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Growth-accelerating effects of alkaline extracts from the pituitary glands of the spring salmon (Oncorhynchus… Jampolsky, Abey 1949

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GROWTH - ACCELERATING EFFECTS OF ALKALINE EXTRACTS FROM THE PITUITARY GLANDS. OF THE SPRING SALMON (Oncorhynchus tsehawytsoha)  Abey  Jampolsky  A Thesis Submitted i n P a r t i a l Fulfilment of The Requirements f o r the Degree of MASTER OF ARTS In the Department of ZOOLOGY  The U n i v e r s i t y of B r i t i s h Columbia Ootober, 1949.  )l  ABSTRACT An alkaline extract from the anterior lobe of the beef p i t u i t a r y proved a potent source of a growth hormone with i n j e c t i o n s to r a t s .  Injections of t h i s extract t o g o l d f i s h resulted i n an  18$  weight increase within eight days, which appears s i g n i f i c a n t when compared to t h e i r controls.  A similar method of preparation  was  used for an extract of the p i t u i t a r y gland of the P a c i f i c Salmon, Onoorhynohus tschawytsoha.  Injections of t h i s extract f a i l e d to  accelerate the growth of t e s t r a t s .  Injections to goldfish  resulted i n a confusing pattern with both increases  and decreases  i n weight being observed, however, the r e s u l t s indicate the presence of a growth-accelerating hormone.  Substantiation  t h i s growth-acoelerating effeot of alkaline extracts of the p i t u i t a r y i s necessary i n view of the c o n f l i c t i n g r e s u l t s obtained.  of fish  Acknowledgments  I would l i k e to take t h i s opportunity to express my appreciation t o Dr. W. A. Clemens, Head of the Department of Zoology, f o r h i s kindness during my studies at the University of B r i t i s h Columbia. I wish t o thank Dr. W. S. Hoar f o r help i n suggestion of the t o p i c , f o r assistance and enoouragement, and f o r the valuable advice he has offered throughout. I also wish t o express my appreciation t o the Department of Zoology, University of B r i t i s h Columbia, f o r the grant of money used i n support of t h i s work.  TABLE OF CONTENTS Pag© Introduction and Review of Literature Materials and Methods  1. 11*  Results E f f e c t s of Extracts on Rats  16.  Potency determination of Beef P i t u i t a r y Extraot  16.  Preliminary Injections of F i s h P i t u i t a r y Extract  17.  F i s h P i t u i t a r y Injection Experiments  18.  E f f e c t s of Extracts on Goldfish  19.  Weight Changes i n Handling the Goldfish  20.  Injections of 1 co. of Anterior Lobe of Beef P i t u i t a r y Extract Goldfish Injected with 1 cc. of Fish P i t u i t a r y Extraot  22. 23.  Discussion  24.  Summary  29.  Tables Table 1 - Potency Assay Determination of Beef Growth Hormone Extraot Using Rats of Group 1 Table 2 - Preliminary Injection of F i s h P i t u i t a r y Extracts t o Rats of Group 1.  31. 32.  Table 3 - Weight Changes Recorded with Rats of Group 2.  33.  Table 4 - Weight Change of Goldfish Handled Only.  34.  Table 5 - Weight Change of Goldfish Injected w i t h 1% Salt Solution. Table 6.- Weight Change o f Goldfish Injected 1 cc. of Anterior Lobe of Beef P i t u i t a r y Extract.  35.  Table 7.- Weight Change of Goldfish Injected with 1 oo. of F i s h P i t u i t a r y Extract.  36.  37.  Figures Figure 1 - Response of Normal Female Rats (group 1) t o hypophyseal Growth Hormone.  39.  Figure 2 - Response of normal female rats (group 2) t o Beef and f i s h hypophyseal growth hormone extract.  40.  Figure 3 * Graph of mean percentage weight gain of g o l d f i s h handled only. Figures represent number of g o l d f i s h which form the indicated averages  41.  Figure 4 - Graph of mean percentage weight gain of g o l d f i s h injected with 1 c c . of 1% s a l t solution. Figures represent number of g o l d f i s h which form the indicated averages 42. Figure 5 - Comparison of the mean percentage weight change of beef p i t u i t a r y extract with f i s h p i t u i t a r y extract injections (1 oo.) t o goldfish.  43.  Figure 6 - Histogram of weight changes observed i n rats (group 2 ) .  44.  Bibliography  45.  GROWTH - ACCELERATING EFFECTS OF ALKALINE EXTRACTS FROM THE PITUITARY GLANDS OF THE SPRING SALMON  (Onoorhynchus tsohawytscha)  INTRODUCTION The p i t u i t a r y gland i s an organ of i n t e r n a l secretion. Exhaustive research has now  been c a r r i e d on i n order that i t s  origins and functions might be better understood.  The  earliest  work had to do with the p i t u i t a r y of mammals beoause of the possible a p p l i c a t i o n of r e s u l t s to human physiology. work followed.  Of the various classes within the  Comparative  vertebrate  series the fishes have come to assume an extremely important p o s i t i o n i n the economics of man.  Although the embryology and  anatomy of the p i t u i t a r y gland of f i s h have been extensively studied l i t t l e attention has been given t o i t s physiological nature.  The present i n v e s t i g a t i o n was  undertaken f o r the purpose  of advancing knowledge i n t h i s f i e l d . History of the Discovery of the Growth Hormone.  The f i r s t investigations of a purely physiological  nature regarding the a c t i v i t y of the p i t u i t a r y gland were those of Oliver and Sohaefer (1895) who found that ..aqueous extracts of the p i t u i t a r y body produced a r i s e i n blood pressure when injected into the blood v e s s e l s . to  Howell .(1898) was the f i r s t  l o c a l i s e t h i s hormonal a c t i v i t y i n the posterior lobe of the  pituitary.  However, i t was not u n t i l 1909 that the growth-  promoting e f f e c t s of the p i t u i t a r y gland were f i r s t recorded (Sohaefer, 1909).  At t h i s time Sohaefer f e d a small amount of  dried powder material, derived from the anterior lobe of ox p i t u i t a r i e s , t o male rats over a period of two months.  A seoond  group, which were used as a control group, were f e d a similar amount of dried powdered material prepared from the t e s t i c l e . Schaefer observed after eight days of the addition of p i t u i t a r y to the food a steady increase i n weight over the seoond group. This increase oontinued u n t i l the end of the experiment. Numerous investigations followed.  Much of the work i s  contradictory and of h i s t o r i c a l interest only. A l d r l o h (1912, '1913) performed two series of experiments.  In 1912 he added 50 mg. of f r e s h dessicated  J  anterior lobe of ox p i t u i t a r y t o the diet of a number of dogs. To another group, which served as h i s " c o n t r o l " , a similar amount of prepared ovary was f e d . He observed that the average weights showed that the "control" group had a tendency t o increase i n weight more rapidly than the. p i t u i t a r y - f e d animals, but  f o r " c e r t a i n reasons" A l d r i c h ooncluded that there was neither  stimulation nor retardation i n growth.  In a second series  3.  A l d r i o h (1913) c a r r i e d out a similar experiment on 10 r a t s divided into two groups of males and females i n each*  As with the dogs,  the r a t "control" group -showed an increasingly greater weight than the p i t u i t a r y group during the three months of experiments. He concluded that ingestion of the anterior lobe impedes growth. A similar set of experiments performed with the posterior lobe had no e f f e c t . Wulzen (1914) f e d young chicks with a weighed amount of unmodified anterior lobe of ox p i t u i t a r y over a period of three months.  In addition they were f e d a balanced chick diet as were a  second group which were also f e d with a similar amount of l i v e r instead of anterior p i t u i t a r y lobes. were of three types: length of wing i n mm.,  Measurements taken weekly  ( l ) average weight i n grams, (2) average (3) average length of foot i n mm.  measurements made on 25 young ohioks showed a d i s t i n c t  A l l the  inhibition  of growth i n the p i t u i t a r y f e d chicks. Evans and Long (1921) f e d one-half gram of ground f r e s h anterior lobe of ox p i t u i t a r y d a i l y t o 54 rats of 21 days of age. A similar diet was given t o l i t t e r mate rats save that one-half gram of raw l i v e r was given instead of the anterior lobe of p i t u i t a r y gland.  