u l a t i o n i n both groups. Stac e y and Pandey (1975) demonstrated the n e c e s s i t y o f p r o s t a g l a n d i n s i n g o l d f i s h at o v u l a t i o n . They showed t h a t i n d o m e t h a c i n , a p r o s t a g l a n d i n i n h i b i t o r , c o u l d s u c c e s s f u l l y b l o c k o v u l a t i o n i n HCG-primed g o l d f i s h and t h a t PGE 1, PGE2 and PGF20( c o u l d overcome t h i s b l o c k and r e s t o r e o v u l a t i o n . As t h e exp e r i m e n t s d e a l t w i t h here were based d i r e c t l y on t h e methods of Stacey and Pandey (1975), i t i s p o s s i b l e t o make a c l o s e comparison between the two s e t s of f i n d i n g s . T h e i r e x periments showed t h a t i n d o m e t h a c i n was c o m p l e t e l y e f f e c t i v e i n b l o c k i n g o v u l a t i o n up t o s i x hours f o l l o w i n g HCG i n j e c t i o n . However, i t was o n l y p a r t i a l l y e f f e c t i v e a t . n i n e h o u r s , and t o t a l l y i n e f f e c t i v e 12 hours a f t e r HCG a d m i n i s t r a t i o n . I t appears t h a t , i n o r d e r t o i n h i b i t o v u l a t i o n w i t h i n d o m e t h a c i n , i t i s n e c e s s a r y to a d m i n i s t e r i t a t l e a s t two t o t h r e e h o u r s p r i o r t o o v u l a t i o n . They a l s o found t h a t i n d o m e t h a c i n would c o n t i n u e t o i n h i b i t o v u l a t i o n up t o a t l e a s t s i x days a f t e r i t s a d m i n i s t r a t i o n . I t appears from Stacey and Pandey*s study t h a t p r o s t a g l a n d i n s y n t h e s i s i s e s s e n t i a l f o r o v u l a t i o n i n the g o l d f i s h ^ They a l s o demonstrated t h e im p o r t a n c e o f t i m i n g i n t h e 116 a d m i n i s t r a t i o n of i n d o m e t h a c i n and p r o s t a g l a n d i n s . The f i n d i n g t h a t i n d o m e t h a c i n must be a d m i n i s t e r e d no l a t e r t han two t o t h r e e hours p r i o r t o o v u l a t i o n , i n o r d e r t o e f f e c t i v e l y b l o c k o v u l a t i o n , suggested two t h i n g s : f i r s t , t h a t p r o s t a g l a n d i n s y n t h e s i s i n c r e a s e s s h o r t l y b e f o r e o v u l a t i o n ; and s e c o n d l y , t h a t t h i s s p e c i f i c p r o s t a g l a n d i n p r o d u c t i o n i s a s s o c i a t e d w i t h o v u l a t i o n . In a d d i t i o n , they observed t h a t p r o s t a g l a n d i n replacement t h e r a p y was most e f f e c t i v e when g i v e n c l o s e to the e x p e c t ed t i m e of HCG-induced o v u l a t i o n . T h i s f u r t h e r s u p p o r t s t h e h y p o t h e s i s t h a t p r o s t a g l a n d i n s y n t h e s i s i s c r u c i a l p r i o r t o o v u l a t i o n . F u r t h e r m o r e , HCG-stimulated f o l l i c l e s were r e s p o n s i v e t o exogenous p r o s t a g l a n d i n s w i t h i n a r e s t r i c t e d p e r i c d , a s f o l l i c u l a r s e n s i t i v i t y had decreased c o n s i d e r a b l y by 15 hours a f t e r HCG a d m i n i s t r a t i o n . The p r e s e n t s t u d y has demonstrated t h a t l e v e l s of PGE1 and PGF2,x change s i g n i f i c a n t l y w i t h i n the p e r i o d of o v u l a t i o n . The plasma P G F 2 o C l e v e l s i n o v u l a t i n g f i s h showed t h e most d r a m a t i c change; the PGF2 0 ( l e v e l s had i n c r e a s e d more than 1 4 - f o l d a f t e r o v u l a t i o n . The r e v e r s e o c c u r r e d i n PGE1 l e v e l s i n t h e plasma, as PGE1 decreased by 2.5 t i m e s over t h e same p e r i o d . These changes appeared t o be r e l a t e d to the r e p r o d u c t i v e s t a t e o f t h e f i s h . The plasma PGF20< l e v e l s i n c r e a s e d o n l y i n t h o s e f i s h which had o v u l a t e d ; they d i d not i n c r e a s e i n t h e two g r a v i d females which d i d not o v u l a t e f o l l o w i n g HCG i n j e c t i o n . The plasma PGE1 l e v e l s were over 2 0 - f o l d g r e a t e r i n t h e g r a v i d , o v u l a t i n g f e m a l e s than i n the n o n - g r a v i d , s a l i n e - i n j e c t e d f e m a l e s . Stacey and Pandey's (1975) st u d y i n d i c a t e d t h a t p r o s t a g l a n d i n s y n t h e s i s i s most c r u c i a l j u s t p r i o r t o o v u l a t i o n . However, the t i m i n g c f the 117 p r o s t a g l a n d i n changes i n t h e b l o o d s u g g e s t s t h a t plasma PGF2 ( <and PGE1 are not d i r e c t l y i n v o l v e d i n i n d u c i n g o v u l a t i o n , as PGF2^ i n c r e a s e d o n l y a f t e r o v u l a t i o n and PGE1 was a t i t s h i g h e s t c o n c e n t r a t i o n n i n e t o 12 h o u r s p r i o r t o o v u l a t i o n . N e v e r t h e l e s s , t h e s e changes seem t o be o c c u r r i n g due t o an a l t e r a t i o n i n t h e r e p r o d u c t i v e s t a t e o f t h e f i s h , and i t i s p o s s i b l e t h a t p r o s t a g l a n d i n s i n the b l o o d are i n v o l v e d i n o t h e r e v e n t s i n t h e o v u l a t o r y p r o c e s s , or i n spawning. PGE1 may i n d i r e c t l y i n f l u e n c e h y d r a t i o n i n t h e ovary by two means: 1) i t may s t i m u l a t e v a s o d i l a t i o n i n the o v a r y , t h e r e b y i n d u c i n g the h y p e r t r o p h y of t h e b l o o d c a p i l l a r i e s i n the f o l l i c u l a r l a y e r b e f o r e t h e onset of o v u l a t i o n , as observed by Yamamato and Yamazaki (1967) 2) i t may a l s o i n c r e a s e t h e v a s c u l a r p e r m e a b i l i t y (Kennedy 1979) w h ich, i n t u r n , would cause an i n c r e a s e i n the uptake of water by t h e gonad. An i n c r e a s e i n water u p t a k e by t h e ovary i s one c f the n e c e s s a r y s t e p s l e a d i n g t o o v u l a t i o n * and i t i s p o s s i b l e t h a t PGE1 p r o d u c t i o n i s i n c r e a s e d i n o r d e r t o a l t e r t h e h y d r a t i o n of t h e ovary p r i o r t o o v u l a t i o n ; I t a l s o appears from th e p r e s e n t s t u d y t h a t HCG i s not i n d u c i n g t h e h i g h l e v e l s of PGE1 observed i n o v u l a t i n g f i s h . However, these f i s h had been s u b j e c t e d t o an i n c r e a s e i n water t e m p e r a t u r e , from 14°C t o 20°C, which may have s t i m u l a t e d PGE1 s y n t h e s i s ; a l t e r n a t i v e l y , PGE1 may be i n c o n s t a n t , h i g h l e v e l s i n g r a v i d g o l d f i s h . T h i s h y p o t h e s i s i s s u p p o r t e d by e v i d e n c e i n t h e s e a s o n a l s t u d y which found t h a t plasma l e v e l s of PGE1 i n the female had i n c r e a s e d s i g n i f i c a n t l y 118 from December t o March. The mean PGE1 l e v e l i n the plasma i n March was about 1C00 pg/ml, and was a p p r o a c h i n g t h a t measured i n the g r a v i d females i n t h e o v u l a t i o n s t u d y (mean of 2600 pg/ml) . The decrease i n PGE1 observed d u r i n g o v u l a t i o n c o u l d be t h e r e s u l t of a s h i f t i n p r o s t a g l a n d i n s y n t h e s i s toward t h e p r o d u c t i o n of PGF2<. PGF2<< i n c r e a s e d i n the plasma a f t e r o v u l a t i o n * y e t t h e e x a c t time of t h i s i n c r e a s e was d i f f i c u l t t o determine. There was some i n d i c a t i o n t h a t the i n c r e a s e was p r o p o r t i o n a l t o the q u a n t i t y o f o o c y t e s o v u l a t e d , and t h a t once t h e m a j o r i t y c f o v u l a t e d o o c y t e s had been removed, the PGF2ff(; l e v e l s d e c r e a s e d . There i s some evi d e n c e i n the l i t e r a t u r e on t h e p o s s i b l e f u n c t i o n s c f PGF2,* i n t h e b l o o d . S t a c e y (1977) found t h a t PGF2<*was very e f f e c t i v e i n s t i m u l a t i n g spawning b e h a v i o u r i n the female g o l d f i s h . P e t e r and B i l l a r d (1976) r e p o r t e d t h a t PGF2(* decreased g o n a d o t r o p i n s e c r e t i o n i n t h e p i t u i t a r y . I t seems p l a u s i b l e t h a t t h e i n c r e a s e i n PGF2o( f o l l o w i n g o v u l a t i o n c o u l d be t r i g g e r i n g spawning b e h a v i o u r , and t h a t t h e h i g h l e v e l s of PGF2C< would have a n e g a t i v e feedback on g o n a d o t r o p i n s e c r e t i o n . These two hypotheses w i l l be d i s c u s s e d i n more d e t a i l l a t e r . A l t h o u g h t h e p r o s t a g l a n d i n s i n t h e plasma do n o t appear t o be p l a y i n g a d i r e c t r o l e i n o v u l a t i o n , the o v a r i a n f l u i d c o n t a i n e d a l l t h r e e p r o s t a g l a n d i n s (PGB 1, PGE 1 and PGF2^) , and t h e s e may be more d i r e c t l y i n v o l v e d i n i n d u c i n g o v u l a t i o n . J a l a b e r t e t a l (1972) found t h a t c o e l c m i c ( o v a r i a n ) f l u i d c o l l e c t e d from t r o u t a t o v u l a t i o n , and used as an i n c u b a t i o n f l u i d , was e f f e c t i v e i n i n d u c i n g i n v i t r o o v u l a t i c n of mature t r o u t o o c y t e s . (GVM ( g e r m i n a l v e s i c l e m i g r a t i o n ) and GVBD 119 ( g e r m i n a l v e s i c l e breakdown) were completed.) J a l a b e r t and S z o l l o z i (1975) l a t e r demonstrated t h a t PGF2^c a t c o n c e n t r a t i o n s o f 1.0 and 5.0 mg/ml c o u l d i n d u c e i n v i t r o o v u l a t i o n of mature t r o u t f o l l i c l e s , but they found PGE2 t o be i n e f f e c t i v e a t the same c o n c e n t r a t i o n s . J a l a b e r t (1976) mentions i n h i s r e v i e w a r t i c l e t h a t PGF2oc.also promotes i n v i t r o o v u l a t i o n of g c l d f i s h o o c y t e s . I n view of the h i g h c o n c e n t r a t i o n of PGF2* p r e s e n t i n g o l d f i s h o v a r i a n f l u i d , i t seems l i k e l y t h a t PGF2,*. c o u l d be r e s p o n s i b l e f o r s t i m u l a t i n g t h e i n v i t r o o v u l a t i o n which J a l a b e r t observed i n t h e o v a r i a n f l u i d medium. I n the p r e s e n t s t u d y , t h e c o n c e n t r a t i o n of PGF2,* i n t h e o v a r i a n f l u i d was o ver 9 ng/ml, and t h e c o n c e n t r a t i o n i n the same i n c u b a t i o n medium which induced 100% o v u l a t i o n i n t r o u t o o c y t e s i n J a l a b e r t and S z o l l o z i ' s (1975) study was about 1000 t i m e s g r e a t e r (1.0 mg/ml). However, doses as low as 0.15 t o 0.30 mg/ml were e f f e c t i v e i n s t i m u l a t i n g p a r t i a l o v u l a t i o n , y e t these' l e v e l s a r e s t i l l o ver 100 times g r e a t e r t h a n the c o n c e n t r a t i o n o f PGF2^ measured i n the o v a r i a n f l u i d of the g o l d f i s h . A l t h o u g h the time r e q u i r e d f o r 100% o v u l a t i o n u s i n g 5.0 mg/ml PGF2 i n the medium was 20 h o u r s , o v u l a t i o n u s u a l l y s t a r t e d w i t h i n an hour of P G F 2 < a d d i t i o n and, i n some c a s e s , 50% o v u l a t i o n had o c c u r r e d w i t h i n t h e i n i t i a l t h r e e hours. The t i m e l a g of the response c o u l d be due t o the time r e q u i r e d f o r the o o c y t e s to absorb PGF2^ from th e medium o r , perhaps, t o s m a l l v a r i a t i o n s i n m a t u r i t y between oocyte p r e p a r a t i o n s . J a l a b e r t and S z o l l o z i (1975) r e p o r t e d t h a t t h e f o l l i c l e s must be c o