STUDIES OF THE 9mm. FUNCTION IN JAPANESE QUAIL (cowamgg cowamx MPONICAI Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY Louis C. Arringfon 1-966 JIIIIIIIIIIIIIIII‘IIIIIIIIIIIIIII i 3 1293 00083 9468 This is to certify that the thesis entitled STUDIES OF THE PINEAL FUNCTION IN JAPANESE QUAIL (QOTURNIX COTURQLE JAPONICA) presented by LOUIS C . ARRINGTON has been accepted towards fulfillment of the requirements for Ph-D. degree in P cience hlajlor professd Date JUly 22, 1966 ABSTRACT STUDIES OF THE PINEAL. FUNCTION“ IN JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA) by Louis C. Arrington The pineal body is one of the few remaining organs of the body which has not been assigned a definite function. The sparse coverage of this gland in textbooks on avian physiology emphasizes the lack of knowledge about the pineal function in birds. The literature has suggested an. involvement of the pineal in the relationship between photo- periods and reproductive performance. It was considered that since-avian reproduction is strongly influenced by light, a determination of pineal function would be of importance to avian physiologists. _ Newly hatched Japanese quail (Coturnix coturnix japonica) were pinealectomized and grown under inhibitory or stimulatory photoperiods. At various ages they were sac— rificed and their body weight, organ weights and age at sexual maturity were compared with unoperated controls. The organs weighed were the pituitary, thyroids, adrenals, Spleen, bursa, testes, ovary and oviduct. The success of the pinealectomy operation was determined by macro- and microscopic examination. Daily injections of melatonin, presumed to be a pineal hormone, were given to immature Coturnix quail to determine its effect on body and organ weights. In addition, adult quail hens were injected daily to observe the effects of melatonin on maintenance of egg production. Louis C. Arrington 'Dhe results of pinealectomy eXperiments indicate that the pineal has little, if any, effect on body weight or on the Aweight of the pituitary, thyroids, adrenals, spleen or bursa. The differences obtained in these parameters were inconsistent and usually nonsignificant. Complete retard- action of growth, and probably atrOphy, of the testes was observed in control quail when grown under photOperiods con- sisting of two hours of light and 22 hours of darkness per day from 3gto h, 5, 6 and 7 weeks of age. Removal of the pineal glands did not prevent or reduce the inhibitory effect_of this photOperiod., Under stimulatory photoperiods (16 hours of light and_8 hours of darkness per day), Srweek old male pinealectomized quail had slightly larger testes. This effect was not present in the 6- and 7-week old males. The possible production of a gonadal inhibitor by the pineal is suggested. However, the results indicate that somewhere near the time of sexual maturity this inhibition ceases due to decreased inhibitor production or decreased response to the inhibitor by the gonads. The reaponses of females in these tests were not consistent. The only significant response was an increased mean ovary weight in the 7-week pinealectomized group. The 5-week data showed a nonsig- nificant difference with the control gonads being slightly larger. The mean ages at which egg laying started were not significantly different. .- a- .9.-— ,f 1W “nil! a a Louis C. Arrington Adult pinealectomized and control quail hens showed “0 difference in the number of days required to respond be' commencing egg production) to a change from inhibitory to stimulatory light cycles. Reversing the change in photOperiods produced no difference in the time of cessation of egg production. Sexually mature pinealectomized males showed less atrOphy of the testes when switched to an in- hibitory light schedule. Due to a large variation in individual results, significance was not obtained, but it is suggested that the presence of thegpineal gland may hasten testicular atrOphy. The results of the pinealectomy experiments suggest that the pineal modifies, rather than controls, the gonadal reSponse to photoperiods. Melatonin injections of 50 to 500 micrograms daily produced a slight retardation of gonadal development in prepuberal quail of both sexes. It appeared that the gonads of male quail at or near the puberal phase of growth were not affected by melatonin injections. Daily doses of 50 to 2000 micrograms of melatonin given for 12 to 2“ days failed to alter the egg-laying capacity of adult Coturnix hens. These results support the suggestions that the pineal produces an inhibition of gonadal growth in prepuberal quail and that the mature quail gonad is less responsive to pineal inhibition. STUDIES OF THE PINEAL FUNCTION IN JAPANESE QUAIL ”K {L Louis CirArrington A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1966 \" \ \w .AcxNowLEnGEenflwns The author expresses his sincere appreciation to the following members of the Department of Poultry Science: to Dr. Robert K. Ringer, for his guidance during the course of study; to Dr. John H. Volford for assistance in the preparation of this manuscript; to Dr. Howard C. Zindel, Chairmanof Poultry Science, for making available the necessary facilities and equipment; and to Dr. Ronald Peterson, Mrs. Sandra L. Pangborn and Mrs. Maryann J. Duke, for thier technical assistance in the laboratory. The author also extends an appreciative acknodledge- ment to the'other members of his graduate program committee for their guidance. These-members are Dr. Joseph Heites,~ Department of-Physiologyi Dr. Esther M. Smith, Department of_Anatomy andeirector of the School of Medical Technology; and Dr. Olaf Hickelson, Departments of Foods and Nutrition, and Biochemistry. Appreciation is expressed to the secretarial staff of the Department of Poultry Science for their contri- butions to the preparation of this thesis. A special word of thanks is extended to his wife, Sandra, for her help and encouragement during the duration of the study. ii TABLE OF CONTENTS INTHRODUCTION . . . . LITERATURE REVIEW._. A. B. Description of the Pineal Gland Functions of the Pineal Gland . Active Pineal Substances. Factors Influencing Pineal Function Summary of Literature . OBJECTIVES . . . . . EXPERIMENTAL PROCEDURE . . . . o A. B. C. D. E. RESULTS. General Management. . . Pinealectomy70peration. Melatonin Injections. . Description of Experiments. Statistical Experiment 1. . Experiment 2. . Experiment 3. . Experiment h. . Experiment 5. . Experiment 6. . Experiment 7. . Treatment . iii Page 28 38 #7 so 51 51 52 51. 