Weighings made every four days showed no s i g n i f i c a n t  difference i n growth between the hypophysis-fed animals and the controls. A 'growth-accelerating' e f f e c t of the hypophysis was reported by Gudernatsch (1918) from observations made on the feeding of albino rats d i f f e r e n t glandular d i e t s .  This conclusion  i s corroborated by Marinus' work (1919). rats into three groups.  He separated 100 young  One group was f e d anterior lobe of the  beef p i t u i t a r y gland, the seoond the pars t u b e r a l i s of the beef p i t u i t a r y gland, and the t h i r d was given beef muscle.  His r e s u l t s  show a 'growth-accelerating' e f f e c t only with those animals f e d the anterior lobe of beef p i t u i t a r y . As already reported Evans and Long (1921), observed that feeding of the anterior p i t u i t a r y lobe caused no s i g n i f i c a n t difference i n growth.  However, i n the same year they c a r r i e d out  another set of experiments i n whioh intraperitoneal i n j e c t i o n s of an anterior p i t u i t a r y lobe extract were made on 38 experimental animals.  A similar number of l i t t e r mate controls were used.  The extract was derived from f r e s h anterior lobes immersed f o r f i v e minutes i n 30$ alobholj  t r i t u r a t e d and centrifuged. The  supernatant l i q u i d was injected f o r a period of two months. Weighings made every f i v e days showed a greater rate of growth of the experimental animals as compared with t h e i r c o n t r o l s . Korenchevsky  (1930) worked with 87 rats of which 76  were young growing rats and 11 were o l d r a t s .  The r a t s were  divided into l i t t e r mate groups of gontrol and experimental animals.  The experimental group was i n t r a p e r i t o n e a l l y injected  d a i l y with an extraot of the anterior lobe of the p i t u i t a r y . A control group were injeoted with a 20$ g l y c e r o l i n saline solution.  The method of preparation of the extract was the  same as that used by Evans and Simpson (1931), except that Korenchevsky  f i r s t ground the glands i n g l y c e r o l instead of  5  d i s t i l l e d water.  The duration of the experiments v a r i e d , f o r  different l i t t e r s , from 6-11 weeks.  The r e s u l t s showed no w e l l  defined influence on the growth of the male r a t s injected w i t h the extract, as oompared with that of the controls i n j e c t e d with glycerol solution. Evans and Simpson (1926), i n j e c t e d 10 male rats with anterior hypophyseal extract, d a i l y , beginning on the 21st day of life.  TVith a single exception the treated animals were from  100-250 grams heavier than t h e i r normal controls at eight months of age. Evans (1935), concludes that the gigantism or accelerated growth that may thus be induced by anterior p i t u i t a r y extracts i n the rat i s an extreme one. The delay i n recognition of the hormone was p a r t l y due t o the experimental work attempted i n feeding experiments.  Many of the e a r l y workers f e d f r e s h anterior lobe  substance which i s an excellent nutriment and as recorded here a number of contradictory r e s u l t s were obtained. (1921) showed the f u t i l i t y of oral dosage.  Evans and Long  Other workers, suoh  as Korenchevsky (1930), who d i d not obtain accelerated growth r e s u l t s with parenteral i n j e c t i o n s , may have seleoted  unreaotive  animals such as young r a t s , or, i f they used responsive  animals  preparations given but onoe or twice a week were i n e f f e c t i v e . Young rats grow so r a p i d l y under s a t i s f a c t o r y conditions that i t i s very d i f f i c u l t t o detect an acceleration of growth which might be assigned t o the action of the hormone i n e a r l y l i f e . Evans and Simpson (1931), had observed that at the age of 5-6 months female rats had reached a stage whioh i s marked  6.  by a slowing i n growth.  This stage forms the 'growth plateau*.  This l e d t o t h e i r adoption of the following prooedure t o determine the potency of an a l k a l i n e extract of the anterior p i t u i t a r y lobe. . From about the 150th day of l i f e female r a t s are weighed at 5 t o 6 day i n t e r v a l s t o ascertain whether the growth plateau has been reached.  At t h i s time the majority of females maintain  t h e i r weight or gain at an average rate of only four to ten grams i n a 20-day period.  From t h e i r numerous observations made from  suoh potency t e s t s they conclude that, while a considerable amount of f l u c t u a t i o n exists i n the responsiveness of animals, only f i v e or Bix individuals are e s s e n t i a l f o r the t e s t .  They further show  that female rats give the best r e s u l t s because >_ ; the plateau whioh they reach at t h i s time i s very d i s t i n o t .  Further, they  are more responsive than males and show less f l u c t u a t i o n of results. Three methods have been widely used i n determining the effeot of the growth hormone on r a t s .  Two  of these depend  on body growth, - (a) increase i n body weight, (b) inorease i n body length, and the t h i r d method i s based on the r a p i d increase i n growth of the oartilagenous epiphyseal discs of the long bones of a young hypophyseotomized r a t .  The method used here i s  dependent upon an observed change i n body weight. Site of Hormone Production.  The mammalian p i t u i t a r y gland can be divided f u n c t i o n a l l y , anatomically, and embryologioally into two parts. There are two evaginations, one from the roof of the primitive  7. stomadeum, ectodermal i n nature, c a l l e d Rathke's pouch. other evagination likewise ectodermal,  The  oomes from the f l o o r of  the t h i r d v e n t r i c l e and remains connected t o the roof of the b r a i n by a s t a l k , the infundibulum. forms the pars nervosa.  This l a t t e r evagination  AVthird portion of the p i t u i t a r y gland  which also presents s p e o i f i o p h y s i o l o g i c a l and d i s t i n c t morphological  c h a r a c t e r i s t i c s i s the pars intermedia which i s  derived from a portion of the primitive stomadeal evagination. The part played by each portion has been the object of Intensive research.  That the growth hormone i s a secretion of the pars  anterior i s unanimously agreed among investigators (Bell,1919). H i s t o l o g i c a l examination of the anterior lobe of the mammalian p i t u i t a r y gland has shown the presence of two main types of c e l l s (Best and Taylor, 1945).  This d i f f e r e n t i a t i o n i s  based on the staining reactions of the c e l l s .  The chromophobe  b e l l s possess no granules, s t a i n l i g h t l y and apparently do not secrete.  The chromophil c e l l s contain large numbers of granules  whioh s t a i n r e a d i l y and are beleived to elaborate the secretion of the anterior lobe (Best and Taylor, 1945).  The chromophil  c e l l s are grouped again i n t o two v a r i e t i e s on the basis of the character of the granules:  (1)  a c i d o p h i l c e l l s which s t a i n more  r e a d i l y with aoid dyes, and  (2) basophil c e l l s which have a  greater a f f i n i t y f o r basic s t a i n s . There has been some divergeance  of opinion as t o the significance of the two  types  of c e l l s present i n the pars a n t e r i o r , but the view which i s held by many workers today was expressed by B e l l (1919).  He  8.  believed that the apparently d i f f e r e n t types of o e l l s represent the same structure i n d i f f e r e n t stages of a c t i v i t y , that i s , the chromophobe c e l l s represent an e a r l i e r stage i n the development of the granular chromophil  cells.  (Best and Taylor, 1945).  Comparative Work on the P i t u i t a r y Gland.  Comparative study of the p i t u i t a r y gland amongst the various vertebrate classes has been largely h i s t o l o g i c a l i n nature. Scruggs (1939), and Kerr (1940^ among others, have made important contributions to the comparative anatomy of the f i s h pituitary,. Perhaps the best comparative d e s c r i p t i o n has been provided recently by Bretschneider and Duyvene de Wit (1947). Comparatively  l i t t l e work has been oarried out on the  growth hormone of vertebrates other than mammals.  