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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 A Thesis Submitted i n Partial Fulfilment of The Requirements for the Degree of MASTER OF ARTS In the Department of ZOOLOGY The University of Br i t i s h Columbia (Oncorhynchus tsehawytsoha) Abey Jampolsky Ootober, 1949. )l ABSTRACT An alkaline extract from the anterior lobe of the beef pituitary proved a potent source of a growth hormone with injections to rats. Injections of this extract to goldfish resulted i n an 18$ weight increase within eight days, which appears significant when compared to their controls. A similar method of preparation was used for an extract of the pituitary gland of the Pacific Salmon, Onoorhynohus tschawytsoha. Injections of this extract f a i l e d to accelerate the growth of test rats. Injections to goldfish resulted in a confusing pattern with both increases and decreases in weight being observed, however, the results indicate the presence of a growth-accelerating hormone. Substantiation of this growth-acoelerating effeot of alkaline extracts of the f i s h pituitary i s necessary in view of the conflicting results obtained. Acknowledgments I would like to take this opportunity to express my appreciation to Dr. W. A. Clemens, Head of the Department of Zoology, for his kindness during my studies at the University of B r i t i s h Columbia. I wish to thank Dr. W. S. Hoar for help i n suggestion of the topic, for assistance and enoouragement, and for the valuable advice he has offered throughout. I also wish to express my appreciation to the Department of Zoology, University of B r i t i s h Columbia, for the grant of money used i n support of this work. TABLE OF CONTENTS Pag© Introduction and Review of Literature 1. Materials and Methods 11* Results Effects of Extracts on Rats 16. Potency determination of Beef Pituitary Extraot 16. Preliminary Injections of Fish Pituitary Extract 17. Fish Pituitary Injection Experiments 18. Effects of Extracts on Goldfish 19. Weight Changes in Handling the Goldfish 20. Injections of 1 co. of Anterior Lobe of Beef Pituitary Extract 22. Goldfish Injected with 1 cc. of Fish Pituitary Extraot 23. Discussion 24. Summary 29. Tables Table 1 - Potency Assay Determination of Beef Growth Hormone Extraot Using Rats of Group 1 31. Table 2 - Preliminary Injection of Fish Pituitary Extracts to Rats of Group 1. 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 with 1% Salt Solution. 35. Table 6.- Weight Change of Goldfish Injected 1 cc. of Anterior Lobe of Beef Pituitary Extract. 36. Table 7.- Weight Change of Goldfish Injected with 1 oo. of Fish Pituitary Extract. 37. Figures Figure 1 - Response of Normal Female Rats (group 1) to hypophyseal Growth Hormone. 39. Figure 2 - Response of normal female rats (group 2) to Beef and f i s h hypophyseal growth hormone extract. 40. Figure 3 * Graph of mean percentage weight gain of goldfish handled only. Figures represent number of goldfish which form the indicated averages 41. Figure 4 - Graph of mean percentage weight gain of goldfish injected with 1 cc. of 1% salt solution. Figures represent number of goldfish which form the indicated averages 42. Figure 5 - Comparison of the mean percentage weight change of beef pituitary extract with fish pituitary extract injections (1 oo.) to 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 pituitary gland is an organ of internal secretion. Exhaustive research has now been carried on i n order that i t s origins and functions might be better understood. The earliest work had to do with the pituitary of mammals beoause of the possible application of results to human physiology. Comparative work followed. Of the various classes within the vertebrate series the fishes have come to assume an extremely important position i n the economics of man. Although the embryology and anatomy of the pituitary gland of f i s h have been extensively studied l i t t l e attention has been given to i t s physiological nature. The present investigation was undertaken for the purpose of advancing knowledge i n this 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 activity of the pituitary gland were those of Oliver and Sohaefer (1895) who found that ..aqueous extracts of the pituitary body produced a rise in blood pressure when injected into the blood vessels. Howell .(1898) was the f i r s t to localise this hormonal activity i n the posterior lobe of the pituitary. However, i t was not u n t i l 1909 that the growth-promoting effects of the pituitary gland were f i r s t recorded (Sohaefer, 1909). At this time Sohaefer fed a small amount of dried powder material, derived from the anterior lobe of ox pituitaries, to male rats over a period of two months. A seoond group, which were used as a control group, were fed 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 pituitary 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 is contradictory and of historical interest only. Aldrloh (1912, '1913) performed two series of experiments. In 1912 he added 50 mg. of fresh dessicated J anterior lobe of ox pituitary to the diet of a number of dogs. To another group, which served as his "control", a similar amount of prepared ovary was fed. He observed that the average weights showed that the "control" group had a tendency to increase i n weight more rapidly than the. pituitary-fed animals, but for "certain reasons" Aldrich ooncluded that there was neither stimulation nor retardation i n growth. In a second series 3. Aldrioh (1913) carried out a similar experiment on 10 rats divided into two groups of males and females i n each* As with the dogs, the rat "control" group -showed an increasingly greater weight than the pituitary 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 effect. Wulzen (1914) fed young chicks with a weighed amount of unmodified anterior lobe of ox pituitary over a period of three months. In addition they were fed a balanced chick diet as were a second group which were also fed with a similar amount of li v e r instead of anterior pituitary lobes. Measurements taken weekly were of three types: (l) average weight i n grams, (2) average length of wing in mm., (3) average length of foot i n mm. A l l the measurements made