55 59 62 62 at. 73 75 78 78 78 —-'—"—'-"—_'_.4_ -, j—l Page DISCUSSION. 0 O 0 O O O O O O O O O O O O 0 O O O O O 82 SUMRY O O I O O 0 O O O O O O O O O O O O O O O O O 97 LITEMTURE CITED. 0 O O O O O O O O O O O O O O O O O 1 00 iv ‘FICHIRE TAEEJ: 10 LIST or FIGURES AND TABLES \ Suggested melatonin metabolism pathway. . . . Body weight and organ weights of n-week old Coturnix quail housed under L2D = 2:22 light cycles at 3 weeks of age as influenced by pinealectomy. O O O O O O O O O O O O Q 0 O 0 Body weight and organ weights of 5-week old Coturnix quail housed under LxD = 2:22 light cycles at 3 weeks of age as influenced by pinealectomy................. Body weight and organ weights of 6-week old Coturnix quail housed under L:D = 2:22 light cycles at 3 weeks of age as influenced by p1n63180t0mYOeooooooooocococe Body weight and organ weights.of 7-week old Coturnix quail housed under LtD = 2:22 light cycles at 3 weeks of age as influenced by pinealeCZtOmYcoooooccooooeoooo Body weight and organ weights of 5-week old Coturnix quail housed under Lin = 16:8 light cycles as influenced by pinealectomy. . . . . Body weight and organ weights of 6-week old Coturnix quail housed under LtD = 16:8 light cycles as influenced by pinealectomy. . . . . Body weight and organ weights of 7-week old Coturnix quail housed under L:D = 16:8 light cycles as influenced by pinealectomy. . . . . Body weight and organ weights of Coturnix quail as influenced by daily melatonin in- jections from 37 to an days of age. . . . . . Body weight and organ weights of male Coturnix quail as influenced by daily mela- tonin injections from #2 to #9 days of age. . Body weight and organ weights of male Coturnix quail as influenced by daily mela- tonin injections from 35 to #9 days of age. . Page 32 62 55 66 67 68 69 70 76 79 80 INTRODUCTION The pineal body or gland is one of the few remaining organs of the body which has not been assigned_a definite function. The most recent textbooks on avian physiology devote only a few sentences to the pineal, stressing the lack of recent work in this area. Even textbooks on general mammalian endocrinology provide little information on the possible role of the pineal. Previous research with both avian and mammalian species has yielded little definitive information due to the inconsistent, and often non~ conclusive, results obtained. Rats have been used in several studies relating the pineal to the effects of light on reproductive parameters. It was considered that since the relationship between light and avian reproductive functions is of economic importance, studies elucidating any pineal involvement would be of importance in interpreting the mechanism by which light mediates its effects on reproduction. This study is an attempt to determine some of the relationships between the pineal gland and physiological parameters in avian species. The importance of the pineal gland in reproductive function was of particular concern. LITERATURE RE VIEW A; Description of the Pineal Gland 1. Embryology I Patten (1951), Romanoff (1960, Spiroff (1958) and Tilney and Uarren (1919) have described the embryological development of the pineal gland in the chicken. Krabbe (1955) discussed_the variations between avian species. Other animals show a similar/pdttern'of development with variations primarily in the appfinrhnce time of each stage. At about 52 hours of incubation the epiphysis appears as an evagination in the mid-dorsal wall of the diencephalon. It is destined to differentiate into the pineal gland; By the 36 somite stage (about 72 hours)iit is a small hemiSpherical protuberance. It continues to proliferate dorso-caudally. By the 8th day, the epiphyseal attach- ment has shifted caudally and the epiphysis has grown out into a long, narrow tube, dilated distally. Proliferation of the ependymal cells in the distal enlargement begins the formation of hollow buds of follicles. Leptomeningeal mesenchyme, containing blood vessels, grows in between the follicles, providing the epiphysis with a connective tissue framework and an extensive vascular network. As the fol- licles and framework develop, the epiphysis continues to grow dorso-caudally, forming a long, very thin stalk. 2. Morphology Quay and Levine (1957) showed that the postnatal growth of the rat pineal has a proliferative phase ex- tending to about two weeks after birth. Studies by Dill and Walker (1966) confirmed this report. A cellular hypertrophy phase occurs during the 10 weeks following birth. Benoit (1950), Cobb and Edinger (1962), Kappers (1965). Spiroff (1958) and Tilney and Warren (1919) describe the pineal in birds. The pineal is fixed to the arch of the diencephalon by a long, thin stalk. The stalk leaves the brain at a point just rostral to the junction of the forebrain and midbrain, maintaining an attachment in the commissural region, over the 3rd ventricle of the brain. The body of the pineal is super— ficially located, usually adhering to the dura and some— times lying in a depression of the cranial roof between the anterior and posterior fossae. It is somewhat teardrop-shaped, although the sides may be partially flattened. The body is situated between the posterior parts of the two cerebral hemisPheres and the cerebellar vermis. Its yellowish color distinguishes the pineal from the surrounding whitish brain tissue. 3. Histology The histology of the avian pineal gland has been described by Basrur and Uinget (1963), Benoit (1950), z. Rappers (1965), Renzoni and Quay (1963),, Spiroff (1958), Stannner (1961) and Tilney and Warren (1919). They demonstrate a specialized, primarily secretory structure, with no clear evidence of photoreceptors. Numerous vesicles are composed of ciliated, cylindrical cells (ependymocytes), small ovoid cells situated between the cylindrical cells (hypendymocytes) and a few peripherally located pinealocytes. These three cell types are of ependymal origin. The thin connective tissue capsule is Vlined with blood vessels and extends between the parenchymal vesicles. This interstitial connective tissue contains lymphocytes, undifferentiated glial cells and a few large epithelioid cells. Spiroff (1958) describes a lympho- cytic invasion of the pineal, seen primarily at 1 week to 6 months of age in domestic fowl. Electron microscopic studies of the pineal body of rats by De Martino 23 31. (196b) revealed that the 7 pinealocytes in impuberal rats are small and clearly out- lined, with few cytoplasmic processes. These processes contain a few granules of irregular shape. In adult rats, the