m p l e t e l y mature, i . e . , GVM and GVBD must be c o m p l e t e d , i n o r d e r f o r PGF2 < <to be e f f e c t i v e , and t h a t Ca++ and Mg++ are 120 a l s o n e c e s s a r y f o r e x p u l s i o n o f t h e o o c y t e . From these d a t a , t h e y s p e c u l a t e d t h a t PGF2c<acts on the smooth m u s c l e - l i k e c e l l of the t h e c a , t h u s s t i m u l a t i n g c o n t r a c t i o n o f the f o l l i c l e and e x p u l s i o n of the o o c y t e . J a l a b e r t (1976) found t h a t PGE2 was i n e f f e c t i v e i n s t i m u l a t i n g i n v i t r o o v u l a t i o n . However, S t a c e y and Pandey (1975) showed t h a t b o t h PGE 1 and PGE2 c o u l d i n d u c e i n v i v o o v u l a t i o n i n t h e g o l d f i s h , and Goetz and Theofan (1979) found t h a t PGE 1 and PGE2, as w e l l as PGF2^ i n d u c e d i n v i t r o o v u l a t i o n of the perch o o c y t e . Goetz and Theofan (1979) r e p o r t e d t h a t i n v i t r o t r e a t m e n t of perch (Perca f l a y e s c e n s ) o o c y t e s w i t h 17-alpha-hydrcxy-20-b e t a - d i h y d r o p r o g e s t e r o n e at 15 C induced GVM and GVEE, and t h a t o v u l a t i o n u s u a l l y o c c u r r e d 33 hours l a t e r . T h i s response was c o m p l e t e l y b l o c k e d f o r 48 hours when i n d o m e t h a c i n was a l s o added to the i n c u b a t i o n medium. However, when PGF^, PGE1 or PGE2 was added t o t h e i n d o m e t h a c i n - p r o g e s t e r o n e t r e a t e d o o c y t e s a f t e r a 35 hour i n c u b a t i o n , the o v u l a t i o n response was r e s t o r e d . Of t h e t h r e e p r o s t a g l a n d i n s t e s t e d , Goetz and Theofan found PGE2 to be t h e most e f f e c t i v e ; a dose of 9.8 ng/ml i n d u c e d about t h e same percentage o v u l a t i o n as would have o c c u r r e d w i t h the 17-°<-OH-20-d i h y d r o p r o g e s t e r o n e w i t h o u t t h e i n d o m e t h a c i n . A h i g h e r c o n c e n t r a t i o n (160 ng/ml) of PGE1 and PGF2 was needed t o e l i c i t a s i m i l a r response. They t h e r e f o r e proposed t h a t s t e r o i d s ( i n p a r t i c u l a r , 17-«:-hydroxy-20-/3-dihydroprogesterone) i n i t i a t e GVM and GVBD and s t i m u l a t e p r o s t a g l a n d i n p r o d u c t i o n w h i c h , i n t u r n , i n d u c e s o v u l a t i o n i n the p e r c h . The mean c o n c e n t r a t i o n of PGF2* measured i n t h e o v a r i a n f l u i d of the g o l d f i s h was 9088 pg/ml, and t h a t of PGE1 was about 15 t i m e s l e s s , a t 636 pg/ml. Goetz 121 and Theofan (1979) f u r t h e r observed t h a t t h r e s h o l d r e s p o n s e s o f 1-5% o v u l a t i o n c o u l d be o b t a i n e d by a d d i n g 0.61 ng/ml of PGE1 o r PGE2 t o t h e i n c u b a t i o n medium c o n t a i n i n g i n d o m e t h a c i n ; however, 9.8 ng/ml of PGE^was r e q u i r e d t o e l i c i t a s i m i l a r r e s p o n s e . A l t h o u g h t h e s e l o w e r c o n c e n t r a t i o n s of PGE1 and PGF2 e <did not i n d u c e a s i g n i f i c a n t o v u l a t i o n r e s p o n s e , t h e y are v e r y c l o s e t o those measured i n t h e o v a r i a n f l u i d of the g o l d f i s h . B r e t o n e t a l (1972) r e p o r t e d an i n c r e a s e i n serum GTH l e v e l s j u s t p r i o r t o o v u l a t i o n i n the g o l d f i s h . Stacey e t a l (1979) have r e c e n t l y found t h a t GTH s t a r t s t o i n c r e a s e a p p r o x i m a t l y e i g h t hours b e f o r e o v u l a t i o n i n the g o l d f i s h , remains h i g h t h r o u g h o u t o v u l a t i o n , t h e n d e c r e a s e s r a p i d l y s h o r t l y a f t e r w a r d s . J a l a b e r t (1976) s p e c u l a t e d t h a t GTH i s n e c e s s a r y i n t h e f i n a l m a t u r a t i o n o f t h e f i s h o o c y t e . He s t a t e d t h a t g o n a d o t r o p i n s t i m u l a t e s 17-<*-OH-20-^-dihydroprogesterone p r o g e s t e r o n e s y n t h e s i s which, i n t u r n , t r i g g e r s the l a t e r s t a g e s of i n t r a f o l l i c u l a r m a t u r a t i o n ( m i g r a t i o n of the n u c l e u s to the ani m a l p o l e , g e r m i n a l v e s i c l e breakdown and i n c r e a s e i n i n t r a f o l l i c u l a r f l u i d ) . As PGF2^ i n c r e a s e s i n t h e b l o o d a f t e r o v u l a t i o n , i t i s u n l i k e l y t h a t P G F 2 o ( i n f l u e n c e s s y n t h e s i s of GTH or s t e r o i d s p r i o r t o o v u l a t i o n . However, PGE1 i s a t a ve r y h i g h c o n c e n t r a t i o n s e v e r a l hours p r i o r t o o v u l a t i o n , which s u g g e s t s t h a t i t c o u l d i n f l u e n c e e i t h e r (or both) of GTH and s t e r o i d s y n t h e s i s . U n f o r t u n a t e l y , t h e f i r s t PGE1 measurement was taken a p p r o x i m a t e l y 15 t o 16 hours a f t e r the water t e m p e r a t u r e had been i n c r e a s e d , and i t i s p o s s i b l e t h a t endogenous GTH l e v e l s were a l r e a d y i n c r e a s i n g by t h i s s t a g e . T h i s makes i t d i f f i c u l t t o s p e c u l a t e , w i t h any c e r t a i n t y , on t h e p o s s i b l e i n t e r a c t i o n o f 122 PGE1 w i t h o t h e r hormones. P e t e r and B i l l a r d (1976) demonstrated t h a t PGE2 and PGF^ suppressed serum GTH when a dose of 2.0 ug was d i r e c t l y i n j e c t e d i n t o t h e t h i r d v e n t r i c l e o f mature female g o l d f i s h . However, GTH l e v e l s i n c r e a s e d s l i g h t l y f o l l o w i n g a 2.0 ug dose o f PGE1. Although lower doses o f PGE1, PGE2 and PGF2c<were w i t h o u t e f f e c t , PGF2o( showed a tendency t o decrease GTH a t t h e l o w e s t dose o f 0.5 ug. They suggested t h a t t h i s e f f e c t was v i a t h e hypothalamus. I t i s p o s s i b l e t h a t t h e i n c r e a s e i n plasma PGF2^ observed a f t e r o v u l a t i o n c o u l d have a n e g a t i v e feedback on GTH s e c r e t i o n . T h e r e f o r e , PGF2^could be t h e c o n t r o l l i n g agent which t u r n s o f f the GTH s e c r e t i o n observed a f t e r o v u a l t i o n . T h i s n e g a t i v e feedback from PGF20< would be v e r y i m p o r t a n t i n f i s h w h i c h , l i k e t h e g o l d f i s h , spawn s e v e r a l t i m e s d u r i n g a season. A c o n t i n u a l h i g h l e v e l of GTH might induce a l l the mature eggs t o o v u l a t e a t the same t i m e , and s u c c e s s i v e spawnings would be i m p o s s i b l e . I t i s i n t e r e s t i n g t o n o t e here t h a t GTH l e v e l s remain h i g h f o r s e v e r a l days i n t r o u t and i n sockeye salmon, and t h a t both o f t h e s e are synchronous spawners (Crim et a l 1975; F o s t i e r e t a l 1978) . The i n v o l v e m e n t o f p r o s t a g l a n d i n s i n spawning b e h a v i o u r o f female g o l d f i s h was f i r s t e s t a b l i s h e d by S t a c e y (1976). An e a r l i e r s t u d y had shown t h a t spawning b e h a v i o u r c o u l d be i n d u c e d i n female g o l d f i s h by t h e i n j e c t i o n o f o v u l a t e d eggs (from a donor female) through the o v i p o r e and i n t o the o v a r i a n lumen (S t a c e y and L i l e y 1974). Stacey (1976) then found t h a t i n d o m e t h a c i n (10 mg/g) c o u l d b l o c k t h i s i n d u c e d spawning b e h a v i o u r when g i v e n e i t h e r 10 h o u r s p r i o r t o , or c o i n c i d e n t 123 w i t h , t h e i n j e c t i o n o f o v u l a t e d eggs. I n a d d i t i o n , he d i s c o v e r e d t h a t PGF2,<. (5 mg/g) was v e r y e f f e c t i v e i n overcoming t h e i n d o m e t h a c i n b l o c k and c o u l d r e s t o r e spawning b e h a v i o u r . The same dose of PGE1 and PGE2 was a l s o t e s t e d and PGE2 was found t o be o n l y m a r g i n a l l y e f f e c t i v e . He a l s o observed t h a t t h e i n j e c t i o n o f PGF2^alone c o u l d i n d u c e spawning b e h a v i o u r i n f i s h h a v i n g v i t e l l o g e n i c o o c y t e s , b u t t h a t t h e r e was no response i n f i s h which had been hypophesectomized t h r e e or f o u r months e a r l i e r . However, when hypophysectomized f i s h were t r e a t e d w i t h salmon g o n a d o t r o p i n o r p i t u i t a r y e x t r a c t f o r two weeks, they responded t o e i t h e r oocyte i n j e c t i o n o r PGF20< a d m i n i s t r a t i o n . Replacement t h e r a p y u s i n g a m i x t u r e o f s t e r o i d s was i n e f f e c t i v e . A l t h o u g h t h e s t e r o i d s used d i d not r e s t o r e the c a p a b i l i t y f o r spawning b e h a v i o u r i n hypophysectomized f i s h , S tacey and L i l e y ' s e a r l i e r work (1974) showed t h a t 17-/5- e s t r a d i o l i n j e c t i o n was e f f e c t i v e i n r e s t o r i n g the spawning r e s p o n s e i n i n t a c t f i s h h a v i n g r e g r e s s e d o v a r i e s . From t h e r e s u l t s of t h e s e two e x p e r i m e n t s , S t a c e y p o s t u l a t e d t h a t egg i n j e c t i o n (or t h e presence o f eggs i n the o v a r i a n lumen) s t i m u l a t e s the r e l e a s e of PGF2^ (perhaps v i a a d i r e c t s t r e t c h response or a f f e r e n t s t i m u l a t i o n ) w h i c h , i n t u r n , t r i g g e r s spawning b e h a v i o u r . He a l s o s t a t e d t h a t both p i t u i t a r y hormones ( e . g . g o n a d o t r o p i n ) and s t e r o i d s were e s s e n t i a l f o r t h e complete s e r i e s o f e v e n t s t o take p l a c e . Again t h e time f a c t o r was shown t o be i m p o r t a n t i n t h i s s t u d y , as a d m i n i s t r a t i o n o f i n d o m e t h a c i n c o u l d b l o c k egg-i n j e c t i o n - i n d u c e d spawning b e h a v i o u r w i t h i n minutes. However, spawning induced by PGF2;* a l o n e c o u l d sometimes l a s t up to two hours. 