on 25 young ohioks showed a distinct inhibition of growth i n the pituitary fed chicks. Evans and Long (1921) fed one-half gram of ground fresh anterior lobe of ox pituitary daily to 54 rats of 21 days of age. A similar diet was given to l i t t e r mate rats save that one-half gram of raw liver was given instead of the anterior lobe of pituitary gland. Weighings made every four days showed no significant difference i n growth between the hypophysis-fed animals and the controls. A 'growth-accelerating' effect of the hypophysis was reported by Gudernatsch (1918) from observations made on the feeding of albino rats different glandular diets. This conclusion is corroborated by Marinus' work (1919). He separated 100 young rats into three groups. One group was fed anterior lobe of the beef pituitary gland, the seoond the pars tuberalis of the beef pituitary gland, and the third was given beef muscle. His results show a 'growth-accelerating' effect only with those animals fed the anterior lobe of beef pituitary. As already reported Evans and Long (1921), observed that feeding of the anterior pituitary lobe caused no significant difference i n growth. However, i n the same year they carried out another set of experiments i n whioh intraperitoneal injections of an anterior pituitary 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 fresh anterior lobes immersed for five minutes i n 30$ alobholj triturated and centrifuged. The supernatant liquid was injected for a period of two months. Weighings made every five days showed a greater rate of growth of the experimental animals as compared with their controls. Korenchevsky (1930) worked with 87 rats of which 76 were young growing rats and 11 were old rats. The rats were divided into l i t t e r mate groups of gontrol and experimental animals. The experimental group was intraperitoneally injected daily with an extraot of the anterior lobe of the pituitary. A control group were injeoted with a 20$ glycerol 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 glycerol instead of 5 d i s t i l l e d water. The duration of the experiments varied, for different l i t t e r s , from 6-11 weeks. The results showed no well defined influence on the growth of the male rats injected with the extract, as oompared with that of the controls injected with glycerol solution. Evans and Simpson (1926), injected 10 male rats with anterior hypophyseal extract, daily, beginning on the 21st day of l i f e . TVith a single exception the treated animals were from 100-250 grams heavier than their normal controls at eight months of age. Evans (1935), concludes that the gigantism or accelerated growth that may thus be induced by anterior pituitary extracts i n the rat i s an extreme one. The delay i n recognition of the hormone was partly due to the experimental work attempted i n feeding experiments. Many of the early workers fed fresh anterior lobe substance which i s an excellent nutriment and as recorded here a number of contradictory results were obtained. Evans and Long (1921) showed the f u t i l i t y of oral dosage. Other workers, suoh as Korenchevsky (1930), who did not obtain accelerated growth results with parenteral injections, may have seleoted unreaotive animals such as young rats, or, i f they used responsive animals preparations given but onoe or twice a week were ineffective. Young rats grow so rapidly under satisfactory conditions that i t is very d i f f i c u l t to detect an acceleration of growth which might be assigned to the action of the hormone i n early 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 led to their adoption of the following prooedure to determine the potency of an alkaline extract of the anterior pituitary lobe. . From about the 150th day of l i f e female rats are weighed at 5 to 6 day intervals to ascertain whether the growth plateau has been reached. At this time the majority of females maintain their weight or gain at an average rate of only four to ten grams in a 20-day period. From their numerous observations made from suoh potency tests they conclude that, while a considerable amount of fluctuation exists i n the responsiveness of animals, only five or Bix individuals are essential for the test. They further show that female rats give the best results because >_ ; the plateau whioh they reach at this time i s very distinot. Further, they are more responsive than males and show less fluctuation of results. Three methods have been widely used i n determining the effeot of the growth hormone on rats. Two of these depend on body growth, - (a) increase i n body weight, (b) inorease i n body length, and the third method is based on the rapid increase i n growth of the oartilagenous epiphyseal discs of the long bones of a young hypophyseotomized rat. The method used here i s dependent upon an observed change in body weight. Site of Hormone Production. The mammalian pituitary gland can be divided functionally, anatomically, and embryologioally into two parts. There are two evaginations, one from the roof of the primitive 7. stomadeum, ectodermal i n nature, called Rathke's pouch. The other evagination likewise ectodermal, oomes from the floor of the t h i r d ventricle and remains connected to the roof of the brain by a stalk, the infundibulum. This latter evagination forms the pars nervosa. AVthird portion of the pituitary gland which also presents speoifio physiological and distinct morphological characteristics i s the pars intermedia which is 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 is a secretion of the pars anterior i s unanimously agreed among investigators (Bell,1919). Histological examination of the anterior lobe of the mammalian pituitary gland has shown the presence of two main types of cells (Best and Taylor, 1945). This differentiation i s based on the staining reactions of the c e l l s . The chromophobe bells possess no granules, stain l i g h t l y and apparently do not secrete. The chromophil cells contain large numbers of granules whioh stain readily and are beleived to elaborate the secretion of the anterior lobe (Best and Taylor, 1945). The chromophil cel l s are grouped again into two varieties on the basis of the character of the granules: (1) acidophil cells which stain