Complete  hypophysectomies of Brown Leghorn chicks have produced r e s u l t s similar t o those with hypophyseotomised mammals (Nalbandov and Card, 1943).  These r e s u l t s by themselves do not necessarily  prove the existence of a growth hormone.  In mammalian studies,  as has already been indicated here, the existence of suoh a hormone was  proved by experimentally producing  gigantism.  However, t o the writer's knowledge t h i s work has not been o a r r i e d out with b i r d s by using an extraot of the p i t u i t a r y gland of that form. A growth response was  observed by Smith and Smith  (1923) i n t h e i r experiments with tadpoles.  Injections of extracts  of the anterior lobe of the ox p i t u i t a r y saw a resultant inorease  9  i n volume of twice t h e i r oontrols supplied with the same d i e t . A l l e n (1929) i n a review of t h i s and similar experiments with tadpoles oonoludes that the pars anterior of the hypophysis contains a growth p r i n c i p l e .  This conclusion was made from  implantation experiments, hypophyseotomies, and i n j e c t i o n s of "beef p i t u i t a r y extracts, but there i s no mention of the preparation of a growth-prinoiple from the frog p i t u i t a r y body. Investigations i n t o the p i t u i t a r y gland of f i s h has mainly been on i t s anatomy and embryology (Scruggs, 1939; Woodman, 1939; Kerr, 1940; Bretsohneider and Duyvene de Wit, 1947). Woodman reports that the development of the p i t u i t a r y gland i n the female A t l a n t i c salmon occurs as the f i s h increases i n size and age and prooeeds t o sea.  She describes the d i f f e r e n t i a t i o n of the buccal  anlage into e o s i n o p h i l i c c e l l s .  This e o s i n o p h i l i c formation i s  suggestive of a growth hormone elaboration by these same o e l l s i n the l i g h t of the present knowledge with regard t o the mammalian p i t u i t a r y (Best and Taylor, 1945), but t o the writer's knowledge the presenoe of a growth hormone has not been reported.  Mathews  (1939) attempted t o c l a r i f y the r e l a t i o n s h i p between the p i t u i t a r y gland and the gonads i n Fundulus.  His i n j e o t i o n experiments were  not conclusive, but he did observe a regression of the testas i n hypophyseotomized forms.  Burger (1941) discusses the p i t u i t a r y  rythm involved i n the normal sexual cycle of Fundulus from similar experiments.  However, he does not note e f f e c t s on body growth i n  hypophyseotomies and implantation experiments. presenoe of a spawning-induoing-principle  Reports on the  i n the p i t u i t a r y of  10. various speoies of f i s h i s found i n the l i t e r a t u r e on hormonal a c t i v i t y of the f i s h p i t u i t a r y gland. made intraparitoneal  injections  Hasler, Meyer and F i e l d (1939)  of fresh-aoetone-dried p i t u i t a r y  glands of the oarp into rainbow trout and obtained mature eggs and sperms s i x or seven weeks i n advance of the normal spawning period i n Wisconsin.  In t h i s same work they make referenoe t o the work of  von Ihering and Azevedo•(1937), who were unable t o induce spawning i n Proohilodus, Astynax and Traohycorystes with f r e s h beef hypophysis. The presence of such a factor  i n the f i s h p i t u i t a r y might be compared  with the maturation factor found i n the mammalian p i t u i t a r y . Azevedo and Canale (1938) induced spawning i n a number of Teleosts using f i s h p i t u i t a r y extracts but were unable t o do so when they used p i t u i t a r y extraots of c a t t l e , sheep and b i r d s .  A definite  speoies s p e o i f i o i t y of gonadotropic hormone i s indicated. A l l t h i s leads t o uncertainty when mammalian p i t u i t a r y a c t i v i t y i s taken t o be i n d i c a t i v e of similar f i s h p i t u i t a r y action.  A great  deal of work i s yet t o be done i n t h i s f i e l d before the various hormonal a o t i v i t i e s of the f i s h p i t u i t a r y are c l e a r l y understood. The object of the present investigation  i s t o determine  whether an alkaline extraot of the p i t u i t a r y gland of the P a o i f i c Salmon, Oncorhynchus tsohawytsoha, contains a p r i n c i p l e which w i l l cause an acceleration of growth when injected mammals.  into other f i s h and  11. MATERIALS AND METHODS Souroe of M a t e r i a l s . The beef p i t u i t a r y glands were obtained from the abbatoirs of the Canada Packers Ltd., Vancouver, B. C ,  and the  writer i s indebted t o t h i s company f o r the removal of the pituitaries.  A t o t a l of approximately 150 beef p i t u i t a r y glands  were obtained. The f i s h p i t u i t a r i e s used were those of the sping Salmon, Onchorhynchus tsohawytscha.  This species was used beoause  of the aooessability, age and size of the f i s h .  The men working  at the docks reported an approximate weight of 30 pounds f o r the f i s h from which the p i t u i t a r y glands were removed.  This i s 10  pounds above the average weight reported by Cobb (1916) f o r t h i s species of f i s h .  This species was abundant, being brought i n d a i l y  throughout the summer t o the docks of the B.C. Packers, Vancouver, and t o the Imperial Cannery, Steveston, B.C., whose men were most h e l p f u l i n providing f i s h heads and f a c i l i t i e s f o r the removal of the p i t u i t a r y gland. At f i r s t the removal was very slow and an average of only 30 t o 35 f i s h heads could be opened and t h e i r p i t u i t a r i e s removed per hour.  However, with practioe a simple  method was devised and l a t e r an average of 70 t o 75 p i t u i t a r i e s were taken out per hour.  A single s l i c e through the head, i n a  dorso-ventral d i r e c t i o n and s p l i t t i n g the upper jaw only, just a l i t t l e . o f f oentre between the two eyes usually exposed the p i t u i t a r y body with one side of i t s containing w a l l out away,  She P i t u i t a r y  12.  body was then l i f t e d out with a blunt-ended k n i f e , i t s connection with the b r a i n breaking e a s i l y .  I f the p i t u i t a r y body was not  thus bared, removal of the s k u l l oartilage revealed the b r a i n which when l i f t e d disclosed the p i t u i t a r y gland l y i n g i n i t s protective socket from whioh i t was then scooped out.  *  Rats were obtained from two sources, the Biology Department at the University of B r i t i s h Columbia,  (19), and the  General B i o l o g i c a l Supply House, Chicago, I l l i n o i s (22). Eighty. #  s i x g o l d f i s h were used from a stook procured from the Goldfish Supply Co., S t o u f f v i l l e , Ont.  A commercially prepared extract,  (Armour Water Soluable extract Anterior P i t u i t a r y Substanoe), was also t e s t e d .  Gauge 22 hypodermic needles were used f o r the rat  i n j e c t i o n s , and gauge 26 needles f o r the g o l d f i s h i n j e c t i o n s . Method of E x t r a c t i o n . The extraction process was begun on both the beef anf f i s h p i t u i t a r i e s w i t h i n several hours of t h e i r removal. P r i o r t o extraction the .glands were kept i n a f r e e z e r .  The  anterior lobes of the beef p i t u i t a r i e s were dissected away, i n gross anatomical dissection, from the posterior lobe and then ground i n a Waring-Blendor t o a l i q u i d consistency, a f t e r whioh they were extracted by the standard method as described by Evans and Simpson (1931), f o r the preparation of an alkaline growth hormone extraot. This method may be summarized as follows: *  Approximately 5,000 f i s h heads were used f o r t h i s study.  13.  Preparation of a Standard Aqueous Alkaline extraot of the Hypophysis. X grams of fresh bovine anterior lobe are ground i n a mortar, with x/2 paste.  grams of clean sand, u n t i l reduced t o a smooth  This mixture i s weighed and 2 oo. of d i s t i l l e d water are  added f o r every 1 gram of ground glands. i s measured and 3/8  The volume of t h i s mixture  of i t s volume of 0.2 N NaOH i s added.  As l i t t l e  as 15 minutes of extraction i s adequate but the extraction can be allowed t o continue i n the ice box 12 hours i f more  convenient.  The a l k a l i n e extraot i s neutralized with 0.2 N Aoetio aoid care being taken to s t i r constantly during the addition of the aoid.  