size and shape of the pinealocytes are less uniform, with numerous cytoplasmic processes extending towards the perivascular spaces. Granules, presumed to be secretory, are more numerous. Anderson (1962) made similar studies of the fine structure of pinealocytes of sheep. The nucleus was described as being large, with infoldings and 5 a prominent nucleolus. The basophilic cytoplasm contains large amounts of mitochondria in addition to clumps of dense particles, numerous cisternae of the endoplasmic reticulum and a system of fine caualiculi. The golgi complex, centrioles,_vesicles, and lipid inclusions were observed in some photo-micrographs. Kappers (1960, 196b, 1965) described the innervation of the pineal gland in fishes, amphibians, reptiles, birds and mammals. Stammerg(196l) gave additional observations from birds. The stalk contains nerves derived from the commissure, which were termed the commissuro—epiphyseal tract. These fibers do not penetrate the bulbous portion of the pineal and are thought to be of no functional significance for the innervation of pinealocytes. The pineal body shows extensive autonomic innervation principally supplied by the two nervi conarii. These are postganglionic fibers, originating in the superior cervical ganglia. The terminal autonomic innervation occurs via perifollicular strands of fibers from which thin single fibers or small groups of fibers branch and penetrate the follicles. Structures thought to be autonomic motor terminals have been observed in relation to the pinealocytes only-—not the vascular walls. This autonomic innervation coming from the superior cervical ganglion appears to be- the major, if not the only, functional nervous supply to the pineal. Sensory in- ”nervation of the pineal has not been shown. , The pineal is highly vascular. Goldman and Hurtman (196%) found that the minimum rate of pineal blood flow per gram of tissue-exceeds that of most endocrine organs, equals that of the neurohypOphysis and is surpassed only by that of the-kidney in the rat. The vasoularization of the mouse pineal was described by Von Bartheld and Hell (195“). Both the afferent and efferent blood pathways are independent from those of the diencephalic choroid plexus and of the nervous-parenchyma. Branches of the posterior cerebral artery supply the pineal after coursing over_the lateral brain stem surface. These branches anastomose with arteries arising from the basilar artery. Only very small vessels which have lost most of the arteriolar character actually enter the pineal. No arterioles were found to reach the pineal body via its stalk. The capillaries of the pineal are typical of those of nervous parenchyma. Vanules are found in the pineal parenchyma just beneath the capsule. They empty the pineal blood into large venous trunks which border immediately on the pineal. The pineal stalk has no veins or venules and is, therefore, not thought to be an important vascular link between the pineal and other parts of the brain. Quay (1959) first reported finding striated.musclel fibers in the rat pineal gland. He found examples in three pineals out ofna series of 1200 being examined. Dill (1963) confirmed this observationflwith a similar report. The occurrence had been reported previously in bovine and human pineals. These pineals appearedehisto- logically-normal except for the presence of this muscle. The significance, if any, of these findings is not.known. B. Functions_of theMPineal Gland 1. Historical_0bservations a hen knew of the existence of the pineal gland before 200 A.D. Kitay and Altschule (195ua), Tilney and warren (1919)”and wurtman~and Axelrod (1965) reviewed some of the early historical writings. Early Greek_ anatomists concluded that the pineal served as a valve over the aqueduct of the cerebrum, regulating the flow of thought. In the 16th century, Galen showed that the‘ pineal was not a valve (he thought that the cerebellar vermi was the thought sphincter), but probably was a gland, perhaps similar to the lymph glands. In the 17th century, Descartes postulated that the pineal contained .the seat of the rational soul. In this theory, the eyes perceived the events of the world and transmitted this information to the pineal via "strings" in the brain. ._u— A A _ ‘7 hi- 'Ihe pineal responded by allowing humors to pass down tubes-to muscles which produced the apprOpriate reaction. These and many other early-investigators presented theories which generally fit into one of three categories: 1. The pineal acts as a valve or sphincter, 2. The pineal is the seat of the soul or_mind or 3. The pineal is a gland with secretory function. r‘ The name 'conarium” was used for the-pineal gland, by - early dissectionists, according to_Galen. This name was derived from the conical shape of the-body (Kitay and Altschule, 195ha).e Other names reported by Rubin_(l952) and Tilney and Warren (1919) include "epiphysis" or_ "epiphysis cerebri,” which_are descriptive of the location in the brain. Some writers called it the "glanula superior“ in contradistinction to the pituitary gland which was called the "glandula inferior." The terms "corpus pineal," ”pineal body" and "pineal gland" resulted from resemblance to a small pine cone. 2. Photoreception vs. Secretory Activity The pineal gland is primarily a photoreceptive organ in fishes, amphibians, and lacertilian reptiles. These organs are described by Rappers (1965) and Tilney and Warren (1919). Dolt (1963) and Pang (1965) have published recent papers dealing with the direct stim- ulation of the pineal by light in rainbow trout and ~killifish, respectively. Kelly (1962) reviewed the work on pineal photoreceptor function. _ Rappers (1965) and Tilney and Warren (1919) reported the lack of discOVery of any photoreceptor cells in the pineal bodies of birds or mammals. Being descendants from -reptiles, birds might be expected to show some vestige of - the parietal eye structure. However, as Cobb and Edinger (1952) state, "...parietal eyes were lost, not-within the a. evolution of birds, but in remote reptilian ancestors some 800 million years before the-first, late Jurassic, appear- ance of feathered animals, and presumably more than 100 million years before the modern type of avian brain was evolved.”w In birds and mammals the specific pineal parenchymal cells, pinealocytes, are secretory. Holmgren (1958) demonstrated the presence of secretory material in the pineal gland of monkeys by aldehyde-fuchsin staining of pineal sections which had been oxidized in performic acid. Quay (1956) used differential staining techniques to demonstrate secretory material in pineal parenchymal cells of rats. Electron microsc0pic studies by Rodin and Turner (1965) showed the presence of granules in vesicles lying in the perivascular spaces of the rat pineal body. 10 3. Clinical Observations. Heubner described the first clinical case in 1898, relating pineal tumors tongonadal function (Kitay, l95hb; Kitay and Altschule, l95ha; Relkins, 1966; Hurtman and Axelrod, 1965).