more readily with aoid dyes, and (2) basophil c e l l s which have a greater a f f i n i t y for basic stains. There has been some divergeance of opinion as to the significance of the two types of cells present i n the pars anterior, but the view which is held by many workers today was expressed by Bell (1919). He 8. believed that the apparently different types of oells represent the same structure i n different stages of activity, that i s , the chromophobe cells represent an earlier stage i n the development of the granular chromophil c e l l s . (Best and Taylor, 1945). Comparative Work on the Pituitary Gland. Comparative study of the pituitary gland amongst the various vertebrate classes has been largely histological 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 description 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 results similar to those with hypophyseotomised mammals (Nalbandov and Card, 1943). These results 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, to the writer's knowledge this work has not been oarried out with birds by using an extraot of the pituitary gland of that form. A growth response was observed by Smith and Smith (1923) i n their experiments with tadpoles. Injections of extracts of the anterior lobe of the ox pituitary saw a resultant inorease 9 in volume of twice their oontrols supplied with the same diet. Allen (1929) i n a review of this and similar experiments with tadpoles oonoludes that the pars anterior of the hypophysis contains a growth principle. This conclusion was made from implantation experiments, hypophyseotomies, and injections of "beef pituitary extracts, but there is no mention of the preparation of a growth-prinoiple from the frog pituitary body. Investigations into the pituitary 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 pituitary gland in the female Atlantic salmon occurs as the f i s h increases i n size and age and prooeeds to sea. She describes the differentiation of the buccal anlage into eosinophilic c e l l s . This eosinophilic formation i s suggestive of a growth hormone elaboration by these same oells i n the light of the present knowledge with regard to the mammalian pituitary (Best and Taylor, 1945), but to the writer's knowledge the presenoe of a growth hormone has not been reported. Mathews (1939) attempted to c l a r i f y the relationship between the pituitary gland and the gonads in Fundulus. His injeotion experiments were not conclusive, but he did observe a regression of the testas i n hypophyseotomized forms. Burger (1941) discusses the pituitary rythm involved i n the normal sexual cycle of Fundulus from similar experiments. However, he does not note effects on body growth i n hypophyseotomies and implantation experiments. Reports on the presenoe of a spawning-induoing-principle i n the pituitary of 10. various speoies of f i s h i s found i n the literature on hormonal activity of the f i s h pituitary gland. Hasler, Meyer and Field (1939) made intraparitoneal injections of fresh-aoetone-dried pituitary glands of the oarp into rainbow trout and obtained mature eggs and sperms six or seven weeks in advance of the normal spawning period i n Wisconsin. In this same work they make referenoe to the work of von Ihering and Azevedo•(1937), who were unable to induce spawning i n Proohilodus, Astynax and Traohycorystes with fresh beef hypophysis. The presence of such a factor i n the f i s h pituitary might be compared with the maturation factor found in the mammalian pituitary. Azevedo and Canale (1938) induced spawning i n a number of Teleosts using f i s h pituitary extracts but were unable to do so when they used pituitary extraots of cattle, sheep and birds. A definite speoies speoifioity of gonadotropic hormone i s indicated. A l l this leads to uncertainty when mammalian pituitary activity i s taken to be indicative of similar f i s h pituitary action. A great deal of work is yet to be done in this f i e l d before the various hormonal aotivities of the f i s h pituitary are clearly understood. The object of the present investigation i s to determine whether an alkaline extraot of the pituitary gland of the Paoific Salmon, Oncorhynchus tsohawytsoha, contains a principle which w i l l cause an acceleration of growth when injected into other f i s h and mammals. 11. MATERIALS AND METHODS Souroe of Materials. The beef pituitary glands were obtained from the abbatoirs of the Canada Packers Ltd., Vancouver, B. C , and the writer i s indebted to this company for the removal of the pituitaries. A total of approximately 150 beef pituitary glands were obtained. The f i s h pituitaries 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 for the f i s h from which the pituitary glands were removed. This i s 10 pounds above the average weight reported by Cobb (1916) for this species of f i s h . This species was abundant, being brought i n daily throughout the summer to the docks of the B.C. Packers, Vancouver, and to the Imperial Cannery, Steveston, B.C., whose men were most helpful i n providing f i s h heads and f a c i l i t i e s for the removal of the pituitary gland. At f i r s t the removal was very slow and an average of only 30 to 35 f i s h heads could be opened and their pituitaries removed per hour. However, with practioe a simple method was devised and later an average of 70 to 75 pituitaries were taken out per hour. A single slice through the head, i n a dorso-ventral direction and splitting the upper jaw only, just a l i t t l e . o f f oentre between the two eyes usually exposed the pituitary body with one side of i t s containing wall out away, She Pituitary 12. body was then l i f t e d out with a blunt-ended knife, i t s connection with the brain breaking easily. If the pituitary body was not thus bared, removal of the skull oartilage revealed the brain which when l i f t e d disclosed the pituitary gland lying 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 Biological Supply House, Chicago, I l l i n o i s (22). Eighty. # six goldfish were used from a stook procured from the Goldfish Supply Co., Stouffville, Ont. A commercially prepared extract, (Armour Water Soluable extract Anterior Pituitary Substanoe), was also tested. Gauge 22 hypodermic needles