124 These observations are very i n t e r e s t i n g i n l i g h t cf the very high concentration of PGF2^(over 9 ng/ml) i n the ovarian f l u i d of the goldfish. In Stacey's (1976) study, ovarian f l u i d was injected along with the eggs and would be equivalent to i n j e c t i n g an exogenous source of PGF2^ This would not, however, refute the stretch hypothesis, as indomethacin could rapidly block spawning following egg i n j e c t i o n * which suggests seme de novo synthesis of prostaglandins. However, stretching alone cannot be the only stimulus which can e l i c i t spawning, since a r t i f i c i a l eggs were much less e f f e c t i v e in inducing spawning (Stacey 1977). It appears that both ovarian f l u i d and stretching are contributing to the release or appearance cf PGF2 at the time of ovulation. Spawning behaviour occurred following an a r t i f i c i a l increase of PGF2j< (presumably in the c i r c u l a t o r y system) when PGF2<x;Was injected i n t r a p e r i t o n e a l l y . This increase in PGF2^ levels can be compared to the increase which was observed i n the plasma of the goldfish at ovulation. From these data, i t i s possible to surmize that the cvarian f l u i d i s a major source of the PGF20<:found i n the blood after ovulation, and that the sharp increase in PGF2^could be c o n t r o l l i n g spawning behaviour. This i s further supported by evidence that removal of a l l ovulated oocytes r e s u l t s in cessation cf spawning (Stacey and L i l e y 1974), and that PGF2^ decreases i n the blood following t h i s removal. The fact that hypophysectomized f i s h and f i s h having regressed ovaries would not respond to either egg i n j e c t i o n or PGF2^administration t i e s in with some of the observations from the seasonal experiments i n the present study. As mentioned 125 p r e v i o u s l y , t h e r e was an i n d i c a t i o n t h a t o v a r i e s which had a g r e a t e r p r o p o r t i o n o f t e r t i a r y o o c y t e s had a g r e a t e r c a p a c i t y f o r P G F ^ s y n t h e s i s , as t h e PGF2<< l e v e l s i n s e v e r a l of these o v a r i e s were very h i g h (over one ng/ g ) , compared t o t h e o t h e r samples taken a t t h e same t i m e . I t i s p o s s i b l e t h a t the f i s h w i t h r e g r e s s e d o v a r i e s , i n S t a c e y ' s e x p e r i m e n t s , d i d not perform the spawning b e h a v i o u r because the o v a r i e s were unable t o s y n t h e s i z e s u f f i c i e n t PGF2 o Cto t r i g g e r t h e response. I n t h i s event, the ovary would need t o have v i t e l l o g e n i c o o c y t e s p r e s e n t i n o r d e r t o produce a h i g h s e c r e t i o n o f PGF2<, and t h e p i t u i t a r y hormones would be e s s e n t i a l f o r PGF2«; s y n t h e s i s because they would promote m a t u r a t i o n and m a i n t a i n v i t e l l o g e n i c o o c y t e s . The s t e r o i d s c o u l d i n f l u e n c e p r o s t a g l a n d i n s y n t h e s i s by e i t h e r d i r e c t s t i m u l a t i o n , o r by t h e maintenance o f v i t e l l o g e n i c o o c y t e s . S t a c e y ' s work i n d i c a t e d t h a t p r o s t a g l a n d i n s were e s s e n t i a l f o r spawning b e h a v i o u r and t h a t PGF2^appeared t o be t h e most i n f l u e n t i a l . The r e s u l t s from my study s u p p o r t t h i s h y p o t h e s i s , and f u r t h e r suggest t h a t t h e o v a r i a n f l u i d i s the source o f PGF 2^ . I n t h e males, p r o s t a g l a n d i n s may be p l a y i n g a r o l e i n s e x u a l development; however, t h e r e s u l t s o f t h i s s t u d y d i d not d e l i n e a t e t h e p r e c i s e f u n c t i o n o f p r o s t a g l a n d i n s d u r i n g e i t h e r m a t u r a t i o n or s p e r m i a t i o n . There was, however, a s i g n i f i c a n t change i n PGE1 l e v e l s f o l l o w i n g HCG i n j e c t i o n i n s p e r m i a t i n g f i s h . I t i s d i f f i c u l t t o r e l a t e these r e s u l t s t o t h o s e o f o t h e r r e s e a r c h e r s , as l i t t l e work has been done i n t h i s a r e a . Yamamato and Yamazaki (1968b) found t h a t salmon 126 g o n a d o t r o p i n (s-GTH) and HCG were e f f e c t i v e i n i n d u c i n g s p e r m i a t i o n i n hypophysectomized male g o l d f i s h . T h i s s u g g e s t s t h a t t h e a c t i o n of HCG on t h e gon a d a l t i s s u e s i s s i m i l a r t o t h a t of s-GTH. I t i s t h e r e f o r e r e a s o n a b l e t o compare the r e s u l t s from t h e p r e s e n t study w i t h f i n d i n g s of Clemens and Grant (1964) and Yamamoto and Donaldson (1968a, b; 1969) who used s-GTH i n most of t h e i r work. Clemens and Grant (1964) showed t h a t t h e p i t u i t a r y i n f l u e n c e d t e s t i c u l a r h y d r a t i o n i n c a r p . The f l u i d i t y cf the semen of t h e s e f i s h i n c r e a s e d t o a t o t a l o f 14-15% i n 24 h o u r s , f o l l o w i n g i n j e c t i o n s o f p i t u i t a r y e x t r a c t s . However, a sharp r i s e of 12% h y d r a t i o n o c c u r r e d d u r i n g t h e i n i t i a l 11 hours of the experiment. I t i s i n t e r e s t i n g t o note t h a t , i n t h e p r e s e n t s t u d y , t h e time of t h i s r i s e i n water c o n t e n t i n the t e s t i s c o i n c i d e d w i t h the peak i n plasma PGE1 measured i n t h e male g o l d f i s h 10 hours a f t e r HCG i n j e c t i o n . T h i s s u g g e s t s t h a t PGE1 may be i n f l u e n c i n g the h y d r a t i o n o f t h e t e s t i s d u r i n g s p e r m i n a t i o n . F u r t h e r e v i d e n c e , from Smith e t a l (1967) and Kennedy (1979), s u p p o r t s t h e h y p o t h e s i s t h a t PGE1 may somehow be a f f e c t i n g the water c o n t e n t o f t h e t e s t i s . I n l i g h t c f the v a s o d i l a t o r q u a l i t i e s of PGE1 (Smith e t a l 1967) , i t i s p o s s i b l e t h a t PGE1 mediates t h e h y p e r t r o p h y o f the b l o o d v e s s e l s i n t h e l o b u l e w a l l s d u r i n g s p e r m i a t i o n , as observed by Yamamoto and Yamazaki (1967). I n a d d i t i o n , i t i s a l s o p o s s i b l e t h a t PGE1 i n f l u e n c e s the uptake o f water by t h e t e s t i s , because PGE1 has been shown t o i n c r e a s e v a s c u l a r p e r m e a b i l i t y (Kennedy 1979). To my knowledge, t h e r e a re no s t u d i e s on the s h o r t - t e r m 127 effects of gonadotropin administration on androgen synthesis to compare with the present prostaglandin study. Furthermore, the function of steroids in spermiation i s not c l e a r l y understood, thus making i t d i f f i c u l t to determine the relationship between prostaglandins and steroids i n spermiation i n f i s h . The function of PGB1 i n the male is v i r t u a l l y unknown. The decrease in plasma PGB1 noted i n the male, was also observed i n a l l the female groups, as described i n the sections on ovulation. It i s possible that the decrease i n PGB1 l e v e l s i s due either to a s h i f t toward the synthesis of PGE1, or simply to a steady depletion of PGE1 i n the blood. PROPOSED MODELS OF THE FUNCTION OF PROSTAGLANDINS CURING OVULATION AND SPERMIATION The following model for the function of PGF2 and PGE1 during ovulation i n the goldfish i s proposed on the basis cf the data from t h i s study and the relationship between prostaglandins and other hormones involved in ovulation, as discussed previously. At present, t h i s model i s very speculative. I t appears that i n the goldfish, PGF2„< i s the most i n f l u e n t i a l at the time of ovulation. PGE1 also seems to be playing an important role; however, i t s function i s less easily defined. Figure 12 shows the proposed mode of involvement of prostaglandins in the events preceding, during and following ovulation. The high levels of PGE1 observed in the plasma up to 12-15 hours prior to ovulation increase the hydration of the ovary f i r s t l y , by increasing vasodilation in the ovary and secondly, by increasing the permeability of blood c a p i l l a r i e s i n the f o l l i c u l a r layer. PGE1 also stimulates the synthesis of 128 gonadotropin- (Peter and B i l l a r d (1976) found PGE1 to increase GTH secretion s l i g h t l y . ) at approximately the same time, gonadotropin secretion* which starts to increase several hours prior to ovulation (Stacey et a l 1979), stimulates ( p a r t i c u l a r l y 17-«t-OH-20-/3-didydroprogesterone) which, i n turn, promotes the f i n a l stages of oocyte maturation (GVM and GVBD). These events promote the production of PGF2 by: 1) breakage of the tissues (such as the m i c r o v i l l i attacking the oocytes) as the f o l l i c l e swells 2) stretching of the smooth muscle-like c e l l s i n the theca (Jalabert and S z o l l o z i 1975) and 3) direct stimulation of PGF2^synthesis by steroids or GTH. Once PGF2^ (or the PGF2^/PGE 1 ratio) reaches a s u f f i c i e n t l y high concentration in the ovarian f l u i d , PGF2^ and/or PGE1 stimulates ovulation by: 1) somehow influencing the protease enzymes which are contributing to the breakdown of the m i c r o v i l l i and the f o l l i c l e 2) promoting the contraction of the f c l l i c l e by stimulating the smooth muscle-like c e l l s i n the theca (most l i k e l y caused by PGF2^ ) 3) by further increasing the hydration of the f o l l i c l e u n t i l i t bursts and releases the oocyte These mechanisms of action of prostaglandins are mediated by the stimulation of the c y c l i c AMP or GMP system. (This has already been suggested to occur in f i s h (Kuc and Watanabe 1978) as well as in mammals (Le Maire et a l 1976b).) 129 FIGURE 12: Schematic r e p r e s e n t a t i o n of the proposed model of the r o l e of p r o s t a g l a n d i n s i n o v u l a t i o n i n the g o l d f i s h . GTH (gonadotropin) , GVM (germinal v e s i c l e migration) and GVBD (geminal v e s i c l e breakdown) . 131 Once ovulation has taken place, PGF2,;<from the ovarian f l u i d i s released into the c i r c u l a t o r y system where i t induces spawning behaviour and feeds back on the p i t u i t a r y to decrease gonadotropin secretion. (It i s necessary for gonadotropin secretion to decrease f o r r e p e t i t i v e spawning to take place; otherwise the oocytes would a l l ovulate at once.) In the males, the function of prostaglandins i s less clear; however, some suggestions can be made on the possible function, mainly of PGE1, during spermiation. The main action of PGE1 appears to be in influencing the hydration of the t e s t i s i n spermiating f i s h . Figure 13 shows some of the possible modes of involvement of prostaglandins in spermiation. Gonadotropin i s involved in the i n i t i a t i o n of spermiation and may also af f e c t hydration of the t e s t i s . (The water content of the t e s t i s i s increased following administration of GTH, Yamazaki and Donaldson (1968a).) Gonadotropin stimulates PGE1 synthesis which, in turn, increases the permeability of the blood vessels th e r i n . The t e s t i s i s then able tc increase i t s water content considerably (an es s e n t i a l step i n the spermiation process). PGE1 may then have some feedback e f f e c t on the p i t u i t a r y , although i t i s d i f f i c u l t to speculate on i t s exact mode of influence, owing to a d i s t i n c t lack of studies i n t h i s area. Obviously, there i s a d e f i n i t e need f o r further research on the involvement of prostaglandins in reproduction i n f i s h . S p e c i f i c a l l y , the following areas should receive immediate consideration: 1) the involvement of the PGE serie s i n t e s t i c u l a r 132 FIGURE 13: Schematic r e p r e s e n t a t i o n of the proposed model o f t h e r o l e of p r o s t a g l a n d i n s i n s p e r m i a t i o n . i n the g o l d f i s h . t HYDRATION TESTIS 134 h y d r a t i o n , 2) the i n t e r a c t i o n o f t h e p r o s t a g l a n d i n s w i t h o t h e r hormones i n v l o v e d i n spe r m a t o g e n e s i s and s p e r m i a t i o n , 3) t h e e x a c t s i t e of p r o s t a g l a n d i n s y n t h e s i s i n t h e ovary, 4) c o n t r o l l e d o b s e r v a t i o n o f t h e i n f l u e n c e of p r o s t a g l a n d i n s of the E s e r i e s on gonadal h y d r a t i o n , as w e l l as t h e i r i n f l u e n c e on enzyme systems i m p o r t a n t d u r i n g o v u l a t i o n , 5) the i n t e r a c t i o n o f p r o s t a g l a n d i n s w i t h o t h e r hormones, p a r t i c u l a r l y the s t e r o i d s and the p i t u i t a r y hormones, 6) t h e e f f e c t o f the h i g h c o n c e n t r a t i o n of i n t h e o v a r i a n f l u i d on t h e o o c y t e s , t h e m s e l v e s . 