~ Heubner's report described precocious bodily and sexual develOpment in a young boy who was found to have a pineal tumor. Subsequent reports of clinical cases associated pineal tumors both with precocious and delayed somatic and sexual deve10pment. These conflicting reports led to three groups of theories concerning pineal function as follows: that the pineal gland stimulates somatic and sexual develOpment; that it inhibits somatic and sexual develOpment and that it has neither effect. Kitay (1954b) reviewed 1&5 reports of verified cases of pineal tumors in boys under 16 years of age. He found that precocious development tended to be associated with tumors of the nonparenchymal tissue, whereas delayed sexual deve10pment tended to be associated with tumors of the parenchymal tissue. In the case of nonparenchymal neo- plasms, the parenchymal tissue is often destroyed, preventing normal function of the pineal. Parenchymal tumors, on the other hand, increase the pineal activity. The secretion and/or release by the normal pineal of a substance having an inhibitory effect on the gonads is indicated by these findings. One must assume that the pineal function was maintained almost normally in cases . L . . ‘ l‘ . H‘E . . K ' - ‘ . ’I ' z. . A ”"193, a -m . a. 1:4: . ‘ ~.. ) v ' mist: ‘ s i! - I ‘. . ‘ . r ' ‘ I ‘ “L |-‘ ‘ . Ill-2'] 1. ‘3'- ‘ _ ~ «In, '- -._,. '. . ' ‘ 11 'where tumors of either type produced no apparent effect on develOpment. In a few cases precocious puberty was observed with parenchymal tumors and delayed development with nonparenchymal tumorse-opposite from the usual results. In the first case, one must assume that parenchymal tumors do not always secrete. In support of this assumption, some pinealomas were found to be composed of cells so undifferentiated and necrotic that secretion was highly unlikely. No evidence was shown to explain the delayed deve10pment with nonparenchymal tumors, which would require increased pineal secretion. h. Results of Pinealectomy Many investigators have removed the pineal gland in order to determine its function. Stalsberg (1965) removed the pineal from 6-day chick embryos with negative results. No changes were observed in pre- and post- hatching survival, incubation period, body weight, skeletal size, testes, ovary or comb characteristics, when compared to unoperated controls. Postnatal pinealectomy of birds and mammals has given contradictory results. Foa (1912, 191“) and Ixawa (1923) observed stimulated growth, gonadal hypertrOphy, increased comb size and early sexual activity in response to pinealectomy of young male chicks. In a similar experiment, Badertscher (1929) failed to obtain any of these results. 12 Shellabarger (1953) and Shellabarger and Breneman (19b9) surgically removed the pineals from male chicks at h days of age. AutOpsy at various ages showed that the testes of the pinealectomized chicks were significantly smaller at 19 days of age, were not different at 28 to ho days of age, were significantly larger at 92 to 70 days of age and were_again not different at 95 days of age. The reason for the pinealectomized birds having smaller testes at 19 days was unclear. The fact that his sham-operated birds actually had slightly larger testes than those of the con- trols would indicate that the trauma of the operation was not reSponsible. He concluded that sometime after 70 days of age, the pineal body has no effect on the testes of chickens. It should be noted that at 9% days, the testes of all birds had gained appreciable weight, indicating that they had started the puberal phase of rapid growth. In another experiment, Shellabarger (1952) again found that pinealectomy reduced the 20-day testes weight of chicks. Andersen and Wolf (193“) and Davis and martin (19h1) extirpated the pineal of very young rats.” They observed no influence on the growth rate, testes weight or age at puberty. In similar pinealectomy experiments, Izawa (1926) obtained accelerated body growth and enlarged testes and epididymides. Dill (1961) pinealectomized 13 rats at 26 days of age, and observed a significant gonadal hypertrophy at 6 weeks after the Operation. Cutting the mid-saggital sinus to serve as sham- operated_controls also resulted in gonadal hypertrOphy (Dill, 1961). The use of these animals as a control could be questioned. Depending on the exact location of the in- cision, there is a possibility that the nervous supply to the pineal may have been disturbed, which would produce a similar effect to that of pinealectomy. Benton and Rusbridge (1933) found no consistent or significant change in testicular weights after removal of the pineal of 26-day old rats. Sullens and Overholser (1951) obtained no differences in somatic or sexual develop- ment between control rats and those which were pinealect- omized at 3 weeks of age for three successive generations. D'Amour and D'Amour (1937) in a similar experiment found no differences in sexual maturity, but observed a distinct weight increase in the third and fourth generations. Although the experimental numbers were small, there was some evidence that this increased growth becomes more marked in successive generations. However, Einhorn and Rowntree (1939) obtained no effect of pinealectomy of successive generations on growth or development of offspring. lb Dandy (1915) removed the pineals of young puppies and was unable to show any evidence of somatic, sexual or mental precocity or retardation, nor any macro” or microscopic changes of the testes or ovaries. Davis and Martin (1991) and Martin and Davis (19b1) pinealectomized cats by electrocautery at 6 to 7 weeks of age. The operated males were heavier and had larger skeletons than did the controls. The pinealectomized animals also showed sexual interest earlier and had larger external genitalia. The females showed no difference in somatic development and estrus was not observed earlier in the pinealectomized group than in the controls. Pinealectomised mothers had small, weak litters. They often showed little maternal instinct and usually had an inadequate milk supply. Hoffman and Beiter (1965a, 1965b) found that pinealectomy prevented the expected atrophy of the testes of mature hamsters placed in a cyclic photOperiod consisting of 1 hour of light per day. Histological analysis showed the testes to be normal in appearance. Bilateral enucleation of pinealectomized hampsters had no effect on gonadal weight or histology. Bilateral enucleation of control or shamuoperated animals resulted in significant atrOphy of the gonads. Both Badertscher (1925) and Fee (191”) were unable to observe any precocious sexual maturity in pinealectomized female chicks. On the other hand, Izawa (1923) obtained — l , ' . .4 i r ‘- - I ‘ ' r 4 A . . ‘ . _ a . I . . v - . .. ' - ~. ’ n l . - I . x i. , t. < ’ b . ' i . . « f -‘ _ ‘v T- . ’ ' ‘ . . ' I ,,- ‘. - ' . _‘ . '-’ . u , - v ' I ' l V ‘ . 