were used for the rat injections, and gauge 26 needles for the goldfish injections. Method of Extraction. The extraction process was begun on both the beef anf f i s h pituitaries within several hours of their removal. Prior to extraction the .glands were kept i n a freezer. The anterior lobes of the beef pituitaries were dissected away, i n gross anatomical dissection, from the posterior lobe and then ground i n a Waring-Blendor to a li q u i d consistency, after whioh they were extracted by the standard method as described by Evans and Simpson (1931), for 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 for this 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 grams of clean sand, u n t i l reduced to a smooth paste. This mixture is weighed and 2 oo. of d i s t i l l e d water are added for every 1 gram of ground glands. The volume of this mixture is measured and 3/8 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 is adequate but the extraction can be allowed to continue i n the ice box 12 hours i f more convenient. The alkaline extraot is 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 is added so that the reaotion of phenol red (1 drop of extraot i n 5 cc. water) is 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. The supernatant f l u i d i s slightly cloudy and pale pink colour. Fresh extraot may be injected into rats without s t e r i l i z a t i o n . If 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 to f a c i l i t a t e grinding of the anterior lobes. Twelve hours of extraction was used in the laboratory eaoh time. After centrifuging a preservative, Merthiolate 1:1,000 was added. One part of Merthiolate was added to 10 parts of extraot. For the f i r s t month of the experiment the alkaline extract was injeoted without sterlization however a Seitz f i l t e r was used thereafter. The extraction method was modified slightly for the f i s h pituitary preparation. An average weight of 0.044 grams p er f i s h pituitary gland was recorded i n six weighings. Thus the salmon glands were about 1/45 of the average weight of the beef pituitaries used. This difference i n size was further magnified by the use of the whole f i s h pituitary whereas only the anterior lobe of the beef pituitary was used. Therefore i n order to lessen the dilution 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 for each gram of gland which was used. The preservative used, and i t s dilution, was the same as with the beef extraction. Experimental Prooedure. Daily intraperitoneal injections were made on rats and goldfish. For rats, the following solutions were used as injection material-, (a) laboratory prepared anterior beef pituitary extract, (b) commercial anterior beef pituitary extract, (o) laboratory prepared f i s h pituitary extract, and (d) a Q% salt solution. Similarly prepared extracts were injected into the goldfish save that the commercial preparation was omitted and a 1% salt solution was used instead of the 6% solution. The salt solutions were both treated in the same manner as were the ground up beef and f i s h pituitaries, and thus served as a control. Weighings were not made to determine whether the rat 'growth plateau', discussed earlier, had been reached. The animals 15. obtained from the biology Department of the University of Br i t i s h Columbia (Group l ) , weighed within the range of those animals whioh had reached the growth plateau indicated i n the normal growth curve for the female rats of Evans and Simpson (1931). The rats obtained from Chicago, (Group 2), were of proper age, (five to six months), and were kept for 1 week before experimental work started i n order that they might become aocl imated to their new surroundings. The rats were weighed every six or seven days. They were numbered by small markings made in their ears with a chicken toe marker. The goldfish weighings presented a problem. The following procedure was followed: eaoh goldfish 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 ruler. It was then placed in a beaker of water, whioh had been previously weighed. After weighing, the difference in weight of beaker alone and beaker plus f i s h was recorded as the weight of the goldfish. The length measured was from the t i p of the snout to the end of the body scales in the t a i l region. However, this method of length measure-ment did not prove sufficiently accurate since measurements of the same goldfish 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. Therefore the length changes w i l l not be reoorded here. 16 The potency assay experiments were carried oat using those rats obtained from the U.B.C. The potency assay was made only for the beef extract and the amount of extract used which gave positive indications of aocelerated growth was used as the criterion for the amount of f i s h extract to be injected. These i n i t i a l injections were carried out for three weeks. Following this the main experiments were carried out over a period of three months. A l l the rat and goldfish injections were made daily eave on the days that the weighings took place. RESULTS Effeots of extracts on Rats. 1« Potency determination of prepared anterior beef pituitary extract. This experiment waB carried out using the 19 rats of group 1. Two different injection materials were used. Both commercially prepared and laboratory prepared extracts were injected i n one and two co. amounts to different animals* The injections were made at approximately the same time eaoh morning. Likewise, the weighings were carried 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 to 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 injections administered over a period of 22 days is 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 injected 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 in 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 this experiment i t was concluded that the most potent injection amount was two cc. of beef pituitary extraot. This is the amount used i n the experiments which followed. Further i t i s evident that Armour Water Soluable extraot Anterior Pituitary Substance does not contain the growth-promoting principle. 