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P r e - o v u l a t o r y changes i n t h e c o n c e n t r a t i o n of p r o s t a g l a n d i n s i n r a b b i t g r a a f i a n f o l l i c l e s . P r o s t a g l a n d i n s . 3, 367-376. L e M a i r e , W.S., L e i d n e r , R. and Marsh, J.M. 1S75a. Pre-and p o s t -o v u l a t o r y changes i n the c o n c e n t r a t i o n of p r o s t a g l a n d i n s i n r a t g r a a f i a n f o l l i c l e s . P r o s t a g l a n d i n s 9,221-225. L e M a i r e , W.J. and Marsh, J.M. 1975b. I n t e r r e l a t i o n s h i p between p r o s t a g l a n d i n s , c y c l i c AMP and s t e r o i d s i n o v u l a t i o n . J . 146 Reprod..Fert. supl. 22, 53-74. Light, R.J. and Samuelsson, B. 1972. I d e n t i f i c a t i o n of prostaglandins i n the gorgonian, Plexaura homonalla Europ. J. Biochem. 28, 232:240. Linder, H. R. , Zor, 0., Bauminger, S., T s a f r i r i , A., Lamprecht, S., Koch, Y., Anteti, S. and Schwartz, A. 1974. 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Tohoku J o u r n a l o f A g r i c u l t u r a l R e s e a r c h . 23, 138-144. O h k i , S., Hanyu, T. , I m a k i , K., Nakazawa, N. and H i r a t a , F. 1974..Radioimmunoassays of p r o s t a g l a n d i n F2-main u r i n a r y m e t a b o l i t e s w i t h p r o s t a g l a n d i n - 1 2 5 1 - t y r o s i n e methyl e s t e r amide. P r o s t a g l a n d i n s ^ 6, 137-148. O r l o f f , J . , H a n d l e r , J.S. and Bergstrom, S. 1965. E f f e c t s of p r o s t a g l a n d i n (PGE1) on the p e r m e a b i l i t y response of t h e to a d b l a d d e r t o v a s o p r e s s i n , t h e o p h y l l i n e and adenosine 3,5-monophosphate. Nature. 205, 397-8. Ogata, H. and Nomura, T. 1975. I s o l a t i o n and i d e n t i f i c a t i o n o f PGE2 from the g a s t r o i n t e s t i n a l t r a c t of shark T r i a k i s S c y l l i a . B i o c h i e m i c a et B i o p h y s i u c s A c t a . 388, 84-9 1. Pandey, S. and Hoar, W.S.. 1972. I n d u c t i o n of o v u l a t i o n i n g o l d f i s h by clomiphene c i t r a t e . Can. J . Z o c l . 50, 1679-1680. P e t e r , R.E.. and B i l l a r d , R. 1976. E f f e c t s o f t h i r d v e n t r i c l e i n j e c t i o n of p r o s t a g l a n d i n s on g o n a d o t r o p i c s e c t e t i o n i n g o l d f i s h C a r a s s i u s a u r a t u s . Gen. and Comp. E n d o c r i n o l . 30, 451-456. Peyraud-Waitzengger, Nomura, T. and Peyraud, C. 1975. C a r d i o v a s c u l a r and v e n t i l a t o r y e f f e c t s of PGE2 i n t h e carp 148 ( C y p r i n u s C a r p i o ) L. . I n t e r n a t i o n a 1 Confe r e n c e on P r o s t a g l a n d i n s - A b s t r a c t s - May 26-30. P i p e r , J.P. and Vane, J.R. 1969. Release o f a d d i t i o n a l f a c t o r s i n a n a p h y l a x i s and i t s antagonism by a n t i - i n f l a m a t o r y drugs. Nature. 223, 29-35. P o w e l l , W. S. , Hammarstrom, S. and Samuelsson, B. 1974a. P r o s t a g l a n d i n F2 a l p h a r e c e p t o r i n o v i n e c o r p o r a l u t e a . Eur. J . Biochem. 41, 103-107. P o w e l l , W.S., Hammerstrom, S., Samuelsson, B. and S j o b e r g , B. 1974b. P r o s t a g l a n d i n F 2 ^ r e c e p t o r i n human c o r p o r a l u t e a . L a n c e t . 1, 1120. Rat n e r , A., W i l s o n , M.C., S r i v a s t a , L., Peake, G.T. 1974. S t i m u l a t o r y e f f e c t s of p r o s t a g l a n d i n E, on r a t a n t e r i o r p i t u i t a r y c y c l i c AMP and l u t e i n i z i n g hormone r e l e a s e . P r o s t a g l a n d i n s . 5, 165-210-Sakena, S.K., E l Sa F o u r y , S. and B a r t k e , A. 1973. PGE2 and PGF2^ decrease plasma t e s t o s t e r o n e l e v e l s i n male r a t s . P r o s t a g l a n d i n s . 4, 235-242. Salmon, H.A. and Karim, S.M.M. 1976. Methods f o r a n a l y s i s of p r o s t a g l a n d i n s . I n " P r o s t a g l a n d i n s : C h e m i c a l and b i o c h e m i c a l a s p e c t s " (S.M.M. Karim, e d i t o r ) , pp. 25-86. U n i v e r s i t y Park P r e s s , B a l t i m o r e , M a r y l a n d . 149 S a n c h e z - E o d r i g u e z , M.r E s c a f f r e , A. M., M a r l o t , S. , Eeinaud, P. 1978. The s p e r m i a t i o n p e r i o d i n t h e rainbow t r o u t ( Salmo g a i r d n e r i ) . Plasma g o n a d o t r o p i n and androgen l e v e l s , sperm p r o d u c t i o n and b i o c h e m i c a l changes i n the s e m i n a l f l u i d . Ann. B i o l . Anim. B i o c h . B i o p h y s . 18,943-948. Sa t o , T., Taya, K., J y u j o , T. , H i r o n o , M. and I g a r a s h i , M. 1974. The s t i m u l a t o r y e f f e c t o f p r o s t a g l a n d i n s on l u t e i n i z i n g hormone r e l e a s e . Am. J . O b s t e t . G y n e c o l . 118, 875-876. S c h r e c k , C.B., 1974. S e a s o n a l androgen and e s t r o g e n p a t t e r n s i n t h e g o l d f i s h C a r a s s i u s a u r a t u s . S h o r t papers and n o t e s , November 1974. 375-378. Shaw, J.E., Eamwell, E.W. 1969. S e p a r a t i o n , i d e n t i f i c a t i c n and e s t i m a t i o n of p r o s t a g l a n d i n s . Methods of b i o c h e m i c a l a n a l y s i s . 17, 235-371. S i n g h , A.D. and S i n g h , T.P. 1976. E f f e c t o f c l o m i d , s e x o v i d and p r o s t a g l a n d i n s on i n d u c t i o n o f o v u l a t i o n and g o n a d o t r o p i n s e c r e t i o n i n a f r e s h w a t e r c a t f i s h , H e t e r o p n e u s t e s f p s s i l i s ( B l o c k . ) E n d o k r i n o l o g i e . 68, 129-136. S i n g h , A.K. And S i n g h , T.P. 1977. T h y r i o d a c t i v i t y and TSH l e v e l i n P i t u i t a r y g l a n d and b l o o d serum i n response t o c l o m i d , s e x o v i d and p r o s t a g l a n d i n t r e a t m e n t i n H eteropneustes f o s s i l i s ( B l o c k ) . E n d o k r i n o l o g i e , 70, 69-76. 150 Smith, E. R. , McMorrow, J.V., C o v i n o , B.G. and L e e , J.B. 1967. Mechanisms of the h y p o t e n s i v e and v a s o d i l a t o r a c t i o n o f p r o s t a g l a n d i n E1. C l i n . Res. 15, 222.. S t a c e y , N. E. and N.R. L i l e y . 1974. R e g u l a t i o n c f spawning b e h a v i o u r i n t h e female g o l d f i s h . Nature. 247, 71-72. St a c e y , N.E. and Pandey S. 1975. E f f e c t s o f i n d o m e t h a c i n and p r o s t a g l a n d i n s on o v u l a t i o n i n g o l d f i s h . P r o s t a g l a n d i n s . 9, 597-607. S t a c e y , N.E. 1976. E f f e c t s o f i n d o m e t h a c i n and p r o s t a g l a n d i n s on the spawning b e h a v i o u r o f female g o l d f i s h . P r o s t a g l a n d i n s . 12, 113-124. S t a c e y , N. 1977. The r e g u l a t i o n o f spawning b e h a v i o u r i n the female g o l d f i s h , C a r a s s i u s a u r a t u s . Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia. S t a c e y , N.E., Cook, A.F., P e t e r , R.E. 1978..Ovulatory s u r g e o f g o n a d o t r o p i n i n the g o l d f i s h , C a r a s s i u s a u r a t u s . Gen. Comp. E n d o c r i n o l . 37, 246-249. T r u e b l o o d , K. N. and Malmberg, E. W. 1949. Chromatographic p r o p e r t i e s of s i l i c i c a c i d - c e l i t e . A n a l y t i c a l C h e m i s t r y . 21, 1055- 1058. Vane, J . R. 1969. The r e l e a s e and f a t e o f v a s o - a c t i v e hormones 151 in the c i r c u l a t i o n . Br. J. Pharmacol. 35, 209-242. Weinheimer, A. J. and Spraggins, E. L. 1969. The occurence of two new prostaglandin derivatives (15-epi-PGA2 and i t s acetate, methyl ester) in the gorgonian Plexaura Lpmcnalla . Tetrahedron Letters. 59, 5185. Wilks, J. , Wentz, A. C. and Jones, G. S. 1973. Prostaglandin F2, concentration in the blood of women during normal menstrual cycles and dysmeorrhea. J. of C l i n . Eudocrinol and Metabolism. 37, 469-471. W i l l i s , A.L. 1969. Eelease of histomine, kinin and prostaglandins during carrogemino-induced inflammation in the rat. In "Prostaglandins, Peptides and Amines" (Martegazza, P. and Horton, E.W., e d i t o r s ) . P 31. London. Academie. Yalow, E. S. and Berson, S. A. .1960. Immunoassay of endogenous plasma i n s u l i n i n man. J . C l i n . Invest. 39, 1157-1175. Yamamoto, K., Nagahamo, Y. and Yamazaki, F. 1966. A method to induce a r t i f i c i a l spawning of goldfish a l l through the year. B u l l . Japan. Soc. Sci. Fisheries. 32, 977-983. Yamamoto, K. and Yamazaki, F. 1966. Ehythm cf development i n the oocyte of the goldfish a l l through the year. Bul l . Japan. Soc. Fisheries. 32, 977-983. 152 Yamamoto, K. and Yamazaki, F. 1967. Hormonal c o n t r o l o f o v u l a t i o n and s p e r m i a t i o n i n g o l d f i s h . Gunma Symp. E n d o c r i n o l . 4, 131-145. Yamazaki, F. 1962. E f f e c t s of hypophysectomy on t h e o v u l a t i o n , o v i p o s i t i o n and s e x u a l b e h a v i o u r i n the g o l d f i s h , C a r a s s i u s a u r a t u s . B u l l . Fac. F i s h e r i e s . Hokkaido Univ. 13, 39-46. Yamazaki, F. 1965. E n d o c r i n o l o g i c a l s t u d i e s on the r e p r o d u c t i o n of the female g o l d f i s h , C a r a s s i u s a u r a t u s L., w i t h s p e c i a l r e f e r e n c e t o t h e f u n c t i o n o f the p i t u i t a r y g l a n d . Mem. Fac. F i s h . Hokkaido Univ. 13, 1-64. Yamazaki, F. and Donaldson, E. M. 1968a. The e f f e c t s of p a r t i a l l y p u r i f i e d salmon p i t u i t a r y g o n a d o t r o p i n on sp e r m a t o g e n e s i s , v i t e l l o g e n e s i s and o v u l a t i o n i n hypophysectomized g o l d f i s h ( C a r a s s i u s a u r a t u s ) . Gen and Comp. E n d o c r i n o l . 11, 292-299. . Yamazaki, F. and Donaldson, E. M. 1968b. Involvement o f g o n a d o t r o p i n and s t e r o i d hormones i n the s p e r m i a t i o n of t h e g o l d f i s h C a r a s s i u s a u r a t u s . Gen. Ccmp. E n d o c r i n o l . 12, 491-497. Yang, N.S.T., March, J.M.and L e M a i r e , W.J. 1973. P r o s t a g l a n d i n changes i n d u c e d by o v u l a t o r y s t i m u l i i n r a b b i t g r a a f i a n f o l l i c l e s . The e f f e c t o f i n d o m e t h a c i n . P r o s t a g l a n d i n s . 4, 395-404. 1 5 3 Zar, J. H. 1 9 7 4 . " B i o s t a t i s t i c a l Analysis". Prentice-Hall Inc. Inglewood C l i f f , N. J. 154 APPENDIX 155 Phosphate B u f f e r S a l i n e JPBS-A)_ - t o make 100 ml o f PBS-A, 0-25 g o f NaCl and 0.05 g c f p-a m i n o s a l i s y c l i c a c i d were d i s s o l v e d i n 80 ml o f a s o l u t i o n of 0.06N Na HPO , and was a d j u s t e d t o a pH of 7.4 by t h e a d d i t i o n of a p p r o x i m a t l y 20 ml of 0.06N KH PO ( t h i s s o l u t i o n was added dropwise as pH 7 was approached). Clinical Assays, Inc. 237 Binney Street. Cambridge. Massachusetts — / 0 2 1 4 2 (617) 492 2526 C A - 5 0 1 3 H P R O S T A G L A N D I N E R A D I O I M M U N O A S S A Y KIT For the quant i tat ive measurement of Prostaglandin A and E in serum or tissue extracts N O T E : STORE THIS KIT AT - 2 ( f C AFTER R E M O V I N G THE T W O V I A L S O F TRIS BUFFER C O N C E N T R A T E February 14, 1975 Radioimmunoassays 151 R a d i o a c t i v e m a t e r i a l T - Not for H u m a n U s e — I n t r o d u c t i o n into F o o d s , B e v e r a g e s , C o s m e t i c s , D r u g s , or M e d i c i n a l s or i n t o P r o d u c t s M a n u f a c t u r e d for C o m m e r c i a l D i s t r i b u t i o n is P r o h i b i t e d — E x e m p t Q u a n t i t i e s S h o u l d not be C o m b i n e d . C L I N I C A L A S S A Y S Division of Travenol Laboratories, Inc. PRECAUTIONARY INSTRUCTIONS T h e u s e r s h a l l s t o r e t h e b y - p r o d u c t m a t e r i a l u n t i l u s e d in the o r i g i n a l s h i p p i n g c o n t a i n e r or in a c o n t a i n e r p r o v i d i n g e q u i v a l e n t r a d i a t i o n p r o t e c t i o n . ' The following precautions should be observed in handling radioactive material: 1. H a n d l i n g s h o u l d p r e c l u d e any p i p e t t i n g by m o u t h . 