1 $ , v . ' V ‘ n ‘ . . . .‘ . . . u . ' ‘ ’ - ‘. ' . 4 ' , ‘ , ‘ . , ' ' , > . . ‘v' I. ‘ ‘ . “ V. ‘ 7 . 1 .I ~ . v A . _ . ‘. . ‘ ‘ , , , , , _ 1 , \ e ( _ ‘ . . I A V ‘ _ . . ," .. . g 4_‘ . , a ‘ I u 15 efilat‘ged gonads and combs in ZOO-day old hens which had beerx pinealectomized at 29 to 35 days of age. Pinealectomy of rats caused increased ovarian develop- ment in experiments by Gittes and Chu (1965), Izawa (1926), Kitay (1951:.) and Wurtman 3 2. (1959, 1960b, 1961). Negative results have been reported by Andersen and Wolf (1939), Benton and Rusbridge (1933) and Wragg (1965). Izawa (1926) observed increased uterine growth following pinealectomy, whereas Wurtman 25 al. (1960b and 1961) and Vragg (1965) found no differences. Reiter 2t El. (1966) showed that pinealectomy could prevent the expected re- gression of the uteri of thiouracilutreated hamsters. Vragg (1965) also reported that pinealectomy had no effect on sexual maturity or the estrous cycle of rats. Andersen and Wolf (1939) confirmed the lack of effect on both sexual maturity and the estrous cycle. FUrther sup- port for the negative results of pinealectomy on the estrous cycle was given by Ifft (1962). Chu 33 31° (1969) and Gittes and Chu (1965), however, observed an increase in the incidence of estrous phases following pinealectomy. Previous references cited contradictory results on the relationship between pinealectomy and growth rates in males and in mixed groups of animals. Malm gt 51° (1959) ob» served significantly greater weight increases in l6 Pinealectomized adult male rats up to the 19th week pOStoperatively. After the 16th week, the weights did not vary, Gittes and Chu (1965) and Kitay (1959a) removed the pineals of immature rats and found no differences in their adult body weight compared with sham-operated or unoperated controls. Newborn female rats were pinealectomized by Wragg (1965) with negative results on body weight at 60 days of age. Gonadal changes in response to pinealectomy has been the primary area of study. However, the effect on other parameters has been noted in conjunction with these studies and in separate experiments. Pinealectomy of 6-day chick embryos produced no changes in parathyroid, thymus or spleen weights at 18 or 63 days post-hatching according to Stalsberg (1965). In addition, there were no differences observed in the weight or the histological appearance of the adenohypOphysis, thyroids or adrenals. D'Amour and D'Amour (1937) obtained no effect on the weights of endocrine organs of rats pinealectomized at l to 3 days of age. Izawa (1923) had similar results with chicks pinealectomized at 29 to 35 days of age and examined at 200 days of age. Adrenal weights were unaffected by pinealectomy in experiments by Shellabarger and Breneman (1999), using chickens; by Andersen and Holf (1939), Isawa (1926) and Hragg (1965), using rats; and by Hoffman and Reiter (1965b) with hamsters. Experiments by Dill (1961), Farrell (1960b) 17 and. Vurtman et a1... (1959, 1960b, 1961) showed a hyper- trephic effect of pinealectomy on the adrenal gland of rats, although Dill's results were not significant, Dandy (1915) and-Davis and Martin (1991) were unable to show any histological alterations of the adrenals in pinealectomized dogs and cats, respectively, Benton and Rusbridge (1933) found no differences in weight or in the relative preportion of cortical and medullary substances in adult rats which had been pinealectomized at 26 days of age; Similar results were found by Panagiotis and Hungerford (1961) with rats on both control and low sodium diets; Both pinealectomized animals and their controls responded to the low sodium regimen with equivalent hypertrophy of cells of the sona glomerulosa; These results were con- firmed by Hurtman gt 21, (1960a). Microscopic examination showed no change in the adrenals of rats which had been pinealectomized for three successive generations, according to Sullens and Overholser (1991)o M31“.3£.El° (1959) reported that pinealectomy failed to alter the phosphorus uptake (a measure of metabolic activity) by the adrenal, Keeler (1961) obtained increased sodium excretion in pinealectomized rats. Tanner and Hungerford (1962) observed the apposite effect; that is, decreased sodium excretion following pinealectomy. Farrell (1960b) pinealectomized dogs, resulting in an acute reduction in aldosterone secretion, however, the steroid 18 ontput returned to or above normal levels within a week. (Ln attempt by Davis (1960) to confirm these results yielded negative results. Kitay (1963) observed increased levels of ACTH in the pituitary, but no change in plasma corticoids following pinealectomy. These seemingly contradictory observations might be partially explained by the proposed existence of two substances in the pineal and/or surrounding tissuem-one being inhibitory and the other stimulatory on adrenal activity. These will be discussed in greater detail in another section. ”Andersen and Wolf (1939), Benton and Rusbridge (1933) , and Vragg (1965) found no weight change in the pituitary after pinealectomizing rats. Shellabarger and Breneman (1999) obtained confirmatory results with chickens. However, Hurtman gt’al. (1959) observed significant pituitary hypertroPhy in pinealectomized rats, while Izawa (1926) reported retardation in the females only. The absence of histological changes in the pituitary following pinealectomy has been reported in rats (Holmes, 1956; Sullens and Overholser, 1991), and dogs (Dandy, 1915). Halm‘gjigl. (1959) found no change in the pituitary metabolic activity of rats compared to sham-Operated controls. Studies of the effect of pinealectomy on thyroid weight (Andersen and Wolf, 1939; Izawa, 1926; Benton and