2. Preliminary injections of f i s h pituitary extracts. This was a preliminary experiment which was carried out on the same rats used for the potency assay. The injections began after the beef extraot injections had stopped. The rats whioh had formerly been used as controls were now injected with two oo. of alkaline f i s h pituitary extraot, and the remainder wSre kept as controls, being handled only. This experiment was carried out over a period of 21 days. Table 2 shows that a l l the animals which had previously been injected with beef pituitary now showed a loss of weight. This i s similar to the findings of many other workers who reported not only a cessation of growth but 18. a loss in weight with the termination of beef pituitary extraot injections (Evans and Simpson 1951). The animals given the commercial preparation a l l show an increase i n weight save for one which did not change i n weight at a l l . This suggests a growth inhibitory effect o£ the commercial extract. Of the five rats whioh were injected with f i s h pituitary extract, three showed small losses ranging from 4.7 to 17.1 grams. The remaining two animals made small gains of 1.8 and 5.2 grams respectively. 3. Fish Pituitary injection experiments. In this experiment 19 rats (group 2), varying i n age from five to six months were used. I n i t i a l l y 12 of these animals were injected with three cc. of f i s h pituitary extract and seven with a 1% salt solution. The f i s h pituitary injections were continued over a period of 23 days on a l l the rats save for a slight change which was made at the end of 11 days of work. At this time two animals from each of two cages were given injections of two cc. of beef extraot. Of the four animals chosen for these new injections two had been administered the f i s h pituitary extraot previously and the other two had been given injections of 6% salt solution. Injeotions continued over another period of 12 days at which time the stock of prepared f i s h pituitary extract was unexpectedly depleted. Injeotions of prepared beef pituitary extraot were continued, however, for another six days. Although these rats were five to six months of age whioh is the age specified for arrival at the growth plateau, 19* examination of their weights with respect to the age-weight curse would suggest that these rats had not as yet reached their growth-plateau. Thus normal and accelerated weight increase greater than that reported for growth-plateau rats may he expected, and suoh is observed to ooour (table S). It w i l l be recalled 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% salt solution i s 22.02 grams. The weight change recorded with injections of 3 cc. of f i s h pituitary extract over a similar period, i s a gain of 18.19 grams per rat. Those animals whioh had been administered injections of 2 co. of beef pituitary extraot over a period of 14 days gave a weight increase of 40.02 grams per rat (figure 8). Again the beef pituitary extraot did seem to have an acceleration effect, on growth, whereas the f i s h pituitary extraot seemed to have no acceleration effect, rather perhaps a suppressing result, as the weight gains recorded were less than those observed with the salt injections (figure 2). Thus, no evidence was obtained for a factor i n salmon pituitary whioh stimulates the growth ofr'rats. 4. Effects of Extracts on Goldfish. Goldfish were placed i n the experimental aquaria for three days before the experiments started. The f i s h were previously marked by clipping their f i n s . In the f i r s t series beef extract injections k i l l e d a l l goldfish whithin a period of nine days. The controls whioh were handled daily, but not injeoted lived. In the experiments 20. whioh followed a total of 76 goldfish received injections of extracts of beef pituitary, f i s h pituitary or 1% salt solution. 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 pituitary extract-injected f i s h , death occurred in one to nineteen days after injections started. Of the 16 goldfish injeoted with the 1% salt solution and the seven goldfish which received no injection, being handled only, just one died (not included in figure 4). The analysis of the data is complicated by the fact that several experiments were oarried out and that the f i s h died at varying periods before the termination of the experiments. In figure 5, the data from different experiments are pooled. The figures on the graph show the number of f i s h forming the average percentage weight change indicated. Since a l l the f i s h did not live throughout the experiments, the figures indicated i n figure 5 may represent both living and dead f i s h . The smaller samples are dead f i s h . Weight changes i n handling the Goldfish. To observe the effects of handling, the weights of nine goldfish were taken periodically over a period of 19 days. During this 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 is observed. This i s completely reversed at the f i n a l weighings, at whioh time an average decrease of five percent is recorded. This latter figure i s similar numerically to the i n i t i a l increase observed. Thus the El overall effect would seem to 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 cc. of 1% Salt solution. Goldfish were injected with a salt solution for two reasons. First a check was necessary to .-ascertain whether the method of injection was compatible with l i f e of the goldfish. Seoondly the effect of the physical act of the needle injection on the weight of the goldfish could thus be followed. This latter, added to the weight effects of handling would enable the segregation of the casual experimental effect from the physiological effects sought. This effect of handling and injection may be placed at a maximum weight change, increase or decrease, of 6% (figures 3,4). Out of a to t a l of 11 goldfish 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 latter goldfish after 50 days had decreased i n weight by an average of. 4.3$ from the weights recorded before the injections began (this is the procedure followed throughout with reports of weight change in percentages). The i n i t i a l weight changes recorded i n this experiment suggests that the effect of injections i s opposite to 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 