2. There s h o u l d be no s m o k i n g or e a t i n g w h i l e r a d i o a c t i v e m a t e r i a l s are b e i n g h a n d l e d . 3 . H a n d s s h o u l d be c o v e r e d w i t h r u b b e r g l o v e s d u r i n g a n d t h o r o u g h l y w a s h e d after h a n d l i n g of r a d i o a c t i v e m a t e r i a l s . 4. S p i l l s s h o u l d be w i p e d up q u i c k l y a n d t h o r o u g h l y a n d t h e c o n t a m i n a t e d m a t e r i a l s a d d e d to r a d i o a c t i v e w a s t e m a t t e r . 5 . W a t e r s o l u b l e w a s t e r a d i o a c t i v e m a t e r i a l c a n be d i s p o s e d of i n t o t h e s a n i t a r y s e w a g e s y s t e m , if the c o n c e n t r a t i o n , after d i l u t i o n w i t h t h e l a b o r a t o r y d i s c h a r g e , d o e s not e x c e e d 4 x 10" 2 m i c r o c u r i e s per liter (1 J 5I) or 10 m i c r o c u r i e s per liter ( 3 H), b a s e d o n a d a i l y a v e r a g e of e f f l u e n t . W h e n e v e r p o s s i b l e , h o w e v e r , d i s p o s a l of r a d i o a c t i v e m a t e r i a l s h o u l d be m a d e t h r o u g h a l i c e n s e d d i s p o s a l s e r v i c e . LICENSING REQUIREMENTS T h e p r o c u r e m e n t of r a d i o a c t i v e m a t e r i a l in t h i s kit i s e x e m p t f r o m N R C or A g r e e m e n t S t a t e l i c e n s i n g r e q u i r e m e n t s . / 5T 2 . I N T R O D U C T I O N The C l i n i c a l Assays, I n c . , Prostaglandin E (PGE) Radioimmunoassay K i t offers a sensit ive method of measuring prostaglandin content inplasma and tissues by measuring the compet i t ive b ind ing of J H labe l led prostaglandin and un labe l led prostaglandin with antibody to prostaglandin . The k i t includes a l l the reagents required in the procedure . Assay of the P G E content is performed by measuring the amount of P rosta -g land in Bj (PGB X ) or Prostaglandin B 2 (PGBs) obtained after conversion of P G E X to P G B : o r P G E 2 to PGB- , by a l k a l i n e treatment. A n a n t i - P G B i rabbit serum is used for the radioimmunoassay. Separat ion of prostaglandin bound to ant ibody and free prostaglandin is ach ieved by prec ip i ta t ing the bound prostaglandin with a second ant ibody . A f te r cent r i fugat ion , the bound rad ioact i v i t y is measured in a l iqu id s c i n t i l l a t i o n counter . To e l iminate protein interference in the assay, a protein denaturation a n d / o r ext ract ion step is performed on the sample prior to assay ing . A l t e r n a t i v e l y , e x t r a c t i o n , fo l lowed by column separation of the various prostaglandins, may be employed (see page 5).= A standard curve is prepared for each group of samples by incubat ing known amounts of prostaglandins. Total time for the assay is a p p r o x i m a t e l y 2 work ing d a y s . This protocol is a modi f icat ion of the procedures of Levine (I) and Gut ie r rez— Cernosek (2) . The assay measures both PCEX and P G E S , s ince both are converted to PGBj and P G B 2 r espect i ve ly , by the a l k a l i n e t reatment . The c ross - react iv i ty curves on page l3 ind icate that P G B 2 cross-reacts 17% with the a n t i - P G B i serum at the 5 0 % binding l e v e l . If quant i f icat ion of P G E j and P G E 2 levels is des i red , it is possible to estimate the re lat ive proportions of P G E X and P G E 2 present by convert ing these prosta -glandins to P G F x i and P G F L J 9 end to P G F 2 a and P G F 2 A respect ively by a sodium bjjrohydride reduct ion (3) . The assay of Fj^a and F 2 a : levels can then be p e r -formed with the h ighly s p e c i f i c antisera of the C l i n i c a l Assays K i t s , C A - 5 0 2 and C A - 5 0 3 , to obtain the re la t i ve proportions of the E1 and E 2 o r i g i n a l l y present. This rat io can then be used in con junct ion wi th the known cross-react i v i t y of P G B 2 wi th the a n t i - P G B x serum suppl ied to c a l c u l a t e the or ig ina l P G E X and P G E 2 l e v e l s . P G A ! and P G A a are a lso converted to their respective P G B isomers by a l k a l i n e treatment. A n assay of the plasma extract with the PGo^ ant iserum, wi th no a l k a l i n e treatment, w i l l measure P G A X at about 14% c r o s s - r e a c t i v i t y , which can then be deducted as equiva lent B x from the assay of the a l k a l i n e - t r e a t e d e x t r a c t . R E A G E N T S This is the first commerc ia l ly a v a i l a b l e k i t for the determinat ion of Prostaglandin levels by radioimmunoassay. The radioimmunoassay k i t contains suf f ic ient r e -agents for 200 assay tubes, i nc lud ing 5 standard curves . The reagents provided are : Reconstituted Vo lume 1 v i a l Prostaglandin ^ Ant iserum, l y o p h i l i z e d 10 ml 1 v i a l Prostaglandin Standard, l y o p h i l i z e d 1 ml 2 v ia ls Rabbit Normal Serum, l y o p h i l i z e d 10 ml each 2 v ia ls G o a t A n t i - R a b b i t Serum, 10 ml each 1 v i a l 3 H labe l led Prostaglandin B 1 ( 2 M , C ! ) , l y o p h i l i z e d 10 ml 2 v ia ls T r i z m a - N a C l Concent ra te , preadjusted to pH 7 . 4 , 10 ml each 300 ml each 1 v i a l G e l a t i n (3g) The reconstituted reagents must be stored frozen at -I0°C to -20°C. Prosta -glandins are stable substances and the k i t may be thawed and frozen repeatedly without a f fect ing the performance of the kit reagents. R E A G E N T S REQUIRED BUT N O T PROVIDED I N KIT G l a c i a l A c e t i c A c i d 0 . I N Sodium hydrox ide ,hydrochlor ic a c i d solutions S U G G E S T E D A P P A R A T U S Precision Pipets: 1.0, 0 . 6 , 0 . 4 , 0 . 1 , 0 . 0 5 ml Cornwal l I ml Repeating Syringe Vortex M i x e r Centr i fuge L iquid S c i n t i l l a t i o n Counter Water Bath (37°C) and Ice Bath pH Meter D ia lys is Apparatus (Opt iona l ) S U G G E S T E D D ISPOSABLE MATERIALS Polypropylene tubes, Fa lcon N o . 2053 , 12x75 mm or equiva lent L iquid S c i n t i l l a t i o n v ia ls S c i n t i l l a t i o n F l u i d , C A - 7 0 2 or equiva lent 2 ml Ca l ib ra ted tubes (Fisher I0 -2 I2 -5B) or equivalent 4 . R E A G E N T P R E P A R A T I O N It is recommended that d i s t i l l e d water be used to reconstitute l y o p h i l i z e d reagents and to prepare a l l the necessary reagents required in this assay. A . Tris Buffer, Reagent A A d d : Contents of one v i a l of Tris Concentrate to 250 ml d i s t i l l ed w a t e r . Bring volume to .300 ml with w a t e r . Storage: Keep re f r igerated . S t a b i l i t y : Two months. B. Isogel Tris Work ing Buffer, Reagent B A d d : O.lOg G e l a t i n to 100 ml Reagent A . Heat and stir unt i l the ge la t in is d i sso l ved . Coo l the s o l u t i o n . Ad just : pH to 7 . 4 i f necessary, wi th O . l N sodium hydroxide o r O . l N hydroch lor ic a c i d . Storage: Keep re f r igerated . S t a b i l i t y : Prepare fresh every two or three d a y s . i C . Prostaglandin Standard A d d : I ml water to the contents of the standard v i a l . M i x gent ly and avoid foaming . Storage: Must ,be kept f r o z e n . S t a b i l i t y : M in imum three months. D . Prostaglandin Antiserum A d d : 10 ml Reagent B to the contents of the antiserum v i a l . M i x gent ly and avo id foaming . Storage: Must be kept f r o z e n . S t a b i l i t y : M in imum three months. E . 3 H Prostaglandin A d d : 10 ml Reagent B to the contents of the 3 H Prostaglandin v i a l . M i x gent ly and avo id foaming . Storage: Must be kept f r o z e n . S t a b i l i t y : M in imum three months. F . Rabbit Normal Serum A d d : 10 ml Reagent B to the contents of a Rabbit Normal Serum v i a l . M i x gent ly and avo id foaming . Storage: Must be kept f r o z e n . S t a b i l i t y : M in imum three months. P G E T O P G B C O N V E R S I O N The extract is made up to 2 . 0 ml in a ca l ibrated tube with the isogel tris buffer (Reagent B). Place 1.0 ml of this solut ion in a 2 ml screw cap v i a l and add 0.1 ml of I N sodium hydroxide (the pH should be adjusted to 12.5 to 12.9 using a pH meter with mic ro -e lect rodes ) . Screw the cap t i g h t l y , mix w e l l and place in a bo i l i ng water bath for 5 minutes. Cool and adjust the pH to approximately 7 .4 with 0.1 ml of I N g l a c i a l a c e t i c a c i d . Note the total volume of the PGB contain ing solut ion for later use in the c a l c u l a t i o n . A S S A Y PROCEDURE Prior to use^thaw the v ia ls conta in ing the prostaglandin ant ibody , p rosta -g land in standard, labe l led prostaglandin , assay samples, normal rabbit serum and goat an t i - r abb i t serum on i c e . Throughout the procedure keep a l l reagents on i c e . The assay procedure includes the preparation of a standard curve where known amounts of prostaglandin are used to compete with a f ixed amount of l abe l led prostaglandin in b inding to a f ixed amount of prostaglandin an t ibody . This standard curve is then used to determine the prostaglandin content of the assay samples from the binding obtained with each sample . The assays are performed in disposable p last ic tubes and each standard curve or assay point is carr ied out in d u p l i c a t e . A standard curve wi th 6 points to cover the range in P G B j of 8 pg to 2 ng is used . The reagents in the k i t are provided in the d i lut ions required to obtain the optimum sensi t iv i ty in the radioimmunoassay. P R E P A R A T I O N O F THE S T A N D A R D C U R V E The PGBj^ prostaglandin standard is furnished at a concentrat ion of 40 ng /ml in isogel tris buf fer , Reagent B. Prior to use in the assay a serial d i l u t i o n in the range of l / l to 1/243 should be prepared. The serial d i lut ions can be stored frozen and used as required to prepare the standard curves . P R E P A R A T I O N O F THE S T A N D A R D D I L U T I O N S 1. Into 5 tubes marked 1 /3 , 1 /9, 1 /27, 1/81 & 1/243 pipet 0 . 6 ml of Reagent B. 2 . The standard suppl ied is considered the l / l d i l u t i o n . Pipet 0 . 3 ml of l / l standard into the 1/3 tube . M i x thoroughly . 3 . N o w take 0 . 3 ml of the solut ion in tube 1/3 and add it to the tube marked 1 /9 . M i x thoroughly . Take 0 . 3 ml of the solut ion in tube 1/9 and add it to the tube marked 1 /27 . M i x thoroughly . Repeat this ser ia l d i l u t i o n to the last tube, 1 /243 . The d i l u t i on concentrations are as fo l lows , per 0 .05 ml : 2000 pg 1/27 74 pg ' / 3 667 pg 1/81 25 pg ' / 9 222 pg 1/243 8 . 2 pg These d i lu t ions of standard should be kept frozen with the remainder of the k i t , and are suf f ic ient for f ive complete standard curves . P R E P A R A T I O N O F THE S T A N D A R D C U R V E A N D A S S A Y SAMPLES 1. Into sixteen tubes numbered 1-16 pipet 1.0 ml of isogeltris buf fer . In a d d i t i o n , p lace an extra 0.1 ml in tubes I and 2 . Tubes I and 2 are background controls and conta in no antiserum or inh ib i to r (standard). Tubes 3 and 4 are b inding controls and contain no inh ib i to rs . Add 0 . 