Busbridge, 1933; Shellabarger and Breneman, 1999), histology (Dandy, 1915; Davis and Martin, 1991; Sullens and Overholser, 1991) u r"-':"r.v — Kw'sil, -r‘—‘,~ *1”! ' 19 ‘nd metabolic activity (Malm _e_t $1., 1959) have yielded negative results in birds and laboratory mammals. Likewise, no effect of pinealectomy has been noted for the thymus (Andersen and Wolf, 1939; Dandy, 1915; Malm at élfi’ 1959; Sullens and Overholser, 1991), pancreas (Dandy, 1915; Sullens and Overholser, 1991), parathyroids (Dandy, 1915), liver (Dandy, 1915), spleen (Dandy, 1915), brain (Izawa, 1926; Malm 33 31., 1959) and other body tissues (Dandy, 1915; Izawa, 1926; Sullens and Overholser, 1991). Two investig- ators measured the eyeballs of animals. Retardation of eye growth was reported in chicks (Izawa, 1923) and rats (Izawa, 1926) following pinealectomy. Benton and Busbridge (1933) found no significant effect in rats. Davis and Martin (1991) noted no behaviour changes in rats, but Judged pinealectomized cats to be more aggressive and belligerento Beiss 3.2 fl" (1963b) observed that pinealectomized rats were more active than their littermate controls. 5. Results of Pineal Extract Injections and Pineal Transplants The close anatomical relationship between the pineal gland and the hypothalamus led to the speculation that the effect of pinealectomy actually resulted from the disturbance of nearby hypothalamic centers known to effect endocrine activities. Studies to determine the effect of 20 PiJIeal extract injections and pineal transplants in both ‘pinealectomized and unOperated animals have been reported by several researchers. Takacs (1935) fed dried calf pineal tissue to young fowl at 10 to 35 milligrams per day levels. After 9 months the experimental birds weighed over 200 percent more than the controls. After 5 months, the difference was still marked, but less pronounced. Similar growth promoting effects of feeding pineal tissue were observed by McCord (1919) in chicks and guinea pigs. He also found that pineals from immature donors gave more striking results. Test chicks fed veal pineal for 1 week grew more than their controls. For the next 9 weeks, they were fed pineal tissue from old cattle and failed to gain more than the controls. Switching back to the veal preparation, gains were again increased over those of the controls. McCord (1919) also noted that while feeding pineal tissue growth was stimulated and the material did not cause the animals to grow beyond normal adult size. As the test animals approached maturity, the treatment became less and less effective. There were also less well-established indic- ations of accelerated mental and sexual develOpment in animals fed the pineal tissue. Lyophilized beef pineal tissue in distilled water was injected into intact and pinealectomized chicks by Shellabarger (1952) at a level of 5 milligrams of pineal 21 material daily. Non-Operated chicks showed increased testis growth, while pinealectomy of chicks prevented the previously obtained inhibition of testis growth. In a subsequent report, Shellabarger (1953) reported the apposite effects; that is, testicular hypertrOphy following ” pinealectomy which was prevented by injections of 5 mil- ligrams of pineal material daily. The author notes that autOpsies were made at different ages in the two tests (20 days in the first experiment and 90, 59 and 60 days in the latter one), suggesting, therefore, that the results might not be in conflict. Four differently prepared pineal extracts were injected into young White Leghorn males by Shellabarger (1953). There were no differences in the testis weights of these experimental groups and their controls. Beiss é£.2li (1963a) determined the phosphorus metabolism in the gonads of mice, rats, rabbits and chickens as an indicator of the action of pineal extracts. Doses of extracts lower than those required for weight changes produced changes in the phosphorus uptake. Pineal extract inhibited the hypertrophy of the testes and combs of chickens usually produced by pituitary extract injections. Evidence was presented for the existence of two antagonistic fractions in the pineal, one inhibiting and the other stimulating gonadal activity. The effect of simple pineal extracts appeared to depend on the age of the test animals. In immature subjects, whose gonadal function is capable of being 22 stimulated, both principles could be active, often leading to contradictory results. In adult animals, only the inhibitory effect has been observed. Bowntree gt :1. (1936) gave successive generations of parent rats daily intraperitoneal injections of pineal extract while observing both the parents and their offspring. Increased irritability, sexual activity and gonadal weights were observed in the injected parents. Maternal instinct levels became quite variable. In the offspring, growthmwas retarded, but somatic and sexual development were accel- erated. These effects became more marked in succeeding generations. The strength and activity of offspring of injected mothers were decreased, especially during the first 15 to 20 days. In this experiment, the controls were not injected with a carrier or placebo. Although the results were not analyzed, the data appear to be sig- nificant. In a later report, Einhorn and Bowntree (1939) gave additional data in support of the previous observations. frequent pineal implants in normal rats did not effect the rate of growth or development of their offspring (Einhorn and Bowntree, 1939). These implants did not persist in the test parents. Lahr (1932) implanted his experimental animals with one or two pineals daily, while the controls were similarly implanted with brain tissue. No difference was found in the growth rates. In both males and females, the gonads (testes and ovaries) were lighter 23 in “eight and sexual maturity was delayed by 3 to 5 days after 31 to 91 days of pineal implantation compared to the control implants. Histological studies showed that the seminiferous tubules of the experimental group were con- sistently smaller and that spermatogenesis was retained. Kozelka (1933) found no differences in body weight or in comb growth when pineals from adults were implanted sub- cutaneously into young chicks (over 28- and 90-day periods) or when pineals from infant chicks or late stage embryos were implanted into half grown birds (over 97- to 59—day periods). All implants were reported to have been absorbed. It should be pointed out that each experimental group (different treatment procedures) consisted of only one