An excess  of aoid i s added so that the reaotion of phenol red (1 drop of extraot i n 5 cc. water) i s yellow.  A l k a l i i s then added cautiously  u n t i l phenol red indicator shows the f i r s t tinge of pink.  The  extract i s centrifuged.  cloudy  and pale pink colour. without s t e r i l i z a t i o n .  The supernatant f l u i d i s s l i g h t l y  Fresh extraot may be injected into rats I f the preparation i s to be stored long i t  should be s t e r i l i z e d by passage through a Seitz f i l t e r . The Waring-Blendor was used t o f a c i l i t a t e grinding of the anterior lobes. laboratory eaoh time. 1:1,000 was extraot. was  added.  Twelve hours of extraction was used i n the After centrifuging a preservative, Merthiolate  One part of Merthiolate was  added to 10 parts of  For the f i r s t month of the experiment the a l k a l i n e extract  injeoted without s t e r l i z a t i o n however a Seitz f i l t e r was  thereafter.  used  The extraction method was modified s l i g h t l y f o r the f i s h p i t u i t a r y preparation.  An average weight of 0.044 grams p er  f i s h p i t u i t a r y gland was recorded i n s i x weighings.  Thus the  salmon glands were about 1/45 of the average weight of the beef p i t u i t a r i e s used.  This difference i n size was further magnified  by the use of the whole f i s h p i t u i t a r y whereas only the anterior lobe of the beef p i t u i t a r y was used.  Therefore i n order t o lessen  the d i l u t i o n of the f i s h extract no sand was added.  Thus 2 cc. of  d i s t i l l e d water were added f o r each gram of gland which was used. The preservative used, and i t s d i l u t i o n , was the same as w i t h the beef extraction. Experimental Prooedure.  Daily intraperitoneal injections were made on rats and g o l d f i s h .  For r a t s , the following solutions were used as  i n j e c t i o n material-,  (a) laboratory prepared anterior beef  p i t u i t a r y extract, (b) commercial anterior beef p i t u i t a r y extract, (o) laboratory prepared f i s h p i t u i t a r y extract, and (d) a Q% s a l t s o l u t i o n .  S i m i l a r l y prepared extracts were  injected into the g o l d f i s h save that the commercial preparation was omitted and a 1% s a l t solution was used instead of the 6% solution.  The s a l t solutions were both treated i n the same  manner as were the ground up beef and f i s h p i t u i t a r i e s , and thus served as a c o n t r o l . Weighings were not made t o determine whether the r a t 'growth plateau', discussed e a r l i e r , had been reached.  The animals  15.  obtained from the biology Department of the University of B r i t i s h Columbia  (Group l ) , weighed w i t h i n the range of those animals whioh  had reached the growth plateau indicated i n the normal growth curve f o r the female rats of Evans and Simpson (1931).  The rats  obtained from Chicago, (Group 2), were of proper age, (five t o six months), and were kept f o r 1 week before experimental work started i n order that they might become aocl imated t o t h e i r surroundings.  new  The rats were weighed every s i x or seven days.  They  were numbered by small markings made i n t h e i r ears with a chicken toe marker.  The g o l d f i s h weighings presented a problem.  following procedure was followed:  The  eaoh g o l d f i s h was taken from  the aquarium by hand, the excess water removed by shaking several times, and the f i s h measured on a metric scale r u l e r .  It was  then placed i n a beaker of water, whioh had been previously weighed. After weighing, the difference i n weight of beaker alone and beaker plus f i s h was recorded as the weight of the g o l d f i s h .  The length  measured was from the t i p of the snout t o the end of the body scales i n the t a i l region. However, t h i s method of length measurement d i d not prove s u f f i c i e n t l y accurate since measurements of the same g o l d f i s h made within a few moments of each other showed differences; as high as 2 mm.  Since the largest difference recorded throughout  the experiment was 4 mm.,  the probable error involved was too great  for an aoceptable growth change. not be reoorded here.  Therefore the length changes w i l l  16  The potency assay experiments were c a r r i e d oat using those rats obtained from the U.B.C.  The potency assay was made  only f o r the beef extract and the amount of extract used which gave positive indications of aocelerated growth was used as the c r i t e r i o n f o r the amount of f i s h extract t o be injected.  These  i n i t i a l i n j e c t i o n s were c a r r i e d out f o r three weeks. Following t h i s the main experiments were c a r r i e d out over a period of three months. A l l the r a t and g o l d f i s h i n j e c t i o n s were made d a i l y eave on the days that the weighings took p l a c e .  RESULTS Effeots of extracts on Rats.  1«  Potency determination of prepared anterior beef p i t u i t a r y extract. This experiment waB c a r r i e d out using the 19 r a t s of  group 1.  Two d i f f e r e n t i n j e c t i o n materials were used.  Both  commercially prepared and laboratory prepared extracts were injected i n one and two co. amounts t o d i f f e r e n t animals*  The  injections were made at approximately the same time eaoh morning. Likewise, the weighings were c a r r i e d out at the same time every seven days over a period of 22 days.  Five animals were kept as  oontrols and were handled only. The greatest weight change i s seen t o occur i n those rats injeoted with 2 co. of laboratory prepared beef extract  17.  (table 1 and figure l ) .  The average weight change per rat with  20 i n j e c t i o n s administered  over a period of 22 days i s 56.95 grams.  Injections of 1 cc. of laboratory prepared beef extraot gave a similar but smaller increase i n weight.  The average weight change  per rat over the 22-day period i s 39.85 grams.  Observations on the  animals i n j e c t e d with one and two oo. of commercial prepared extraot show a small gain i n only one animal, four rats showed a deorease and one did not record a change i n weight.  The control animals  were handled only and a l l showed an increase i n weight whioh ranged from 2.4 grams to 10.4 grams, (table l ) . From t h i s experiment i t was  concluded that the most  potent i n j e c t i o n amount was two cc. of beef p i t u i t a r y extraot. This i s the amount used i n the experiments which followed.  Further  i t i s evident that Armour Water Soluable extraot Anterior P i t u i t a r y Substance does not contain the growth-promoting p r i n c i p l e . 2.  Preliminary i n j e c t i o n s of f i s h p i t u i t a r y e x t r a c t s . This was  a preliminary experiment which was c a r r i e d  out on the same r a t s used f o r the potency assay.  The i n j e c t i o n s  began a f t e r the beef extraot i n j e c t i o n s had stopped. whioh had formerly been used as controls were now  The r a t s  i n j e c t e d with  two oo. of a l k a l i n e f i s h p i t u i t a r y extraot, and the remainder wSre kept as controls, being handled only. c a r r i e d out over a period of 21 days.  This experiment was  Table 2 shows that a l l the  animals which had previously been i n j e c t e d with beef p i t u i t a r y now  showed a loss of weight.  many other workers who  This i s similar t o the findings of  reported not only a cessation of growth but  18.  a loss i n weight with the termination of beef p i t u i t a r y extraot injections (Evans and Simpson 1951).  The animals given the  commercial preparation a l l show an increase i n weight save f o r one which d i d not change i n weight at a l l .  This suggests a growth  i n h i b i t o r y effect o£ the commercial extract.  Of the f i v e r a t s  whioh were i n j e c t e d with f i s h p i t u i t a r y extract, three showed small losses ranging from 4.7 t o 17.1 grams.  The remaining two  animals made small gains of 1.8 and 5.2 grams r e s p e c t i v e l y . 3.  F i s h P i t u i t a r y i n j e c t i o n experiments. In t h i s experiment 19 rats (group 2), varying i n  age from f i v e t o s i x months were used.  