6 ml of the isogel tris buffer to the sample assay tubes, b e g i n -ning wi th tube 17. Each sample is run in d u p l i c a t e . 2 . To tubes 5 and 6 add 0 . 0 5 ml of l / l standard; mix w e l l . To tubes 7 and 8 add 0 .05 ml of 1/3 standard. To tubes 9 and 10 add 0 .05 ml of 1/9 standard. To tubes 13 and 14 add 0 .05 ml of 1/81 standard. To tubes 15 and 16 add 0 .05 ml of 1/243 standard. 3 . Add 0 .4 ml of each a l k a l i n e - t r e a t e d sample extract to dup l ica te tubes, beginning with tube 17. 4 . Add 0 . 0 5 ml o f 3 H labe l led prostaglandin to a l l tubes. 5 . Stopper tubes I and 2 . Incubation time is counted from the add i t ion of ant iserum. A d d 0 . 0 5 ml of antiserum beginning wi th tube 3 . M i x thoroughly on a vortex a g i t a t o r . 6 . Incubate the tubes for not less than one hour in a water bath at 37°C. 7 . A f te r i n c u b a t i o n , add 0.1 ml of Normal Rabbit Serum and 0.1 ml of G o a t A n t i - R a b b i t Serum to a l l tubes. M i x thoroughly and incubate 18-20 hours at 4°C . This incubat ion may be extended, i f greater b ind ing is des i red . 8 . A t this point the tubes contain the fo l lowing reagents: Tube Tris Buffer (ml) Inhibitor (ml) Tracer (ml) Prostaglandin Ant iserum (ml) Rabbit Normal Serum (ml) G o a t A n t i Rabbit Serum (ml) 1,2 I.I -- 0 . 0 5 -- 0.1 0.1 3 , 4 1.0 ~ 0 . 0 5 0 . 0 5 0.1 0.1 5-16 1.0 0 . 0 5 0 . 0 5 0 .05 0.1 0.1 17-18 0 . 6 0 . 4 0 0 . 0 5 0 . 0 5 0.1 0.1 e t c . 8 . A f te r this i ncubat ion , centr i fuge the tubes at 4°C for 30 minutes at 1600 G . 9 . Decant the supernatant from tubes I and 2 into two s c i n t i l l a t i o n v ia ls marked and T 2 . 10. Line the bottom of a suitable test tube rack With paper towels or other absorbent m a t e r i a l . A f te r decant ing the supernatant from each tube into a waste conta iner , p lace it upside down on this rack to d r a i n . In a few minutes, dry the inside of each tube care fu l l y with a folded strip of f i l te r paper or other suitable mate r i a l , taking care not to touch the precip i tate at the bottom. 11. A d d 1.0 ml N a O H ( O . l N ) to each tube , inc lud ing tubes I and 2 , mixing on vortex mixer , to dissolve the p r e c i p i t a t e . Decant the solut ion into a s imi lar ly numbered s c i n t i l l a t i o n v i a l , h i t t ing the rims of the tube and v i a l together f i rmly to insure maximal transfer. 12. Add 10 ml of s c i n t i l l a t i o n f lu id ( C A - 7 0 2 ) to each v i a l , i nc lud ing Tx and T 2 . M i x thoroughly . 13. Count each v i a l for 2 to 5 minutes. V i a l s Jx and T 2 should conta in 7 -10 ,000 C P M . B a c k g r o u n d c o u n t s . The a v e r a g e c o u n t s i n v i a l s I a n d 2. T o t a l c o u n t s . The a v e r a g e c o u n t s i n v i a l s ll a n d T s . C o r r e c t e d T o t a l c o u n t s . T c = T - B K G . T o t a l b o u n d . The a v e r a g e c o u n t s i n v i a l s 3 a n d 4, o r t he g e l t r i s c o n t r o l . C o r r e c t e d T o t a l b o u n d . B c = B - B K G . These c o u n t s r e p r e s e n t t h e t o t a l a m o u n t o f r a d i o i s o t o p e t h a t has b e e n b o u n d by the a n t i b o d y . In t h e s t a n d a r d c u r v e i t is d e f i n e d as 100% b i n d i n g . T u b e c o u n t s . The u n c o r r e c t e d c o u n t s fo r a t u b e n . O u e n c h C o r r e c t i o n B e t a p a r t i c l e d e t e c t i o n i n a l i q u i d s c i n t i l l a t i o n c o u n t e r d e p e n d s o n the o p t i c a l c h a r a c t e r i s t i c s o f the s a m p l e . T h e c o l o r o r t r a n s p a r e n c y o f the s a m p l e a f f e c t the e f f i c i e n c y w i t h w h i c h b e t a r a d i a t i o n c a n be d e t e c t e d . E a c h s a m p l e may q u e n c h d i f f e r e n t l y . S i n c e the s a m p l e s h a v e b e e n e x t r a c t e d , t h e i n c u b a t i o n s o l u t i o n s a r e l i k e l y to h a v e s i m i l a r o p t i c a l c h a r a c t e r i s t i c s a n d p r o b a b l y w i l l ' n o t r e q u i r e a q u e n c h c o r r e c t i o n . Q u e n c h c o r r e c t i o n c a n b e p e r f o r m e d b y u s i n g a n A u t o m a t i c E x t e r n a l S t a n d a r d as p r e s c r i b e d b y the s c i n t i l l a t i o n c o u n t e r m a n u f a c t u r e r . Q u e n c h c o r r e c t i o n c a n a l s o b e p e r f o r m e d b y u s i n g a n i n f e r n a l s t a n d a r d . A f t e r c o u n t i n g e a c h v i a l , a d d 0.01 ml o f t r a c e r (or l a b e l l e d p r o s t a -g l a n d i n ) t o e a c h v i a l , i n c l u d i n g t o t a l v i a l s 11 a n d T 2 . M i x w e l l a n d c o u n t a g a i n . The e f f i c i e n c y n o r m a l i z e d c o u n t s a r e c o m p u t e d u s i n g t h e a v e r a g e c o u n t s fo r the t o t a l v i a l s ( T 1 ) a n d t h e n e w c o u n t s f o r t h e v i a l ( C P M 1 ) as f o l l o w s : N o r m a l i z e d C P M = C P M n x Ij L C P M ' - CPM„ n n N o r m a l i z e d C P M s h o u l d b e u s e d f o r a l l c a l c u l a t i o n s . S t a n d a r d C u r v e T h e s t a n d a r d c u r v e is o b t a i n e d b y p l o t t i n g t h e p e r c e n t b i n d i n g for e a c h c o n c e n t r a t i o n o f i n h i b i t o r . T h e p e r c e n t b o u n d is C P M n - B K G 10 Figure I. A T Y P I C A L P R O S T A G L A N D I N S T A N D A R D C U R V E Pros rag I and in L e v e l , pg A typ ica l standard curve is shown in Figure 1. The percent b ind ing w i l l range from about 9 8 % at 8 .2 pg to 9 % at 2 . 0 n g . To improve a c c u r a c y , tubes hav ing b ind ing higher than 9 5 % should be repeated at a higher concentrat ion of extract and tubes hav ing b ind ing be low 10% should be repeated at a lower concentrat ion of e x t r a c t . 3 . K i t Binding C a p a c i t y The reagents have been se lected to provide a constant b ind ing over a long period o f t i m e . A not iceab le change or trend in the percent of total b ind ing should be considered an ind icat ion that either k i t reagents or prepared reagents have de te r io ra ted . Total b ind ing should be above 3 0 % . B e % T O T A L B I N D I N G = — - X 100 T c 4 . Sample Assay A f t e r determining the amount of prostaglandin present in the extract a l iquot used in the assay, it is necessary to convert this amount to a concentrat ion of the o r ig ina l sample . Cor rect ion factors i nc lude : a . The amount of sample o r i g i n a l l y used in the ext ract ion procedure . b . A n ext ract ion e f f i c i e n c y f a c t o r . c . A d i l u t i on factor for the f ract ion of extract used as inhib i tor in the assay. C O M M E N T S Dia lys is E f f i c iency In mult ip le experiments performed in our laborator ies, the d ia lys is e f f i c i e n c y has been shown to approach 4 6 % after 10 hours of continuous a g i t a t i o n o f the c e l l s . Due to evaporat ion and other losses, the actua l recovery of material has, in genera l , been less and varies from experiment to exper iment . We have found it helpful in our laboratories to inc lude with every d ia lys is experiment a set of ce l ls conta in ing tracer m a t e r i a l . A n equal amount of tracer is pipetted at the same time into a s c i n t i l l a t i o n v i a l , held as a control and def ined as 100% in computing d ia lys is e f f i c i e n c y . S i m i l a r l y , other extract ion methods may be checked for ext ract ion e f f i c i e n c y by adding tracer to the sample before e x t r a c t i o n . A f te r the e x t r a c t i o n , and just prior to the radioimmunoassay, an a l iquot is removed and counted to g ive the appropriate ext ract ion e f f i c i e n c y f a c t o r . ! Dia lys is Apparatus The d ia lys is step recommended in this procedure may be performed using a Karush-type Chamber assembly, such as those a v a i l a b l e from Be l lco G l a s s , P . O . Box B, 340 Edrudo Road, V i n e l a n d , N e w Jersey , 08360 ( c a t . n o . 3213, I ml c a p a c i t y ) . To obtain the . e f f i c iency character ist ics ind icated above , this type of c e l l requires continuous a g i t a t i o n . Semi -permeable ce l lu lose membranes are a v a i l a b l e from a var iety of sources. We have found Fisher D i a l y z e r Tubing (catalog n o . 8 - 6 6 7 C ) to be sat is factory . This tubing must be treated before use by b o i l i n g about 5 feet of it in 500-1000 ml of d i s t i l l ed water contain ing a pinch of EDTA and sodium b icarbonate , unt i l the tubing is soft and odorless. This usually requires three passes. O n c e the tubing is ready, it may be stored at 4°C under d i s t i l l ed water unt i l used . Ant ibody Cross React iv i ty The a n t i - P G B / antiserum used in this k i t cross-reacts with PCAX, P G A 2 , P G B S , P G E ! , P G E 2 , P G F x a and PGFgry. A set of Inhibit ion Curves is shown on Figure 2 for a n t i - P G B ! ant iserum. REFERENCES 1. Lev ine , L . , G u t i e r r e z Cernosak, R . M . , and V a n V u n a k i s , H e l e n . Spec i f i c i t i es of Prostaglandins , F j t Y , and F 2 o; A n t i g e n - A n t i b o d y React ions . J . B i o l . C h e m . 246y N o . 22 , 6782, 1971. 2 . G u t i e r r e z Cernosak, R . M . , M o r r i l l , L . M . , and L e v i n e , L . Prosta -g land in ?^nt Levels in Peripheral Sera of M a n . Prostaglandins, I, N o . I, 71, 1972. 3 . L e v i n e , L . , H i n k l e , P . M . , V o e l k e l , E . F . , and Tashj ian, A . H . , J r . Prostaglandin Production by Mouse Fibrosarcoma Ce l ls in Cu l tu re : Inhib i t ion by Indomethacin and A s p i r i n . B i o c h . and B i o p h . Res. C o m m . , 47, N o . 4 , 888, 1972. 4 . J u b i z , W . and F r a i l e y , J . Prostaglandin E Genera t ion During Storage of Plasma Samples. Prostaglandins 7, N o . 4 , 339 , 1974. 5 . J a f f e , B . M . and Behrman, H . R . (1974). Prostaglandins E, A , F, in "Methods of Hormone Radioimmunoassay, " Jaffe and Behrman, e d s . , p . 2 2 , A c a d e m i c Press, N e w Y o r k . 1 / 6 ? < > f; Clinical Assays, Inc. 237 Binney Street. Cambridge, Massachuse t t s^/02142 (617) 492-2526 C A - 5 0 3 S H P R O S T A G L A N D I N F g o r R A D I O I M M U N O A S S A Y KIT For the Quant i ta t i ve Measurement of Prostaglandin F 2 a in Plasma or Tissue Extracts N O T E : STORE THIS KIT AT -20°C AFTER R E M O V I N G THE T W O V I A L S O F TRIS BUFFER C O N C E N T R A T E February 14, 1975 Radioimmunoassays I 70 Radioact ive material — Not for Human Use — Introduction into F o o d s , Beverages, C o s m e t i c s , Drugs, or M ed ic ina l s or into P roducts Manufactured for C o m m e r c i a l D ist r ibut ion is Proh ib i ted — Exempt Quant i t ies Shou ld not be C o m b i n e d . C L I N I C A L A S S A Y S Division ofTravenol Laboratories, Inc. PRECAUTIONARY INSTRUCTIONS The user shal l store the by-product material unt i l used in the or ig inal s h i p p i n g conta iner or in a conta iner providing equivalent radiat ion protect ion . The following precautions should be observed in handling radioactive material: 1. Hand l ing s h o u l d preclude any p ipett ing by mouth . 