bird. Kitay and Altschule (1995b) observed decreased ovary weights in response to three experimental plans involving the intraperitoneal injection of extracts from bovine pineals. Bats injected for 2 weeks from 32 days of age had ovarian reductions of borderline significance when the equivalent of 50 milligrams of pineal powder was injected daily. Doubling the dosage (100 milligrams of pineal powder daily) produced highly significant inhibition. Bats given the higher dosage starting at 95 days of age showed only borderline significance, indicating a decreasing effect with animals approaching maturity. Hurtman 23 21. (1961) showed that the increase in ovarian weight of rats placed in continuous light was prevented by the intraperitoneal 29 iniecfiion of bovine pineal extracts. However, the uterus hypertrOpied as usual under these conditions. Wurtman it; El. (1959) also found that pineal extract would prevent hypertrOphy of the ovary following pinealectomy. The effect of pineal extract on the intact animal varied with the dose. Daily injections of 0.3 milliliters caused ” significant gonadal atrophy (P < .05), while 1.0 milli- liters dosage levels caused atrophy, significant at the 0.1 percent level. Soffer it ii. (1965) observed the inhibition of the uterine stimulatory effect of both human chorionic gonadotropin and human menopausal gonadotropin by the injection of bovine pineal extracts in laboratory mice. The extract failed, however, to produce any such inhibition against oestrone. Bats have a post-reproductive period at about 1 to 2 years of age, in which they show persistent estrus. Meyer £5 El. (1961) altered this persistent state of estrusby injecting 1 or 2 milliliters of a protein-free bovine extract; but they found no influence on the rogue larity of the estrous cycle in younger adult animals given 0.5 or 1.0 milliliter injections. Exposure to continuous light induces prolonged estrus in rats. Ifft (1962) found that daily 1 milliliter injections of bovine pineal extract did not alter this occurrence, but daily 2 milliliter injections caused a marked increase in the diestrus phase of the estrous cycle. Two milliliters of a brain extract (control injection) was ineffective. (III: I! 25 Iffd:(l962)_also transplanted pineals from.newborn ‘rats into the kidney capsules of adult rats in continuous light. These animals in which the transplanted pineal appeared viable (histological examination) were considered the experimental group while those which did not persist were used as controls. No differences could be shown in the estrous cycles of the two groups. The possibility exists that the transplants which failed to survive the 30 to 50 days of testing, the controls, actually were active for some portion of this time, thus influencing the early results. Moszkowska (1958) caused precocious puberty in female rats by grafting an anterior pituitary from a mature male rat on the ovary. The additional grafting of four pineals clearly decreased this effect. The intramuscular transplantation of 10 or 20 isogeneic pineals into pinealectomized rats consistently reversed the ovarian hypertrophy seen in sham-transplant, pinealectomized control animals (Gittes and Chu, 1965). Histologically, the pineal tranSplants showed rich vascularization and good overall appearance. However, the authors did show that pineal HIOMT enzyme activity was very low. They suggested that this enzyme level was probably dependent on nervous innervation of the pineal. Holmes (1957) described successful intraocular transplants of pineals in adult rats and rabbits. AutOgraphs attached to the iris, became vascularized and showed persistence of healthy tissue. Homografts of adult tissue became 26 Vascularized and developed, but shoed evidence of an antigenic reaction. Ebels at El“ (1965) obtained two active pineal fractions; one capable of stimulating pituitary rsn excretion and the other capable of diminishing pituitary FSH excretion in iitig. Moszkowska (1963) observed an inhibitory effect on FSH excretion when pineals were included in a synthetic incubating medium for pituitary glands. Significant pituitary atrophy resulted when Hurtman it 51. (1959) injected 26-day old rats with protein-free bovine pineal extract. Little work has been done on the effects of pineal extracts on factors other than the gonads and adrenals. Anton-Tay (1965) found that extract of pineal glands can depress thyroid function, presumably through a blockage of the synthesis and/or release of pituitary TSH. Thyroidal response to cold was markedly decreased in rabbits given injections of pineal extracts. Beiss it ii. (1963a) suggested that the pineal contains inhibitory and stimulatory factors which act not only on the gonads, but also on the pituitary, thyroid and adrenal glands. An inhibitory factor might also reduce the metabolic activity of the liver. Kitay (1963) found no effect of pineal gland extracts on the weight of adrenal glands. Daily doses of 0.3 27 willileters of bovine pineal extract caused insignificant adrenal atrOphy in immature rats, according to Hurtman E: $1. (1959). although increasing the dosage level to 1.0 milliliters daily caused significant atrOphy. Dill (1961) obtained adrenal hypertrophy by injecting an aqueous extract of beef pineal glands in immature rats. This was accompanied by a pronounced rise in plasma and adrenal corticosterone. However, he also observed adrenal enlargement when a control extract of calf brain was injected, although this treatment did not affect the corticosteroid levels. An acetone extract of beef pineals showed no effect on the factors measured. Since Hurtman é£_él. (1960a) found that bovine pineal extract administrations caused no change in the size and lipid content of the adrenal zona glomerulosa, the urinary potassium or the sodium and water intake, they suggested that the pineal hormone probably did not have a specific effect on the secretion or action of aldosterone. Lommer (1966) reduced the corticosteroid production in incubated slices of bovine adrenal cortex through the addition of a hexane extract of bovine pineal tissue. It was suggested that the pineal contains one or more substances which inhibit the llmhydroxylation of corticosteroids. Lucie gt_al. (1961) added homogenized diencephalon tissue (including the pineal gland) to a medium in which quartered rat adrenals were incubated. 