effects are of the same order numerically. Weighings of the 11 goldfish noted above did not 22. proceed after the observations of the f i r s t five days. Figures 3 and 4 indicate that the effects 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 latter 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 to handling. This early expression of weight decrease is similar to the weight deoreases recorded i n the other "control" experiments. Injections of 1 oc. of Anterior Lobe of Beef Pituitary Extraot. This extraot is the same as that which proved potent in the rat experiments. (This injection amount would appear sufficient i n the light of the large difference i n mass between the rat and goldfish). As already noted the beef-injected goldfish were dead within a short time after the injections began. In two experiments, comprising a total of 18 goldfish, 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 third experiment three goldfish showed an average i n i t i a l increase i n weight of 9.8$. However, two of these latter f i s h showed a small i n i t i a l decrease, while the th i r d recorded a very large increase (table 6, exp. 3), A seoond weight observation could be made on only 9 beef pituitary extract-injected f i s h (table 6J. Of this t o t a l , six remained of the 18 goldfish noted above i n experiments 1 and 2. The f i n a l weight changes of each of these six goldfish showed a continued increase i n weight save for one f i s h . The f i n a l increase recorded was an average of 18$. As to the goldfish of experiment 3 the two whioh showed an i n i t i a l decrease did continue to do so for a period of approximately two weeks i n whioh time three weighings were made. These data are summarized i n figure 5. Goldfish injected with 1 cc. of Fish pituitaiy extraot. As with the beef injected goldfish death occurred here within 19 days. Of the 39 f i s h injected with f i s h pituitary extract 26 showed an increase and 13 an i n i t i a l decrease, (table 7). For the f i s h pituitary injections four groups of f i s h were used. In only two of these groups is there a definite trend' seen regarding the i n i t i a l change in 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 in 85$ of this number, yielding an average increase of 11.5$. Of these 19 goldfish 10 lived long enough to enable a second set of weight-change observations, these showed that six f i s h had increased in weight and four had decreased i n weight to give an average increase of 5.7$. Highly oontradiotory results are recorded i n the rest of the experiments on the f i s h extraot-injected goldfish (table 7, exps. 1, 3, 4). Out of a total of 20 specimens i n i t i a l weight increases are observed for nine f i s h , 10 show an i n i t i a l decrease, and one f i s h did 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 five had deoreased i n weight. The average f i n a l weight change for these eight f i s h is a decrease of 1.4$. DISCUSSIOM To the writer's knowledge no attempt has been made to derive a growth-stimulating factor from the f i s h pituitary. Proceeding on the premise that the pituitary gland of f i s h , possessing endocrine activity, does elaborate a growth hormone, a standard method was used for the extraction pf the growth hormone of the anterior lobe of the f i s h pituitary gland. Figure 6 shows that a smaller average gain i n weight i s evident i n the fish-injeoted-rats than i n those injected with a saline solution. In both instanoes the amount gained is similar to that whioh would be recorded by normal growing rats at this 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 principle i n the latter extraot. It can be stated that there are no indications of the presence of a growth hormone i n an alkaline extraot preparation of the pituitary gland of the Paoifio Salmon, on administration to rats. This, however, does not necessarily prove the absence of growth hormone in f i s h pituitary The presence of a speoies hormone specificity has been discussed by several workers. Hasler, Meyer and Field 25. (1939), were able to induce rainbow trout to spawn pre-maturely using a hormonal extract of the pituitary gland of carp, while pregnant mare serum and the f o l l i c l e stimulating fraction prepared from the sheep pituitary f a i l e d to induoe premature spawning, von Ihering and Azevedo (1937) were unable to induce spawning i n Froohilidus, Astyanax, and Traohyoorystes with prolan or fresh beef hypophysis. Cardosa (1934), injectingf£sh hypophysis, induced spawning prematurely i n Proohilodus. Easier and Meyer (1942), report the refractoriness of goldfish to thyroid preparation of mammalian origin. In the light of the above i t would seem that a species specificity may exist with the growth hormones. The existenoe of a variation i n quantity of the anterior pituitary hormones from species to species has become definitely established (Creassr and G-orbman, 1939). If this failure of the rat to .Tesact totiie growth hormone extraot of the f i s h pituitary could be ascribed entirely to this quantitative v a r i a b i l i t y i n growth hormone fractions, i t could be construed that the species specificity i s of negligible importance. However, results of hormone exchanges between phylogenetioally distant speoies cannot be successfully explained on a quantitative basis (Creasar and Gorbman, 1939). The method used for the extraction of the f i s h pituitary growth hormone has, for the lack of a known method, been the same =as that used for the extraction of a potent growth hormone of the beef pituitary. That such a method w i l l produce a potent extract of the f i s h pituitary growth hormone is not known. Thus i t is impossible to ascertain, at this time, specifically. 