I n i t i a l l y 12 of these  animals were injected with three c c . of f i s h p i t u i t a r y extract and seven with a 1% s a l t s o l u t i o n .  The f i s h p i t u i t a r y injections were  continued over a period of 23 days on a l l the r a t s save f o r a s l i g h t change which was made at the end of 11 days of work.  At t h i s time  two animals from each of two cages were given i n j e c t i o n s of two c c . of beef extraot. Of the four animals chosen for these new i n j e c t i o n s two had been administered the f i s h p i t u i t a r y extraot previously and the other two had been given injections of 6% s a l t s o l u t i o n . Injeotions continued over another period of 12 days at which time the stock of prepared f i s h p i t u i t a r y extract was unexpectedly depleted.  Injeotions of prepared beef p i t u i t a r y extraot were  continued, however, f o r another s i x days. Although these rats were f i v e t o s i x months of age whioh i s the age s p e c i f i e d f o r a r r i v a l at the growth plateau,  19* examination of t h e i r weights with respect t o the age-weight  curse  would suggest that these r a t s had not as yet reached t h e i r growthplateau.  Thus normal and accelerated weight increase greater than  that reported f o r growth-plateau rats may he expected, and suoh i s observed t o ooour (table S).  It w i l l be r e c a l l e d that the rats of  group 1 showed a maximum weight increase of only 10 grams over a three week period.  Over a period of 23 days the average weight  increase per animal injected with three cc. of 5% s a l t i s 22.02 grams.  solution  The weight change recorded with injections of  3 cc. of f i s h p i t u i t a r y extract over a similar period, i s a gain of 18.19 grams per r a t . Those animals whioh had been administered injections of 2 co. of beef p i t u i t a r y extraot over a period of 14 days gave a weight increase of 40.02 grams per rat (figure 8). Again the beef p i t u i t a r y extraot d i d seem t o have an acceleration e f f e c t , on growth, whereas the f i s h p i t u i t a r y extraot seemed t o have no acceleration e f f e c t , rather perhaps a suppressing r e s u l t , as the weight gains recorded were less than those observed with the salt injections (figure 2 ) .  Thus, no evidence was obtained f o r a  f a c t o r i n salmon p i t u i t a r y whioh stimulates the growth ofr'rats. 4.  E f f e c t s of Extracts on Goldfish. Goldfish were placed i n the experimental aquaria f o r  three days before the experiments started.  The f i s h were previously  marked by clipping t h e i r f i n s . In the f i r s t series beef extract i n j e c t i o n s k i l l e d a l l g o l d f i s h whithin a period of nine days.  The controls whioh  were handled d a i l y , but not injeoted l i v e d .  In the experiments  20.  whioh followed a t o t a l of 76 goldfish received i n j e c t i o n s of extracts of beef p i t u i t a r y , f i s h p i t u i t a r y or 1% salt s o l u t i o n .  The deaths  which occurred i n the f i r s t experiment with beef extract injeotions proved a forerunner of that whioh was to follow.  In a series of 60  beef and f i s h p i t u i t a r y extract-injected f i s h , death occurred i n one t o nineteen days after injections started.  Of the 16 g o l d f i s h  injeoted with the 1% s a l t solution and the seven g o l d f i s h which received no i n j e c t i o n , being handled only, just one died (not included i n figure 4 ) .  The analysis of the data i s complicated by the f a c t  that several experiments were oarried out and that the f i s h died at varying periods before the termination of the experiments. 5, the data from d i f f e r e n t experiments are pooled. the  The figures on  graph show the number of f i s h forming the average percentage  weight change indicated. the  In f i g u r e  Since a l l the f i s h d i d not l i v e throughout  experiments, the figures indicated i n figure 5 may represent both  l i v i n g and dead f i s h .  The smaller samples are dead f i s h .  Weight changes i n handling the G o l d f i s h .  To observe the e f f e c t s of handling, the weights of nine g o l d f i s h were taken p e r i o d i c a l l y over a period of 19 days. During t h i s time the goldfish were handled only momentarily each day.  The changes i n weight are recorded i n table 4 and figure 3.  In 88$ of the oases an i n i t i a l inorease i s observed.  This i s  completely reversed at the f i n a l weighings, at whioh time an average decrease of f i v e percent i s recorded.  This l a t t e r  i s similar numerically t o the i n i t i a l increase observed.  figure Thus the  El o v e r a l l e f f e c t would seem t o be a decrease i n weight.  In some  other animals handling apparently produces a small i n i t i a l weight increase (Evans and Simpson, 1931). Injection of Goldfish with I c c . of 1% Salt  solution.  Goldfish were injected with a salt solution f o r two reasons.  F i r s t a check was necessary to .-ascertain whether the  method of i n j e c t i o n was compatible with l i f e of the g o l d f i s h . Seoondly the effect of the physical act of the needle i n j e c t i o n on the weight of the g o l d f i s h could thus be followed.  This l a t t e r ,  added t o the weight e f f e c t s of handling would enable the segregation of the casual experimental e f f e c t from the physiological sought.  effects  This effect of handling and i n j e c t i o n may be placed at a  maximum weight change, increase or decrease, of 6% (figures 3,4). Out of a t o t a l of 11 g o l d f i s h 73$ shoved an i n i t i a l average decrease i n weight of 3,7% (table 5, exp. 1 and figure 4 ) . In a separate experiment two saline injected f i s h recorded an i n i t i a l increase of the same magnitude (table 5, exp. 2 ) .  However,  the l a t t e r g o l d f i s h after 50 days had decreased i n weight by an average of. 4.3$ from the weights recorded before the injections began (this i s the procedure followed throughout with reports of weight change i n percentages).  The i n i t i a l weight changes recorded i n t h i s  experiment suggests that the e f f e c t of injections i s opposite t o the increase i n the i n i t i a l weight recorded with only the handling of the f i s h .  These opposed i n i t i a l e f f e c t s are of the same order  numerically.  Weighings of the 11 g o l d f i s h noted above did not  22.  proceed a f t e r the observations of the f i r s t f i v e days.  Figures 3  and 4 indicate that the e f f e c t s of both handling and injeotions are to  cause a subsequent decrease i n weight after the i n i t i a l change.  This l a t t e r i s also borne out by the records i n tables 4 and 5. Perhaps the i n i t i a l weight decrease observed with the salt injected f i s h (table 5 exp. 1 and figure 4) i s due merely t o handling.  This  early expression of weight decrease i s similar to the weight deoreases recorded i n the other "control" experiments. Injections of 1 oc. of Anterior Lobe of Beef P i t u i t a r y Extraot. This extraot i s the same as that which proved potent i n the r a t experiments.  (This i n j e c t i o n amount would appear  s u f f i c i e n t i n the l i g h t of the large difference i n mass between the  r a t and g o l d f i s h ) .  As already noted the beef-injected g o l d f i s h  were dead w i t h i n a short time after the injections began.  In two  experiments, comprising a t o t a l of 18 g o l d f i s h , a l l showed an i n i t i a l increase with an average of 19,4$ recorded (table 6, exps. 1, 2 ) .  In a t h i r d experiment three goldfish showed an average  i n i t i a l increase i n weight of 9.8$.  However, two of these l a t t e r  f i s h showed a small i n i t i a l decrease, while the t h i r d recorded a very large increase (table 6, exp. 3),  A seoond weight observation  could be made on only 9 beef p i t u i t a r y extract-injected f i s h (table 6J.  Of t h i s t o t a l , s i x remained of the 18 g o l d f i s h noted  above i n experiments 1 and 2.  The f i n a l weight changes of each of  these s i x g o l d f i s h showed a continued increase i n weight save f o r one f i s h .  The f i n a l increase recorded was an average of 18$.  