2. There shou ld be no s m o k i n g or eat ing whi le radioact ive mater ia ls are being hand led . 3. Hands s h o u l d be covered with rubber g loves dur ing and thoroughly w a s h e d after handl ing of radioact ive mater ia ls . 4. S p i l l s shou ld be wiped up qu ick ly and thoroughly and the contaminated mater ia ls added to radioact ive waste matter. 5. Water so lub le waste radioact ive material can be d i s p o s e d of into the sanitary sewage s y s t e m , if the concent ra t ion , after d i lu t ion with the laboratory d ischarge , does not exceed 4 x 1Cr 2 m ic rocur ies per liter ( 1 2 5 l ) or 10 m ic rocur ies per liter ( 3 H), based on a dai ly average of eff luent. Whenever p o s s i b l e , however, d i s p o s a l of radioact ive material s h o u l d be made through a l i censed d i s p o s a l serv ice. LICENSING REQUIREMENTS The procurement of radioact ive material in th is kit is exempt f rom N R C or Agreement State l i cens ing requirements. Ill 2 I N T R O D U C T I O N The C l i n i c a l Assays, I n c . , Prostaglandin F~£y(PGF2Q,) Radioimmunoassay K i t offers a sensitive method o f measuring prostaglandin content in serum or plasma by measuring the compet i t ive b inding of 3 H labeled prostaglandin and un labeled prostaglandin wi th ant ibody to prostaglandin . The k i t includes a l l the r a d i o -immunoassay reagents required in the procedure . Separat ion o f prostaglandin bound to ant ibody and free prostaglandin is ach ieved by prec ip i ta t ing the bound prostaglandin wi th a second antibody b ind ing r e a c t i o n . A f t e r cent r i fugat ion , the bound rad ioact i v i t y is measured in a s c i n t i l l a t i o n counter . To e l iminate protein interference in the assay, a protein denaturation a n d / o r ex t ract ion step is performed o n the sample prior to assay ing . A l t e r n a t i v e l y , e x t r a c t i o n , fo l lowed by column separation of the various prostaglandins, may be e m p l o y e d . A standard curve is prepared for each group o f samples by incubat ing known amounts of prostaglandins. Total time for the assay is approximately two working days . This protocol is a modi f icat ion of the procedures of Jaffe (1), Levine (2), C a l d w e l l (3), and H i c k l e r (4). Through the introduct ion o f a new sensitive a n t i - P G F s a serum and a very high s p e c i f i c a c t i v i t y H prostaglandin F 8 q, , (February, 1975) the assay can now de fect as low as 10 pg of i n h i b i t o r . Wherever possib le , a sample s i ze should be selected wh ich w i l l d i rect the assay to a more sensitive portion of the standard curve . R E A G E N T S This is the first commerc ia l l y a v a i l a b l e k i t for the determinat ion of prostaglandin leve ls by radioimmunoassay. The radioimmunoassay k i t contains suf f ic ient reagents to perform 200 assays i nc lud ing 5 standard curves . The reagents provided are : F ina l Vo lume I v i a l Prostaglandin Ant iserum, l y o p h i l i z e d 10 ml 1 v i a l Prostaglandin Standard , l y o p h i l i z e d 5 ml 2 v ia l s Rabbit Normal Serum, l y o p h i l i z e d 10 ml, each 2 v i a l s G o a t A n t i - R a b b i t Serum 10 m l , each 1 v i a l 3 H labeled Prostaglandin Fsft (2p.Ci), l y o p h i l i z e d 10 ml 2 v i a l s Tris Buffer C o n c e n t r a t e , 10 ml each 300 m l , each I v i a l G e l a t i n (3g) The reconstituted reagents must be stored f rozen at -I0°C to -20°C. Prostaglandins are stable substances and the k i t may be thawed and frozen repeatedly without a f fect ing the performance o f the k i t reagents . The buffer solut ion is stored at 4 °C . R E A G E N T S REQUIRED BUT N O T P R O V I D E D I N KIT 0 . I N Sodium hydroxide and hydroch lor ic a c i d solutions S U G G E S T E D A P P A R A T U S Prec is ion pipers: 1 .0, 0 . 5 , 0 . 1 , 0 . 0 5 ml Vor tex M i x e r Cent r i fuge C o r n w a l l I ml Repeating Syringe L iqu id S c i n t i l l a t i o n Counter Wate r Bath (37°C) and Ice Bdth pH Mete r D ia l ys i s apparatus (opt ional) S U G G E S T E D D I S P O S A B L E MATERIALS Polypropylene tubes, Fa lcon N o . 2053 , 12x75 mm or equiva lent L iqu id s c i n t i l l a t i o n v ia l s S c i n t i l l a t i o n F l u i d , C A - 7 0 2 or equ iva lent D ia l ys i s membrane, Fisher 8 - 6 6 7 C or equ iva lent /75 R E A G E N T P R E P A R A T I O N It is recommended that d i s t i l l e d water be used to reconstitute l y o p h i l i z e d reagents and to prepare a l l the accessory reagents required in this assay. A. Tris Buffer, Reagent A Conta ins : The Tris Buffer Concent rate contains Tr izma (0.36g) and sodium ch lor ide (2 .5g) in 10 ml water (30xconcentrate) , preadjusted to pH 7 . 4 . Add: Contents of I v i a l of tris concentrate to 250 ml d i s t i l l e d wate r . Bring volume to 300 ml w i th w a t e r . Storage: Keep re f r ige ra ted . S t a b i l i t y : Two months B . Isogel Tris Work ing Buffer, Reagent B Add: O.lOg G e l a t i n to 100 ml Reagent A . Heat and stir unt i l the g e l a t i n is d i s s o l v e d . C o o l the s o l u t i o n . Ad just : pH to 7 . 4 i f necessary, w i th 0 . I N sodium hydroxide or 0 . I N hydroch lo r ic a c i d . Storage: K e e p refr igerated . S t a b i l i t y : Prepare fresh every two or three d a y s . i C . Prostaglandin Standard , 24 ng /m l Add: 5 ml Reagent B to the contents of the standard v ia l . M i x gent ly to avoid foaming Storage: Must be kept f r o z e n . S t a b i l i t y : M i n i m u m three months D . Prostaglandin Ant iserum Add: 10 ml Reagent B to the contents o f the antiserum v i a l . M i x gent ly to avo id foaming . Storage: Must be kept f r o z e n . S t a b i l i t y : M i n i m u m three months E . 3 H Prostaglandin (2MCi) Add: 10 ml Reagent B to the contents o f the 3 H Prostaglandin v i a l . M i x gent l y to avo id foaming . Storage: Must be kept f rozen . S t a b i l i t y : M in imum three months. F . Rabbit Normal Serum Add: 10 ml Reagent B to the contents of a Rabbit Normal Serum v i a l . M i x gent ly to avo id f oaming . Storage: Must be kept f rozen . S t a b i l i t y : M in imum three months. G. G o a t A n t i - R a b b i t Serum Conta ins : 10 ml G o a t A n t i - R a b b i t Serum wi th preservative Storage: Must be kept f rozen S t a b i l i t y : M i n i m u m three months E X T R A C T I O N O F P R O S T A G L A N D I N S Plasma samples are recommended for the measurement of prostaglandins in blood . However , the presence of platelets in plasma has been shown to be responsible for the generat ion of P G E during storage of the sample both at 4°C and under f reezing condit ions (5) . To avo id spuriously high va lues , plasma samples should be frozen and ana lyzed w i th in a week . S ince proteins b ind prostaglandins in compet i t ion wi th ant ibod ies , i t is necessary to ext ract the prostaglandins from plasma and-t issues. W i th any e x t r a c t i o n , the recovery e f f i c i e n c y should be monitored by adding several thousand C P M o f the 3 H PGFaa to representative samples. Some typ ica l ext ract ion methods are summarized be low : 1 . Ext ract ion of 1 .0 ml sample a l iquot (or more i f a v a i l a b l e ) with 3 ml petroleum ether to remove neutral l i p i d s . A f te r removal of the ether phase, add 3 . 0 ml o f an ethyl acetate : isopropanol : 0 . 2 N HCI (3 :3 :1 ; v / v / v ) s o l u t i o n . Vor tex for 15 seconds t w i c e , and add 2 . 0 ml o f e thy l acetate and 3 . 0 ml w a f e r . A f te r m i x i n g , the phases are separated by centr i fugat ion . The organic phase (3 out of 3 . 5 ml) is transferred to a polypropylene test tube and d r ied at 55°C in an a i r stream (6) . The residue may require add i t iona l c leanup before if is suitable for the radioimmunoassay. A s i l i c i c a c i d column separation is i n d i c a t e d , i f separation of the prostaglandins into P G A - P G B , P G E , and P G F fractions is desired (see page 24, ref . (6)). A l t e r n a t i v e l y , d ia lys is of the extract may be performed for further pu r i f i ca t ion without separating the P G fract ions (see p p . 11, 12 of this p ro toco l ) . 2. Ext ract ion o f 5 ml plasma with 10 ml methy la l : a l coho l (3 :1 , v / v ) . A f te r filtering the p r e c i p i t a t e , and evaporat ing the f i l t r a t e , the residue is d issolved in isogel tris buffer and d i a l y z e d overnight (7) . ( M e t h y l a l is a v a i l a b l e from Fisher S c i e n t i f i c C o . , C a t . N o . M - 2 2 2 ) . 3. Tissue samples should be processed immediate ly . They are homogenized i n a mixture of 1.0 ml of a phosphate buffer sal ine and 3 . 0 ml of the above ethy l aceta te : isopropanol : HCI e x t r a c t i o n so lut ion in a mechan ica l homogen ize r . The samples are then processed as in 1 . above : 2 . 0 ml ethy l acetate and 3 . 0 ml water are added , mixed and the organ ic phase separated and dr ied (6) . Another protocol for the ext ract ion o f tissues by successive par t i t ion ing in ethy l a c e t a t e , n - h e x a n e , and aqueous methanol may be found in (8) . 4. Samples of b i o l o g i c f l u ids , such as aqueous humor and cerebrospinal f l u i d , w h i c h conta in small amounts of p ro te in , can be measured d i r e c t l y without e x t r a c t i o n . The d i lu t ions concentrat ion are as fo l lows , per 100 micro l i te rs : 1/1 2400 pg 1/27 8 8 . 9 pg 1 /3 800 pg 1/81 2 9 . 6 pg 1 /9 267 pg 1 /243 9 .2 pg These d i lut ions o f standard should be kept frozen wi th the remainder of the k i t , and are suf f ic ient for f ive complete standard curves . P R E P A R A T I O N O F THE S T A N D A R D C U R V E A N D A S S A Y S A M P L E S 1. Into s ixteen tubes numbered 1 - 1 6 pipet 0 .5 ml o f isogeltr is buf fer . In a d d i t i o n , p l a c e an extra 0.1 ml in tubes 1 and 2 . Tubes 1 and 2 are background controls and conta in no antiserum or inh ib i tor (standard). Tubes 3 and 4 are b inding controls and conta in no inh ib i to rs . A d d the appropriate volume of isogeltr is buffer to tubes 17 and u p . The total volume of Reagent B and sample ext ract should be 0 . 6 m l . The sample s ize is the volume wh ich is estimated to conta in su f f i c ien t P G F 2 Q ! so that the leve l fa l ls on a sensit ive part of the standard c u r v e . If the P G F S Q ; l eve l cannot be est imated , then two di f ferent s ize assay samples may be taken wi th the appropriate ad justment made in the size of the volume of the buffer s o l u t i o n . 2 . To tubes 5 and 6 add 0 .1 ml o f 1/1 standard; mix w e l l . To tubes 7 and 8 add 0.1 ml of 1 /3 s tandard . To tubes 9 and 10 add 0.1 ml of 1 /9 s tandard . To tubes 13 and 14 add 0.1 ml of 1/81 standard . To tubes 15 and 16 add 0 .1 ml o f 1 /243 s tandard . 3 . A d d the appropriate volume of sample extract in Reagent B solut ion to dup l i ca te tubes, beg inn ing w i th tube 17 . 4 . A d d 0 . 0 5 ml o f 3 H l a b e l l e d prostaglandin to a l l tubes. 5. Stopper tubes 1 and 2 . Incubat ion time is counted from the add i t ion of ant iserum. A d d 0 . 0 5 ml of ant iserum beginn ing wi th tube 3 . M i x thoroughly on a vortex a g i t a t o r . 6. Incubate the tubes for not- less than one hour in a wafer bath at 37°C . 