26. the failure of the f i s h pituitary extracts to cause an acceleration of growth i n the rats. The injection of the beef pituitary extract into the goldfish gave a comparatively large i n i t i a l increase i n weight after the f i r s t period of injeotions. Figures 3 and 4 indicate a va r i a b i l i t y of six peroent i n weight increase or decrease due to handling and injection. This limit i s substantially exceeded by the weight increases recorded with the beef pituitary injected goldfish. In two experiments which were performed on a total of 18 goldfish an i n i t i a l average weight increase of 19.4$ i s recorded. Of this group six remained alive long enough for 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$. These observations seem significant when compared to the results of the f i s h pituitay injected goldfish and the controls (figure 5). The individual results obtained with the f i s h pituitary injected f i s h are highly contradictory to one another as 26 record i n i t i a l increases and 13 record decreases. However, i f two experimental groups containing 19 specimens were viewed alone a positive indication of an i n i t i a l weight gain i s seen. The average i n i t i a l gain for these two groups Is 11.5$. This figure 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 effects. Of the remainder there is an even s p l i t between the number whioh gained wight and those which lost weight i n i t i a l l y . These results do suggest a balance between the weight gaining prooesses and the weight decreasing effects, whioh may be interpreted as the balance between the activity of a potent growth hormone and the weight changes due to the method of administration, both of whioh have been noted here. However before such a growth hormone activity can be conclusively accepted as present i n the f i s h pituitary 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 goldfish i s striking when i t is considered that only one of the salt injected f i s h died. Nixo-Niooscio (1940) states that i t is the albumen content of the hypophysis of oattle whioh i s responsible for the death of the goldfish. But this does not account for the death of the goldfish caused by the f i s h pituitary injeotions. Although i t is not stated i n the report on Nixo-Niooscio 1s work, i t may 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 to the activity of the injected material. Thompson (1937), was unable to develop an anti-hormone to the gonadotropic hormone from sheep pituitary gland by injecting i t into sheep. Thus, although Thompson notes that many observers are agreed that anti-hormones may be obtained after suitable injection of pituitary extracts into animals, he suggests that hormones may be antigenic. Such a possibility is greatly inoreased where heterologous species are concerned, as i n this work here. The problem here regarding the failure of the f i s h pituitary extract to cause an accelerated growth of the rats, i s whether we are dealing with a true species specif ic-ity/or whether the recorded observations are really the result of the production of protein antibodies brought about by the use of hormone protein mixtures derived from heterogeneous speoies. A thi r d alternative is suggested i n view of the absence of a known method for the extraction of a potent f i s h pituitary growth hormone. Does the method used for a potent extraction of the beef pituitary produce equally as potent a f i s h pituitary extract, which i s the method used i n this work? The results observed here with injections of the goldfish are not positively indioative and further research i n this f i e l d w i l l be needed before a positive manifest activity of a growth hormone extract of the f i s h pituitary gland w i l l be secured. 29 SUMMARY An alkaline extraot, prepared by the method of Evans and Simpson (1931), from the anterior lobe of the beef pituitary, proved to be a potent source of a growth hormone when adiministered to female rats. Daily intraperitoneal injeotions of this extract into goldfish resulted i n an 18.0$ weight increase within eight days. The goldfish which oontinued to live beyond this f i r s t weighing continued to show a weight increase of 11.5$. This weight increase appears significant i n view of the decreased weight recorded with the salt, injeoted goldfish. A similar method was used for the preparation of a growth hormone extraot from the pituitary gland of the Pacific Salmon, Onoorhynohus tschawytsoha. Injections of this extract into female rats five to six months of age f a i l e d to accelerate the growth of the animals. Injections to goldfish resulted i n a oonfusing pattern. Both increases and decreases were observed. However i f two of the experimental groups containing a total of 19 goldfish were examined, an 11$ increase i n weight is seen following the f i r s t period of injeotions. This suggests a lesser potency of the f i s h pituitary extract, and the occurrence of contradictory results between individual goldfish as the balance between the growth-accelerating effect of the hormone and the weight decreasing effeot of the method of injection. Further elucidation as to the occurrence of a speoies specificity here must await the development of a known potent method for extraction of the growth hormone of the f i s h pituitary. In order to substantiate this claim of a growth promoting effeot i n an alkaline extraot of the pituitary gland of the Pacific Salmon more data on this 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 I n i t i a l Over 3 Week Ho. Injection Material Weight (g was) Period. 60 2 cc. Prep. Beef 274.0 / 57.7 98 » it ti 223.5 / 70.6 7 it ii n 207.1 / . 4 9 . 6 4 it it ii 211.3 / 49.9 53 1 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 5 2 co. Comm. Beef 208.0 - 11.8 8 it ti ti 211.0 - 7.9 43 1 oc. Comm. Beef 241.7 83 it ti ti 244.0 / 14.1 3 Ti H ft 225.1 - 20.8 9 it 11 it 204.3 - 19.1 54 Control 258.0 / 3.1 45 n 209.5 / 10.4 77 •ii 200.0 / 8.8 1 ft 212.2 / 7.2 61 it 217.4 / 2.4 TABLE 2 - Preliminary Injeotion of Fish Pituitary Extracts to Rats of Group 1. (Following Beef Pituitary Injection Experiment). Weight Change I n i t i a l Over 3 Week Ro. Injection Material Weight (grams) Period, (grams) 60 Control 274.0 - 16.3 92 II 223.5 - 36.4 7 n 207.1 - 24.0 4 tt 211.3 - 23.2 53 n 241.7 - 8.1 111 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 / 0.3 83 it 244.0 3 II 225.1 / 14.0 9 n 201.3 / 16.0 54 2oo. Fish Pituitary 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). I n i t i a l Mo. Weight Weight Change Weight Change over over 7 23 days with days with 6% Salt Fish Pituitary no HairLung 3 oo. 5 oo. Weight Change over 18 Days with Beef Pituitary Injections 2 co. 2 5.49.0 / 11.0 /19.6 3 161.4 / 12.9 /1.2 4 170.0 / 12.0 5 137.9 / 5.1 6 148.7 / 12.0 7 180.1 / 4.3 /2.9 10 150.5 / 17.7 /29.3 11 176.7 / 10.9 / 23.7 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 /20.9 31 119.0 / 8.8 /21.0 32 162.1 / 16.9 33 166.4 / 10.4 /16.5 34 151.0 / 13.9 /24.1 35 155.3 / 8.0 / 5.7 36 157.2 / 8.5 /49.0 /44.9 /70.8 /44.5 TABLE 4. Weight Change of Goldfish Handled Only. No. I n i t i a l Weight (grams) No. Days First Weight Change grams % Second Weight Change grams % No. Days 1. 