As t o the g o l d f i s h of experiment 3 the two whioh showed an i n i t i a l decrease d i d continue t o do so f o r a period of approximately two weeks i n whioh time three weighings were made. summarized  These data are  i n figure 5.  Goldfish injected with 1 c c . of Fish p i t u i t a i y  extraot.  As with the beef i n j e c t e d g o l d f i s h death occurred here within 19 days.  Of the 39 f i s h injected with f i s h p i t u i t a r y  extract 26 showed an increase and 13 an i n i t i a l decrease, (table 7 ) . For the f i s h p i t u i t a r y injections four groups of f i s h were used.  In only two of these groups i s there a d e f i n i t e  trend' seen regarding the i n i t i a l change i n weight after the f i r s t period of injeotions (table 7, exps. 2 and 5 ) . With 19 injected specimens an i n i t i a l weight increase i s observed i n 85$ of t h i s number, y i e l d i n g an average increase of 11.5$.  Of these 19  g o l d f i s h 10 l i v e d long enough t o enable a second set of weightchange observations, these showed that s i x f i s h had increased i n weight and four had decreased i n weight t o give an average increase of 5.7$. Highly oontradiotory r e s u l t s are recorded i n the rest of the experiments on the f i s h extraot-injected g o l d f i s h (table 7, exps. 1, 3, 4 ) .  Out of a t o t a l of 20 specimens  i n i t i a l weight increases are observed f o r nine f i s h , 10 show an i n i t i a l decrease, and one f i s h d i d not record a change. Of those that survived injeotions beyond the f i r s t weighings,  24. three had inoreased i n weight at death, and f i v e had deoreased i n weight.  The average f i n a l weight change f o r these eight f i s h i s  a decrease of  1.4$.  DISCUSSIOM To the w r i t e r ' s knowledge no attempt has been made to derive a growth-stimulating f a c t o r from the f i s h p i t u i t a r y . Proceeding on the premise that the p i t u i t a r y gland of f i s h , possessing endocrine a c t i v i t y , does elaborate a growth hormone, a standard method was used f o r the extraction pf the growth hormone of the anterior lobe of the f i s h p i t u i t a r y gland. Figure 6 shows that a smaller average gain i n weight i s evident i n the f i s h - i n j e o t e d - r a t s than i n those injected with a saline s o l u t i o n .  In both instanoes the amount  gained i s similar to that whioh would be recorded by normal growing rats at t h i s stage i n l i f e .  In addition-the: recorded  gain i n weights of the beef-extract injected rats shows the presence of a growth-accelerating p r i n c i p l e i n the l a t t e r extraot.  It can be stated that there are no indications of the  presence of a growth hormone i n an a l k a l i n e extraot preparation of the p i t u i t a r y gland of the P a o i f i o Salmon, on administration to r a t s .  This, however, does not necessarily prove the absence  of growth hormone i n f i s h p i t u i t a r y The presence of a speoies hormone s p e c i f i c i t y has been discussed by several workers.  Hasler, Meyer and F i e l d  25.  (1939), were able to induce rainbow trout t o spawn pre-maturely using a hormonal extract of the p i t u i t a r y gland of carp, while pregnant mare serum and the f o l l i c l e stimulating f r a c t i o n prepared from the sheep p i t u i t a r y f a i l e d t o induoe premature spawning,  von Ihering and Azevedo (1937) were unable t o induce  spawning i n F r o o h i l i d u s , Astyanax, and Traohyoorystes with prolan or f r e s h beef hypophysis.  Cardosa (1934), injectingf£sh hypophysis,  induced spawning prematurely i n Proohilodus.  Easier and Meyer  (1942), report the r e f r a c t o r i n e s s of g o l d f i s h to t h y r o i d preparation of mammalian o r i g i n .  In the l i g h t of the above i t would seem that  a species s p e c i f i c i t y may The existenoe  e x i s t with the growth hormones. of a v a r i a t i o n i n quantity of the anterior  p i t u i t a r y hormones from species to species has become d e f i n i t e l y established (Creassr and G-orbman, 1939).  I f t h i s f a i l u r e of the  rat t o .Tesact totiie growth hormone extraot of the f i s h p i t u i t a r y could be ascribed e n t i r e l y to t h i s quantitative v a r i a b i l i t y i n growth hormone f r a c t i o n s , i t could be construed that the s p e c i f i c i t y i s of n e g l i g i b l e importance.  species  However, r e s u l t s of  hormone exchanges between phylogenetioally distant speoies cannot be successfully explained on a quantitative basis (Creasar Gorbman, 1939).  and  The method used for the extraction of the f i s h  p i t u i t a r y growth hormone has, f o r the lack of a known method, been the same =as that used for the extraction of a potent growth hormone of the beef p i t u i t a r y .  That such a method w i l l produce  a potent extract of the f i s h p i t u i t a r y growth hormone i s not known. Thus i t i s impossible t o ascertain, at t h i s time, s p e c i f i c a l l y .  26.  the  f a i l u r e of the f i s h p i t u i t a r y extracts t o cause an acceleration  of growth i n the r a t s . The i n j e c t i o n of the beef p i t u i t a r y extract into the  g o l d f i s h gave a comparatively large i n i t i a l increase i n weight  after the f i r s t period of i n j e o t i o n s .  Figures 3 and 4 indicate a  v a r i a b i l i t y of s i x peroent i n weight increase or decrease due to handling and i n j e c t i o n . the  This l i m i t i s substantially exceeded by  weight increases recorded with the beef p i t u i t a r y injected  goldfish.  In two experiments which were performed on a t o t a l of  18 g o l d f i s h an i n i t i a l average weight increase of 19.4$ i s recorded. Of t h i s group s i x remained a l i v e long enough f o r the observation of a second weight change.  The f i n a l recorded weight change of these  six f i s h averaged a percentage gain of 18$.  These observations  seem s i g n i f i c a n t when compared t o the r e s u l t s of the f i s h p i t u i t a y injected g o l d f i s h and the controls (figure 5 ) . The i n d i v i d u a l r e s u l t s obtained with the f i s h p i t u i t a r y injected f i s h are highly contradictory to one another as 26 record i n i t i a l increases and 13 record decreases. However, i f two experimental groups containing 19 specimens were viewed alone a positive i n d i c a t i o n of an i n i t i a l weight gain i s seen. The average i n i t i a l gain f o r these two groups Is 11.5$.  This  f i g u r e i s likewise greater than the percentage of error possible,  27. as suggested i n figures 3 and 4, i n evaluating the physiological growth e f f e c t s .  Of the remainder there i s an even s p l i t between  the number whioh gained wight and those which l o s t weight  initially.  These r e s u l t s do suggest a balance between the weight gaining prooesses and the weight decreasing e f f e c t s , whioh may be interpreted as the balance between the a c t i v i t y of a potent growth hormone and the weight changes due t o the method of administration, both of whioh have been noted here.  However before such a growth hormone  a c t i v i t y can be conclusively accepted as present i n the f i s h p i t u i t a r y extraot as prepared, a great deal of research w i l l be necessary. The death of the beef and f i s h injected g o l d f i s h i s s t r i k i n g when i t i s considered that only one of the salt i n j e c t e d f i s h died.  Nixo-Niooscio (1940) states that i t i s the albumen  content of the hypophysis of oattle whioh i s responsible f o r the death of the g o l d f i s h .  But t h i s does not account f o r the death of  the g o l d f i s h caused by the f i s h p i t u i t a r y i n j e o t i o n s .  Although  i t i s not stated i n the report on Nixo-Niooscio s work, i t may 1  be that death i s due to a form of anaphylaxis. The injected protein i n both the beef and f i s h extracts aoting as an antigen. Twombley (1936), makes an interesting observation regarding the formation of antibodies to the injected foreign material.  He suggests that antibodies would form which would act  as a protective substance t o the a c t i v i t y of the injected material. Thompson (1937), was unable t o develop an anti-hormone t o the gonadotropic hormone from sheep p i t u i t a r y gland by i n j e c t i n g i t  into sheep.  