7. A f t e r i n c u b a t i o n , add Q . l ml o f Normal Rabbit Serum and 0 .1 ml of G o a t A n n -Rabb i t Serum to a l l tubes . M i x thoroughly and incubate 18 -20 hours at 4 °C. This incubat ion .may be ex tended , i f greater b ind ing is d e s i r e d . /76 8 A t this point the tubes contain the fo l l ow ing reagents: G o a t , Rabbit A n t i Isogeltris Prostaglandin Normal Rabbit Buffer Inhibitor Tracer Ant iserum Serum Serum Tube (ml) (ml) (ml) (ml) (ml) (ml) 1,2 0 . 6 — 0 .05 — 0.1 0.1 3 , 4 0 . 5 — 0 . 0 5 0 . 0 5 0.1 0.1 5 - 1 6 0 . 5 0.1 0 . 0 5 0 . 0 5 0.1 0 .1 17-18 Total volume 0 . 6 ml 0 . 0 5 0 . 0 5 0 .1 0.1 e t c . See Text 8 . A f t e r this i n c u b a t i o n , centr i fuge the tubes at 4 C for 30 minutes at 1600 G . 9 . Decant the supernatant from tubes 1 arid 2 into two s c i n t i l l a t i o n v ia ls marked T x and T g . 10 . L ine the bottom of a suitable test tube rack wi th paper towels or other absorbent m a t e r i a l . A f te r decant ing the supernatant from each tube into a waste conta iner , p lace i t upside down on this rack to d r a i n . In a few minutes, dry the inside of each tube care fu l l y wi th a fo lded strip o f f i l ter paper or other suitable mate r i a l , taking care not to touch the prec ip i tate at the bottom . c 1 1 . A d d 1.0 ml N a O H (0 . I N ) to e a c h tube, i n c l u d i n g tubes 1 and 2 , mixing on a vortex mixer , to dissolve the p r e c i p i t a t e . Decant the solut ion into a s imi la r l y numbered s c i n t i l l a t i o n v i a l , h i t t ing the rims o f the tube and v i a l together f i rmly to insure maximal transfer. 12 . A d d 10 ml of s c i n t i l l a t i o n f lu id ( C A - 7 0 2 ) to e a c h v i a l , i n c l u d i n g 1 and T 3 . M i x thoroughly . 13 . Count each v i a l for 2 to 5 minutes. V i a l s T and T s should conta in 6 - 8 , 0 0 0 C P M . 9 C A L C U L A T I O N S B K G Background counts . The average counts in v ia ls I and 2. T Total counts . The average counts in v ia ls Tj and T 2 . T c Cor rected Total counts . T C = T - B K G . B Total b o u n d . The average counts in v ia ls 3 and 4, or the gel tris control B c Cor rected Total b o u n d . B c = B - B K G . These counts represent the total amount of radioisotope that has been bound by the a n t i b o d y . In the standard curve it is def ined as 100% b i n d i n g . C P M n Tube counts . The uncorrected counts for a tube n . 1 . Q u e n c h Cor rec t ion Beta par t ic le d e t e c t i o n in a l iqu id s c i n t i l l a t i o n counter depends on the o p t i c a l character is t ics of the sample . The co lor or transparency of the sample a f fect the e f f i c i e n c y with wh ich beta radiat ion can be d e t e c t e d . Each sample may quench d i f f e r e n t l y . S ince the samples have been e x t r a c t e d , the incubat ion solutions are l i k e l y to have s imi lar o p t i c a l character is t ics and probably w i l l not require a quench c o r r e c t i o n . Q u e n c h cor rect ion can be performed by using an Automat ic External Standard as prescribed by the s c i n t i l l a t i o n counter manufacturer . Q u e n c h cor rect ion can also be performed by using an internal standard. A f t e r count ing each v i a l , add 0.01 ml of tracer (or l abe l led p ros ta -g land in ) to each v i a l , i n c l u d i n g total v ia l s Tj and T 2 . M i x w e l l and count a g a i n . The e f f i c i e n c y normal ized counts are computed using the average counts for the total v ia ls (T 1) and the new counts for the v i a l ( C P M ' ) as fo l lows : N o r m a l i z e d C P M = C P M n x ' ' " 1 C P M ' - CPM„ N o r m a l i z e d C P M should be used for a l l c a l c u l a t i o n s . 2. Standard Curve The standard curve is obta ined by p lot t ing the percent b ind ing for each concent rat ion of i n h i b i t o r . The percent bound is C P M n - B K G 10 A t y p i c a l s t a n d a r d c u r v e is s h o w n i n F i g u r e 1 . The p e r c e n t b i n d i n g w i l l r a n g e f r o m a b o u t 9 8 % at 9 . 4 p g to 9 % at 2 . 4 n g . To i m p r o v e a c c u r a c y , t ubes h a v i n g b i n d i n g h i g h e r t h a n 9 5 % s h o u l d b e r e p e a t e d a t a h i g h e r c o n c e n -t r a t i o n o f e x t r a c t a n d tubes h a v i n g b i n d i n g b e l o w 1 0 % s h o u l d b e r e p e a t e d at a l o w e r c o n c e n t r a t i o n o f e x t r a c t . F i g u r e I. A T Y P I C A L P R O S T A G L A N D I N S T A N D A R D C U R V E P r o s t a g l a n d i n L e v e l , pg 3 . K i t Binding C a p a c i t y The reagents have been se lected to provide a constant b ind ing over a long period of t i m e . A not iceable change or trend in the percent of total b ind ing should be considered an i nd ica t i on that 'e i ther k i t reagents or prepared reagents have de te r i o ra ted . Total b ind ing should be above 30 %. B c ^ % T O T A L B I N D I N G = - — X 100 4 . Sample Assay A f te r determining the amount of prostaglandin present in the extrai a l iquot used in the assay, it is necessary to convert this amount to a concentrat ion of the or ig ina l sample . Co r rec t ion factors i n c l u d e : a . The amount of sample o r i g i na l l y used in the ext ract ion p rocedure . b . A n ext ract ion e f f i c i e n c y f a c t o r . c . A d i l u t i o n factor for the f ract ion of extract used as inh ib i to r in the assay. C O M M E N T S D ia lys is E f f i c i ency In mul t ip le experiments performed in our laborator ies , the d ia lys is e f f i c i e n c y has been shown to approach 4 6 % after 10 hours of continuous aa i ta t i on of the c e l l s . Due to evaporat ion and other losses, the ac tua l recovery of mater ial has , in g e n e r a l , been less and varies from experiment to exper iment . W e have found it he lp fu l i n our laboratories to inc lude wi th every d ia lys is experiment a set of ce l l s conta in ing tracer m a t e r i a l . A n equal amount of tracer is p ipetted at the same time into a s c i n t i l l a t i o n v i a l , he ld as a control and def ined as 100% i n computing d ia lys is e f f i c i e n c y . S i m i l a r l y , other e x t r a c t i o n methods may be checked for e x t r a c t i o n e f f i c i e n c y by adding tracer to the sample before e x t r a c t i o n . A f te r the e x t r a c t i o n , and just prior to the radioimmunoassay, an a l iquot is removed and counted to g ive the appropriate ext ract ion e f f i c i e n c y f a c t o r . 180 II 3. K i t B ind ing C o p a c i t y The reagents have been se lected to provide a constant b ind ing over a long per iod of t i m e . A no t iceab le change or trend in the percent of total b ind ing should be considered an i n d i c a t i o n that either k i t reagents or prepared reagents have d e t e r i o r a t e d . Total b ind ing should be above 30 %. B c % T O T A L B I N D I N G = X 100 4 . Sample Assay A f te r determin ing the amount of prostaglandin present in the extract a l i quo t used in the assay, it is necessary to convert this amount to a concent ra t ion of the o r ig ina l sample . C o r r e c t i o n factors i n c l u d e : a . The amount of sample o r i g i n a l l y used in the e x t r a c t i o n p rocedure . b . A n ext ract ion e f f i c i e n c y f a c t o r . c . A d i l u t i o n factor for the f rac t ion of ext ract used as inh ib i to r in the assay. C O M M E N T S D i a l y s i s E f f i c i e n c y In mul t ip le experiments performed in our laborator ies , the d ia l ys i s e f f i c i e n c y has been shown to approach 4 6 % after 10 hours of continuous a q i t a t i o n of the c e l l s . Due to evaporat ion and other losses, the a c t u a l recovery of mater ia l has , in g e n e r a l , been less and varies from experiment to exper iment . W e have found it he lpfu l in our laborator ies to i nc lude wi th every d ia lys is experiment a set of ce l l s c o n t a i n i n g t racer mater ia l . A n equal amount of t racer is p ipetted at the same time into a s c i n t i l l a t i o n v i a l , he ld as a contro l and def ined as 100% in computing d ia lys is e f f i c i e n c y . S i m i l a r l y , other e x t r a c t i o n methods may be c h e c k e d for e x t r a c t i o n e f f i c i e n c y by add ing tracer to the sample before e x t r a c t i o n . A f t e r the e x t r a c t i o n , and just pr ior to the radioimmunoassay, an a l i quo t is removed and counted to g ive the appropr iate e x t r a c t i o n e f f i c i e n c y f a c t o r . 12/ 12 Dia lys is Apparatus The d ia lys is step recommended in this procedure may be performed using a Karush-type Chamber assembly, such as those a v a i l a b l e from Be l lco G l a s s , P . O . Box B, 340 Edrudo Road, V i n e l a n d , New Jersey 08360 ( C a t . N o . 3213, 1 ml c a p a c i t y ) . To obta in the e f f i c i e n c y character ist ics ind icated above , this type of c e l l requires continuous a g i t a t i o n . Semi -permeable ce l l u lose membranes are a v a i l a b l e from a var iety of sources. We have found Fisher D i a l y z e r Tubing (Cata log N o . 8 -667C) to be sat is factory . This tubing must be treated before use by b o i l i n g about 5 feet of it in 500 -1000 ml of d i s t i l l e d water conta in ing a p inch of EDTA and sodium b icarbonate , unti l the tubing is soft and odor less. This usually requires three passes. O n c e the tubing is ready, it may be stored at 4° C under d i s t i l l ed water unti l used . Ant ibody C ross -React i v i t y The a n t i - P G F 2 a serum in this k i t cross-reacts s ign i f icant ly wi th P G F i a and should , therefore, not be used to d i f ferent iate between P G F 2 a and P G F x a : . A t 5 0 % binding the cross - react ion wi th P G E i and E s is 1:5000, and with P G A , and A s less than 1 :10 ,000 . 13 ,14 -D ihyd ro -15 -ke top ros tag land in F 2 a ( P G F 2 a metabolite) cross-reacts 0 . 3 % . REFERENCES 1. J a f f e , Bernard M . , Smith , J a y W . , N e w t o n , W i l l i a m T . , and Parker , C h a r l e s W . Radioimmunoassay for Prostaglandins. Sc ience 171, 494 , 1971. 2 . Lev ine , Lawrence and Van V u n a k i s , H e l e n . A n t i g e n i c A c t i v i t y of Prostaglandins. B iochemica l and Biophysical Research Communications 4 1 , 1171, 1970. 3 . C a l d w e l l , Burton V . , Burnstein, Sumner, Brock, W i l l i a m A . and Speroff, L e o n . Radioimmunoassay of the F Prostaglandins. J . C l i n . E n d o c r . , 33 , 171, 1971. 4 . H i c k l e r , Roger B . , Prostaglandin Symposium of the Worcester Foundation for Experimental B i o l o g y . Ramwel l , P . W . , and Shaw, J . , editors, Interscience, N . Y . , 279 , 1968. 5 . J u b i z , Wo and F r a i l e y , J . Prostaglandin E Generat ion Dur ing Storage of Plasma Samples. Prostaglandins 7 , N o . 4 , 339, 1974. ' 6 . J a f f e , B . M . and Behrman, H . R. (1974). Prostaglandins E , A , F , in "Methods of Hormone Radioimmunoassay," Jaffe and Behrman, e d s . , p . 22 , A c a d e m i c Press, N e w Y o r k . J 7 . Gut ie r rez Cernosak, R . M . , M o r r i l l , L . M . , and Lev ine , L. Prostaglandin F 2 a Levels in Peripheral Sera of M a n . Prostaglandins, I , N o . 1, 7 1 , 1972. 8 . Humes, J . L . and Strausser, H . R . Prostaglandins and C y c l i c Nuc leo t ides in Mo loney Sarcoma Tumors. Prostaglandins, 5 , N o . 2 , 183, 1974. 

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