11.1 8 > / 0.4 / 3.6 - 0.6 - 5.4 19 4. 10.6 * 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 / 5.6 - 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 / 0.8 - 0.7 - 5.8 15 35. TABLE 5. Weight Change of Goldfish Injected with 1% Salt Solution. 59. 62. 65. 9.0 8.9 8.0 Experiment 2. 9. 10.0 52. 6.2 9. 52. 9. 52. Fir s t Seoond I n i t i a l No. Weight Change Weiglht Change No. No. Weight (grams) Days grams % g?ams % Days Experiment 1. 32. 8.5 » * 35. 13.3 38. 10.0 41. 17.9 44. 8.9 47. 10.6 50. 8.4 53. 9.3 56. 11.0 5 - 0.3 - 3.5 5 5 - 0.8 - 8.0 5 - 1.8 -10.0 5 - 0.5 - 5.6 5 - 0.6 - 5.7 5 - 0.3 - 3.6 3 - 0.3 - 3.2 5 / 0.1 / 0.9 5 / 0.1 / 1.0 5 - 0.3 - 3.4 5 - 0.2 - 2.5 7 / 0.1 / 1.0 7 / 0.6 / 7.3 Third L8 - 0.4 - 4.0 18 30 30 - 0.3 - 3.7 F i f t h / 0.1 / 1.0 - 0.8 - 9.7 - 0.2 - 2.0 15 / 0.1 / 1.2 15 Fourth - 0.8 - 8.0 24 - 0.2 - 2.4 24 36. TABLE 6. Weight Change of Goldfish Injected With 1 oo. of Anterior Lobe of Beef Pituitary Extract. Ho. I n i t i a l Weight (grams) Fi r s t Ho. Weight Change Days grams % Second Weight Change Ho. grams % Days Experiment 1. -2. 12.5 8 3. 13.5 8 6. 9.2 5 7. 13.2 6 8. 10.3 5 12. 10.2 7 14. 10.1 5 15.' 10.0 5 Experiment 2. 31." 14.9 5 34. 12.8 5 37. 10.4 1 43. 7.7 5 46. 6.5 2 49. 6.0 5 52. 10.7 5 58. 9.5 5 61. 13.2 5 64. 9.7 5 Experiment 3. 95. 12.1 7 96.* 18.4 7 91. 8.5 7 / 4.3 / 2.5 / 2.4 / 2.4 / 1.9 / 1.4 / 2.8 / 2.0 / 3.0 / 3.1 / 0.4 / 1.2 / 0.9 / 1.3 / 3.9 / 0.9 / 0.9 / 2.0 - 0.3 - 0.4 / 3.0 / 34.4 / 18.5 / 26.1 / 18.2 / 18.4 / 13.7 / 27.7 / 20.0 / 20.1 / 24.2 / 3.8 / 15.6 / 13:8 / 21.7 / 36.4 / 9.5 / 6.8 / 20.6 / 4.5 / 33.3 / 1.6 / 10.7 / 3.2 / 25.0 / 0.4 / 6.7 / 3.7 / 34.6 - 0.3 - 3.2 10 6 8 6 6 8 2.5 - 0.7 - 5.8 12 3.2 - 1.1 8.9 12 35.3 / 1.5 / 17.6 12 Third 95. 18 - 0.3 - 2.5 96. 15 - 1.6 - 13.0 91. 14 / 1.0 / 11.8 37. TABLE 7. Weight Change of Goldfish Injected with 1 oo. of Fish Pituitary Extraot. I n i t i a l Weight No. No. (grams) Days Fir s t 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 Experiment 2. 30. 8.4 33. 14.9 36. 8.6 39. 13.3 42. 14.5 45. 8.3 48. 16.7 51. 15.5 54. 12.3 57. 9.4 60. 9.1 63. 6.6 Experiment 3. 6 9 9 9 3 5 5 5 5 5 5 5 5 5 4 5 / 1.6 / 11.7 - 0.6 - 4.0 - 1.7 - 12.7 / 1.9 / 0.8 / 4.0 - 0.1 / 2.1 / 0.1 / 1.0 / 2.1 / l.S / 1.5 / 0.2 / 12.8 / 9.3 / 30.0 - 0.7 / 25.3 / 0.6 / 6.4 / 17.1 / 16.0 * 16.5 / 3.0 - 1.3 - 0.6 - 1.8 / 0.6 / 3.1 / 1.3 / 1.2 - 0.2 - 0.7 - 10.4 - 5.8 - 13.4 / 7.0 / 23.3 / 9.0 / 14.4 - 1.2 - 4.5 14 14 14 9 9 9 7 10 12 1.7 0.5 1.6 13.7 4.8 12.0 17 19 18 / 1.3 / 13.8 9 - 0.5 - 7.6 10 81. 9.9 7 / 1.9 19.0 / 2.1 / 21.2 8 72. 16.9 7 - 1.0 - 5.9 - 2.4 - 14.2 12 - 3.4 - 20.1 14 70. 12.1 7 / 3.6 / 29.8 - 0.2 - 1,7 11 3. 10.3 7 - 0.4 3.9 - 0.9 - 8.7 8 Experiment 4. 74. 7.6 6 1 0.9 / 11.8 1. 11.0 7 / 1.8 / 16.4 2. 6.6 6 / 0.6 / 9.1 93.. 5.4 6 / 0.7 / 13.0 3. 10.4 7 - 1.0 9.6 98. 8.1 7 - 0.4 - 4.9 89, 9.9 7 - 0.1 - 1.0 94. 8.1 4 / 0.1 1.2 35. 12.0 7 - 2.3 - 19.2 19. 9.3 6 - 0.4 - 4.3 7. 12.0 7 - 0.2 - 1.7 82. 7.3 7 / 0.4 5.5 - 0.1 / 0.6 / 0.6 / 2.0 - 0.9 / 7.4 / 5.0 / 27.4 10 10 13 12 / 0.2 / 1.7 16 (continued on next page) 38. TABLE 7. Weight Change of Goldfish Injected with 1 cc. of Fish Pituitary Extraot. (continued from page 37). I n i t i a l F i r s t Seoond Third Weight Ho. Weight Change Weight Change Ho. Weight Change Ho. Ho. (grams) Days ggams % grams % Days grams % Days Experiment 5. 6. 7.2 7 / 1.0 / 14.0 9. 8.1 5 / 0.4 / 4.9 8. 6.4 4 / 0.7 / 10.9 1. 11.0 3 / 2.7 / 24.3 / 0.4 / 3.6 5 4. 7.6 4 / 0.8 / 10.5 5. 6.5 4 / 1.4 / 21.5 2.. 11.8 6 / 0.3 / 2.5 - 0.1 - 0.8 9 39 IO\ :  3 To 7s Jo Dd,c/s  Figure 1. Response of Normal Female Rats (group 1) to hypophyseal beef Growth Hormone, a} 2 oo. commercial extract of growth hormone. 2 rats. b) 1 cc. commercial extract of growth hormone. 4 rats. c) Control, handled only. 5 rats. dj 1 cc. laboratory prepared growth hormone. 4 rats, e; 2 oo. laboratory prepared growth hormone, 4 rats. 1t\e&T\ Peircetxtaje (Jetgkt Increase Tigure' Z~. Response of normal female rats (group 2) to Beef and Fish 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. saline. — _ io rats injected with 3 cc. f i s h growth hormone. 41. Figure 3. Graph of mean percentage weight gain of goldfish handled only. Figures represent number of goldfish whioh form the indicated averages. Percentage k/cight Cha .Tv.jc ov r-O o » —7K / \ / \ / \ r leu Figure 4. Graph of mean percentage weight gain of goldfish injected with 1 oo. of 1% salt solution. Figures represent number of goldfish which form the indicated averages. 43 Figure 5. Comparison of the mean percentage weight change of beef pituitary extract with fi s h pituitary extraot injections ( l co.) to goldfish. Horizontal lines give limits of variation, Q%t recorded with handling and saline injections of goldfish. Figures represent number of goldfish which form the indioated average. Goldfish injected with beef pituitary extraot. — - Goldfish injected with f i s h pituitary extract. 44 Figure 6. Histogram of weight changes observed i n rats (group 2). Bars (horizontal) - change in weight per day per rat over an eight' day period. Time interval for acclimatization of rats (handled only). Bars (angled) - change i n weight per day per rat injected with"6$ salt solution over a 23 day period. Bars (stippled) - change in v/eight per day per rat injected with f i s h pituitary extract over a 23 day period. Bars (plain) - change in weight per day per rat injected with beef pituitary extract over a 14 day period. 45 Bibliography Aldrioh. Amer. J. Physiol., 30:1912. (Quoted from Wulzen, 1914). AHrioh. Amer. J. Physiol., 31:1913. (Quoted from Wulzen, 1914). Allen, B. M. The influence of the thyroid gland and hypophysis upon growth and development of amphibian larvae. Quart. Rev. 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