Thus, although Thompson notes that many observers are  agreed that anti-hormones may be obtained after suitable i n j e c t i o n of p i t u i t a r y extracts into animals, he suggests that hormones may be antigenic.  Such a p o s s i b i l i t y i s greatly inoreased where  heterologous species are concerned, as i n t h i s work here. The problem here regarding the f a i l u r e of the f i s h p i t u i t a r y extract t o cause an accelerated growth of the r a t s , i s whether we are dealing with a true species specif ic-ity/or whether the recorded observations are r e a l l y the result of the production of protein antibodies brought about by the use of hormone p r o t e i n mixtures derived from heterogeneous  speoies. A t h i r d alternative  i s suggested i n view of the absence of a known method f o r the extraction of a potent f i s h p i t u i t a r y growth hormone.  Does the  method used f o r a potent extraction of the beef p i t u i t a r y  produce  equally as potent a f i s h p i t u i t a r y extract, which i s the method used i n t h i s work?  The r e s u l t s observed here with injections of  the g o l d f i s h are not p o s i t i v e l y indioative and further research i n t h i s f i e l d w i l l be needed before a positive manifest a c t i v i t y of a growth hormone extract of the f i s h p i t u i t a r y gland w i l l be secured.  29  SUMMARY An a l k a l i n e extraot, prepared by the method of Evans and Simpson (1931), from the anterior lobe of the beef p i t u i t a r y , proved t o be a potent source of a growth hormone when adiministered t o female r a t s .  Daily i n t r a p e r i t o n e a l injeotions of t h i s extract  into g o l d f i s h resulted i n an 18.0$ weight increase within eight days. The g o l d f i s h which oontinued to l i v e beyond t h i s f i r s t weighing continued t o show a weight increase of 11.5$.  This weight increase  appears s i g n i f i c a n t i n view of the decreased weight recorded with the salt, injeoted g o l d f i s h . A similar method was used f o r the preparation of a growth hormone extraot from the p i t u i t a r y gland of the P a c i f i c Salmon, Onoorhynohus tschawytsoha.  Injections of t h i s extract into female  rats f i v e to s i x months of age f a i l e d t o accelerate the growth of the animals.  Injections to g o l d f i s h r e s u l t e d i n a oonfusing  Both increases and decreases were observed. experimental  pattern.  However i f two of the  groups containing a t o t a l of 19 g o l d f i s h were examined,  an 11$ increase i n weight i s seen following the f i r s t period of injeotions.  This suggests a lesser potency of the f i s h p i t u i t a r y  extract, and the occurrence of contradictory r e s u l t s between i n d i v i d u a l g o l d f i s h as the balance between the  growth-accelerating  effect of the hormone and the weight decreasing effeot of the method of i n j e c t i o n .  Further e l u c i d a t i o n as t o the occurrence of a speoies  s p e c i f i c i t y here must await the development of a known potent method for  extraction of the growth hormone of the f i s h p i t u i t a r y .  In  order t o substantiate t h i s claim of a growth promoting effeot i n an  alkaline extraot of the p i t u i t a r y gland of the P a c i f i c Salmon more data on t h i s problem w i l l be required.  31.  TABLE 1 - Potency Assay Determination of Beef Growth Hormone Extract Using Rats of Group 1 . Weight Change Over 3 Week Period.  Ho.  Injection Material  Initial Weight (gwas)  60  2 c c . Prep. Beef  274.0  /  57.7 70.6  98  »  it  ti  223.5  /  7  it  ii  n  207.1  /.49.6  4  it  it  ii  211.3  /  49.9  00.  Prep. Beef  241.7  /  40.7  111  tt  "n  II  237.5  /  53.2  6  n  it  it  229.9  /  31.2  2  n  it  it  202.5  /  34.3  208.0  -  11.8  211.0  -  7.9  244.0  /  14.1  225.1  -  20.8  204.3  -  19.1  258.0  /  3.1  209.5  /  10.4  200.0  /  8.8  212.2  /  7.2  217.4  /  2.4  53  5 8 43  1  2 co. Comm. Beef it  ti  ti  1 oc. Comm. Beef  83  it  ti  ti  3  Ti  H  ft  9  it  11  it  54  Control  45  n  77  •ii  1  ft  61  it  241.7  TABLE 2 - Preliminary Injeotion of F i s h P i t u i t a r y Extracts t o Rats of Group 1.  (Following Beef P i t u i t a r y I n j e c t i o n  Experiment).  Weight Change Over 3 Week Period, (grams)  Ro.  Injection Material  Initial Weight (grams)  60  Control  274.0  - 16.3  223.5  - 36.4  92  II  7  n  207.1  - 24.0  4  tt  211.3  - 23.2  n  241.7  -  111  ti  237.5  - 29.9  6  it  229.9  -  8.0  2  it  202.5  -  7.2  5  it  208.0  { 11.3  8  ti  211.0  / 10.9  43  it  241.7  /  83  it  244.0  53  3 9 54  II  n 2oo. F i s h P i t u i t a r y  8.1  0.3  225.1  / 14.0  201.3  / 16.0  258.0  - 17.1  45  ti  ti  tt  209.5  - 13.0  77  tt  it  n  200.0  /  1.8  1  ti  tt  ti  212.2  -  4.7  61  ti  ii  ii  217.4  /  5.2  33. TABLE 3.  Weight Changes Recorded with Rats of Group 2. (grams).  Weight Change Weight Change over over 7 23 days with Fish Pituitary I n i t i a l days with 6% Salt 5 oo. Mo. Weight no HairLung 3 oo.  Weight Change over 18 Days with Beef P i t u i t a r y Injections 2 co.  /19.6  2  5.49.0  /  11.0  3  161.4  /  12.9  4  170.0  /  12.0  5  137.9  /  5.1  6  148.7  /  12.0  7  180.1  /  4.3  10  150.5  /  17.7  11  176.7  /  10.9  12  167.8  /  9.2  /24.7  13  156.3  /  13.2  /16.5  14  132.0  /  7.8  /20.9  17  172.9  /  2.8  /33.6  20  182.3  /  10.2  /31.3  30  138.0  /10.0  31  119.0  /  8.8  32  162.1  /  16.9  33  166.4  /  10.4  34  151.0  /  13.9  35  155.3  /  8.0  36  157.2  /  8.5  /1.2 /49.0  /44.9 /2.9 /29.3 /  23.7  /20.9 /21.0 /70.8 /16.5 /24.1 /  5.7 /44.5  TABLE 4.  No.  Weight Change of Goldfish Handled Only.  Initial Weight (grams)  1.  11.1  4.  10.6  No. Days 8 >  First Weight Change grams %  Second Weight Change grams %  / 0.4 /  3.6  - 0.6  - 5.4  19  No. Days  8  / 0.2  /  1.9  - 0.2  - 1.9  19  *  5.  13.9  8  / 1.0 /  7.2  - 1.2  - 8.6  19  9.  10.6  8  - 0.5  -  4.7  / 0.1 / 0.9  19  11.  11.2  8  / 1.3  / 11.6  - 0.5  - 4.5  19  13.  12.5  8  / 0.7 /  - 0.8  - 6.4  19  17.  13.0  8  / 1.4 / 10.8  - 1.0  - 7.7  19  20.  13.5  9  / 1.5 / 11.1  - 0.6  - 4.4  15  23  12.1  9  / 0.1 /  - 0.7  - 5.8  15  5.6  0.8  35.  TABLE 5.  No.  Weight Change of Goldfish Injected with 1% Salt  Initial Weight (grams)  No. Days  First Weight Change grams %  Solution.  Seoond Weiglht Change g?ams %  No. Days  Experiment 1. 32. »  8.5  5  - 0.3  - 3.5  *  35.  13.3  5  38.  10.0  5  - 0.8  - 8.0  41.  17.9  5  - 1.8  -10.0  44.  8.9  5  - 0.5  - 5.6  47.  10.6  5  - 0.6  - 5.7  50.  8.4  5  - 0.3  - 3.6  53.  9.3  3  - 0.3  - 3.2  56.  11.0  5  / 0.1  / 0.9  59.  9.0  5  / 0.1  /  62.  8.9  5  - 0.3  - 3.4  65.  8.0  5  - 0.2  - 2.5  1.0  Experiment 2. 9.  10.0  7  / 0.1 / 1.0  - 0.2  - 2.0  15  52.  6.2  7  / 0.6  / 0.1  / 1.2  15  / 7.3  Third  Fourth  9.  L8  - 0.4  - 4.0  - 0.8  - 8.0  24  52.  18  - 0.3  - 3.7  - 0.2  - 2.4  24  Fifth 9.  30  / 0.1  / 1.0  52.  30  - 0.8  - 9.7  36. TABLE 6.  Weight Change of Goldfish Injected With 1 oo. of Anterior Lobe of Beef P i t u i t a r y E x t r a c t .  Ho.  Initial Weight (grams)  Ho. Days  First Weight Change grams %  Second Weight Change grams %  Ho. Days  Experiment 1. -  2. 3. 6. 7. 8. 12. 14. 15.'  12.5 13.5 9.2 13.2 10.3 10.2 10.1 10.0  8 8 5 6 5 7 5 5  / / / / / / / /  4.3 2.5 2.4 2.4 1.9 1.4 2.8 2.0  / / / / / / / /  34.4 18.5 26.1 18.2 18.4 13.7 27.7 20.0  5 5 1 5 2 5 5 5 5 5  / / / / / / / / / /  3.0 3.1 0.4 1.2 0.9 1.3 3.9 0.9 0.9 2.0  / / / / / / / / / /  7 7 7  - 0.3 - 0.4 / 3.0  / 4.5  / 33.3  10  20.1 24.2 3.8 15.6 13:8 21.7 36.4 9.5 6.8 20.6  / 1.6 / 3.2  / 10.7 / 25.0  6 8  / 0.4 / 3.7 - 0.3  / 6.7 / 34.6 - 3.2  6 6 8  2.5 3.2 35.3  - 0.7 - 1.1 / 1.5  -  Experiment 2. 31." 34. 37. 43. 46. 49. 52. 58. 61. 64.  14.9 12.8 10.4 7.7 6.5 6.0 10.7 9.5 13.2 9.7  Experiment 3. 95. 96.* 91.  12.1 18.4 8.5  Third 95. 96. 91.  18 15 14  - 0.3 - 2.5 - 1.6 - 13.0 / 1.0 / 11.8  5.8 8.9 / 17.6  12 12 12  37.  TABLE 7.  Weight Change of Goldfish Injected with 1 oo. of F i s h P i t u i t a r y Extraot.  No.  Initial Weight No. (grams) Days  First Weight Change grams %  Second Third Weight Change No. Weight Change No. grams % Days grams % Days  Experiment 1. 21. 22. 24. 28.  13.7 12.4 10.4 13.4  6 9 9 9  / 1.6 - 0.6  / 11.7 - 4.0  - 1.7  - 12.7  3 5 5 5 5 5 5 5 5 5 4 5  / / / / / / / / / /  1.9 0.8 4.0 0.1 2.1 0.1 1.0 2.1 l.S 1.5 0.2  / / / / / / / / * /  7 7 7 7  / / -  1.9 1.0 3.6 0.4  6 7 6 6 7 7 7 4 7 6 7 7  1 / / / / /  0.9 1.8 0.6 0.7 1.0 0.4 0.1 0.1 2.3 0.4 0.2 0.4  - 1.3 - 0.6 - 1.8  - 10.4 - 5.8 - 13.4  14 14 14  / / / / -  0.6 3.1 1.3 1.2 0.2 0.7  / 7.0 / 23.3 / 9.0 / 14.4 - 1.2 - 4.5  9 9 9 7 10 12  / 1.3  / 13.8  9  - 0.5  -  1.7 0.5 1.6  13.7 4.8 12.0  17 19 18  - 3.4  - 20.1  14  / 0.2  /  16  Experiment 2. 30. 33. 36. 39. 42. 45. 48. 51. 54. 57. 60. 63.  8.4 14.9 8.6 13.3 14.5 8.3 16.7 15.5 12.3 9.4 9.1 6.6  12.8 9.3 30.0 0.7 25.3 0.6 6.4 17.1 16.0 16.5 3.0  7.6  10  2.1 / 21.2 2.4 - 14.2 0.2 - 1,7 0.9 - 8.7  8 12 11 8  - 0.9 / 7.4  10 10  / 0.6 / 5.0 / 2.0 / 27.4  13 12  Experiment 3. 81. 72. 70. 3.  9.9  16.9 12.1 10.3  - 19.0 5.9  /  29.8 3.9  / / / /  11.8 16.4 9.1 13.0 9.6 4.9 1.0 1.2 19.2 4.3 1.7 5.5  / -  Experiment 4. 74. 1. 2. 93.. 3. 98. 89, 94. 35. 19. 7. 82.  7.6 11.0 6.6 5.4 10.4 8.1 9.9 8.1 12.0 9.3 12.0 7.3  -  -  - 0.1 / 0.6  (continued on next page)  1.7  38.  TABLE 7. Weight Change of Goldfish Injected with 1 c c . of F i s h P i t u i t a r y Extraot.  Ho.  Initial Weight Ho. (grams) Days  (continued from page 37).  First Weight Change ggams %  Seoond Weight Change grams %  Ho. Days  / / / / / / /  / 0.4  / 3.6  5  - 0.1  - 0.8  9  Experiment 5. 6. 7.2 9. 8.1 8. 6.4 1. 11.0 4. 7.6 5. 6.5 2.. 11.8  7 5 4 3 4 4 6  1.0 0.4 0.7 2.7 0.8 1.4 0.3  / / / / / / /  14.0 4.9 10.9 24.3 10.5 21.5 2.5  Third Weight Change Ho. grams % Days  39  IO\  3  Figure 1.  :  To  7s Dd,c/s  Jo  Response of Normal Female Rats (group 1) t o hypophyseal beef Growth Hormone, a} 2 oo. commercial extract of growth hormone. 2 r a t s . b) 1 c c . commercial extract of growth hormone. 4 r a t s . c) Control, handled only. 5 r a t s . dj 1 c c . laboratory prepared growth hormone. 4 r a t s , e; 2 oo. laboratory prepared growth hormone, 4 r a t s .  1t\e&T\ P e i r c e t x t a j e (Jetgkt I n c r e a s e  Tigure' Z~.  Response of normal female rats (group 2) t o Beef and F i s h hypophyseal growth hormone extract* — 2 rats injected with 3 co. f i s h extract followed by beef growth hormone. 2 rats injected with 3 co. saline followed by beef growth hormone. - 5 rats injected with 3 cc. s a l i n e . — _ io rats injected with 3 cc. f i s h growth hormone.  41.  Figure 3.  Graph of mean percentage weight gain of g o l d f i s h handled only. Figures represent number of g o l d f i s h whioh form the indicated averages.  Percentage  »  k/cight  —7K  / \  / /  ov  Cha.Tv.jc  \ \  rr O  o  Figure 4.  leu  Graph of mean percentage weight gain of g o l d f i s h injected with 1 oo. of 1% salt s o l u t i o n . Figures represent number of g o l d f i s h which form the indicated averages.  43  Figure 5.  Comparison of the mean percentage weight change of beef p i t u i t a r y extract with f i s h p i t u i t a r y extraot injections ( l co.) t o g o l d f i s h . Horizontal l i n e s give l i m i t s of v a r i a t i o n , Q% recorded with handling and saline injections of g o l d f i s h . Figures represent number of g o l d f i s h which form the indioated average. t  Goldfish injected with beef p i t u i t a r y extraot. — -  Goldfish injected with f i s h p i t u i t a r y extract.  44  Figure 6.  Histogram of weight changes observed i n rats (group 2). Bars (horizontal) - change i n weight per day per rat over an eight' day period. Time i n t e r v a l f o r acclimatization of rats (handled only). Bars (angled) - change i n weight per day per r a t injected with"6$ s a l t solution over a 23 day period. Bars (stippled) - change i n v/eight per day per rat injected with f i s h p i t u i t a r y extract over a 23 day period. Bars (plain) - change i n weight per day per r a t injected with beef p i t u i t a r y extract over a 14 day period.  45  Bibliography  A l d r i o h . Amer. J . Physiol., 30:1912. (Quoted from Wulzen, 1914). AHrioh. Amer. J . Physiol., 31:1913. (Quoted from Wulzen, 1914). A l l e n , B. M.  The influence of the t h y r o i d gland and hypophysis upon growth and development of amphibian larvae. Quart. Rev. B i o l . , 4:325-372, 1929.  Azevedo, P. de e L. Canale. A hipofise e sua aoao nas gonados dos peixes neotropicos. Arq. Inst. B i o l . (Sao Paulo)., 9:165-186, 1938. (Summary B i o l . Abst.). B e l l , W.  B.  The P i t u i t a r y , 1919. Ltd.  Best, C. H. and Taylor, N. B. P r a c t i c e , 1945.  The Macmillan Company of Canada  Physiologioal Basis of Medical Williams and Wilkins Co.  BretSchneider, L. H. and H. Duyvene de Wit. Monograph-Sexual Endoorinology of Non-Mammalian Vertebrates. E l s e v i e r Pub. Co., 1947. Burger, J.. W,  Some experiments on the e f f e c t s of hypophyseotomies and p i t u i t a r y implantations on the male Fundulus h e t e r o o l i t u s . B i o l . B u l l . , 80:31-36, 1941.  Cardosa, D. M.  Arch. Inst. B i o l . (Sao Paulo), 5:133, 1934. from Hasler,-Meyer and F i e l d , 1939).  Cobb, J . N.  P a o i f i c salmon f i s h e r i e s . Third E d i t i o n . the Commissioner of F i s h e r i e s , 1921.  (Quoted  Report of  Creaser, C. W. and Gorbman, A. Species s p e c i f i c i t y of thegonadotropic f a c t o r s i n vertebrates. Quart. Rev. B i o l . , 14:311-331, .1939. Evans, H. M.  The growth hormone of the anterior p i t u i t a r y . Amer. Med. Ass., 104:1232-1237, 1935.  J.  Evans, H. M. and Long, J . A. The e f f e c t of the anterior lobe administered i n t r a p e r i t o n e a l l y upon growth, maturity, and oestrus cycles of the r a t . Anat. R e c , 21:62-63, 1921. Evans, H. M. and Long, J . A. The e f f e c t of feeding the anterior lobe of the hypophysis on the oestrus cycle of the r a t . Anat. R e c , 21:62, 1921.  46.  Evans, H. M. and Simpson, M. E. E f f e c t s of anterior hypophyseal extracts on the male. Anat. R e c , 32:206, 1926. Evans j H. M. and Simpson, M. E., Hormones of the anterior hypophysis. Amer. J . Physiol., 98:511-546, 1931. Gudernatsch, J . F. Feeding r a t s on glands of internal secretion. Anat. R e c , 14:35-36, 1918. Hasler, A. D. and Meyer, R. K. Respiratory responses of normal and castrated g o l d f i s h to t e l e o s t and mammalian hormones. J . exp. Zool., 91:391-404, 1942. Hasler, A. D., Meyer, R. K. and F i e l d , H. M. Spawning induoed prematurely i n trout with the a i d of p i t u i t a r y glands of the carp. Endoorinology, 25:978-983, 1939. Howel. J . exp. Med., 3:1898.  (Quoted from Sohaefer, 1909}  Ihering, R. von and P. de Azevedo. Zool. Anz., 120:71, 1937. (Quoted from Hasler, Meyer and F i e l d , 1939). Kerr, T.  On the histogenesis of some t e l e o s t p i t u i t a r i e s . roy. Soc. Edirib., 60:224-240, 1940.  Proc.  Korenchevsky, V. The influence of the hypophysis on metabolism, growth and sexual organs of male rats and r a b b i t s . Bio-Chem. J . , 24:383-393, 1930. Marinus, C. J . The e f f e c t of feeding pars t u b e r a l i s and pars anterior proprior of bovine p i t u i t a r y glands upon the early development of the white r a t . Am. J . Physiol., 49:238-247, 1919. Matthews, S. A. The relationship between the p i t u i t a r y gland and the gonads i n Fundulus. B i o l . B u l l . , 76:241-250, 1939. Kalbandov, A. V. and Card, L. E. Effect of hypophysectomy of growing chicks. J . exp. Zool., 94:387-413, 1943. Nixo-Nicoscio, N. V. P r o c Moscow Zool., Park, 1:178-184. (English Summary). Oliver and Sohaefer. J . Physiol., 18: 1895. Schaefer, 1909).  (Quoted from  Sohaefer, E. A. The functions of the p i t u i t a r y body. S o c London, 81:442-468, 1909.  Proc. roy.  47. Scruggs, W. M.  The e p i t h i l i a l components of the teleost p i t u i t a r y gland as i d e n t i f i e d by a standardized method of selective staining. J . Morph., 65:187-213, 1939.  Smith, P. E. and Smith, I. P. The function of the lobes of the hypophysis as indicated by replacement thereapy with different portions of the ox gland. Endoorinology, 7:579-591, 1923. Woodman, A. S. Wulzen, R.  The p i t u i t a r y gland of the A t l a n t i c Salmon. J . Morph., 65:411-435, 1939.  The anterior lobe of the p i t u i t a r y body i n i t s relationship t o the g rowth of b i r d s . Am. J . Physiol., 34:127-139, 1914.  

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