28 The 8Beretion of aldosterone was increased without in- creasing that of corticosterone. A commercial pineal powder and two extracts of pineal failed to affect the secretion of either aldosterone or corticosterone. Taylor (1960) suggested that the subcommissural area may be a locus of stimulation, either humeral or neural, whereas the pineal is probably the source of inhibitory influences on aldosterone regulatory mechanisms. In preliminary studies, Everitt andeuang (1962) found evidence of an adrenocortical inhibitory factor in an acetone insoluble extract of sheep pineals. C. Active Pineal Substances l. Adrenalglomerulotropin and Anticorticotropin In 1959, Farrell (1959a) observed that the pineal complex contained a factor capable of stimulating aldosterone secretion. Further studies (Farrell, 1959b, 1960a, 1960b, 1960c) added support to this observation. Farrell first preposed the name "glomerulotrOpin" for the active factor which he presumed to be a hormone type ~of substance. The name was derived from the fact that aldosterone is secreted by the zona glomerulosa, therefore, the active substance should be exerting its effect on that portion of the adrenal. The possibility of confusion was recognized in that the name could imply an effect on the glomerulus of the kidney. Thus, the suggested name was changed to "adrenoglomerulotrOpin." During efforts to purify adrenoglomerulotropin, an inhibitory factor was 29 found. This new fraction seemed to inhibit both aldosterone and to a lesser extent, cortisol. This substance was termed "anticorticotropin." This factor appears to compete with pituitary corticotropin and with adrenoglomerulotroPin; the rate of adrenal steroid secretion being dependent on the balance between these factors. Farrell and McIsaac (1961) reported the isolation and identification of adrenoglomerulotropin two years after it was first proposed to exist. It was identified as 1-methyl-6—methoxy-l,2,3,9-tetrahydro-2-carboline, which was derived from 5-methoxytryptamine. Taylor and Darrell (1963) pointed out that this may not be the exact structure of the natural substance, but submicro- gram doses failed to increase the secretion of cortisol. Panagiotis and Hungerford (1961) observed a loss of stainable fat in histological sections of pineal glands following sodium restriction. This was thought to indicate an augmented activity in the pineal cells, probably in releasing a hormone (adrenoglomerulotropin) involved in salt retention. Machado and da Silva (1963) showed that rats in~ jected with pineal body extract exhibited strong sodium retention, comparable to that obtained by injecting 10 micrograms of aldosterone. Adrenalectomized animals did not show these results indicating that the extract 30 acted through the adrenal, rather than directly to cause sodium retention. It was assumed that the pineal extract contained adrenoglomerulotropin, which acted on the adrenal gland. Some doubt remains as to the exact source of adrenoglomerulotropin. Its presence in pineal ex- tracts does not exclude the possibility that it actually comes from adjacent structures which are removed with the pineal. Since pinealectomy and midbrain lesions do not prevent all experimental hyperaldosteronism, Keeler (1961) suggested that the anterior central midbrain area and the pineal cannot be the only sources of the adreno- glomerulotropic hormone. Tanner and Hungerford (1962) noted an increase in sodium retention of pinealectomized rats. If the adrenglomerulotropic effect had been re- moved by pineal abalation one would have expected the opposite result; increased sodium excretion. Their results suggested the removal of a factor which inhibits aldosterone secretion. Studies previously cited demonstrated a lipid soluble factor in pineal extracts which inhibits aldosterone activity. Fabre gt El° (1965) found ubiquinone in the lipid fraction of pineal extracts and indicated that it reduced aldosterone secretion rates following intravenous infusion of the pineal lipid fraction into intact dogs. Ii 5 ‘1' 31 _,2. Melatonin Lerner it :1. (1958) first isolated the active factor from bovine pineal glands that lightens amphibian skin color and inhibits MSH. They suggested that this substance be called "melatonin." No melatonin activity was found in bovine pituitaries, hypothalamus, thymus, thyroids, adrenals, ovaries, testes or eyes. The following year they identified the structure of melatonin as N-acetyl-S-methoxytryptamine (Lerner ii 21.. 1959a). The isolation of melatonin from other tissues will be described later. Axelrod and Veissbach (1960) isolated_the enzyme hydroxyindole-O-methyl transferase (HIOMT) from the pineals of several mammalian species, and found it to be present only in the pineal. The suggested melatonin metabolic scheme is shown in Figure 1. This was worked out from the findings of many authors (Axelrod and Weissbach, 1960, 1961; Kopin 3: Eli’ 1960, 1961; Lerner et al., 1960; McIsaac st 51., 1965; weissbach and Axelrod, 1960; Heissbach 22 21., 1960, 1961). Tryptophan is the precursor of the melatonin pathway. Under the influence of tryptophan hydroxylase (not shown in Figure 1), it is converted to 5-hydroxytrypt0phan, which becomes 5-hydroxytryptamine or serotonin in the presence of the 5-hydroxytryptophan decarboxylase (5-HTPD) enzyme. Serotonin is acetylated in the presence of acetyl-coenzyme A to form N-acetylserotonin. The N—acetylserotonin reacts with 32 Figure I.--Suggested Melatonin Metabolism Pathway S‘HVDROXYTRYPTOPHAN S-HTPD (decarbo ylatlon) i (oxidation) SEROTONIN S-HYDROXYINDOLE (S-hydroxytryptamine) HAO ACETIC ACID AeCoA S-adenOSyl- methionine (acetylation) HIOHT N-ACETYLSEROTONIN (methoxy|ation) S-adenOSyl- methionine S-HETHOXYINDOLE ACETIC ACID HIOHT (deamination) (methoxylation) DAm (deacetylation) HELATON m ——7-—————) S-HETHOXYTRYPTAHINE (N-acetyl-S-methoxy- DAc tryptamine) ENZYMES NH S-HTPD - S-hydroxytryptophan (hydroxylatiOn) decarboxylase Ac Co A I Acetyl coenZyme A NIGHT - Hydroxyindole -0- 6-HYOROXYHELATONIN U"k"°"" methyl transferase (N-acetyl-S-methoxy- NH - Hicrosomal hydroxylase 6-hydroxytryptamlne) MAO - Monoamine oxidase DAc - Deacetylase (conjugation) DAm - Deaminase SULFATE GLUCOSIDURONIC ACID CONJUGATE CONJUGATE 7* I) 'i I" . ' ‘A I ’ \¥'\:h' m. . I. ' ' \I ‘.' W" k. , ' ._ ._ -l: a. . u A‘ . 5 « “fiilfi..k tn,l - _.. , r , ' L ‘ "-..I .- _ ~~ . ' A t , 2’; m- g, h“ ‘ 33 S—adenosylmethionine in the presence of a methylating enzyme, hydroxyindole-O-methyl transferase (HIOMT), to form melatonin. Three metabolites of melatonin have been described. The primary one. s 6-hydroxyme1atonin, formed in the presence of microsomal hydroxylase enzyme. The 6-hydroxymelatonin is then conjugated with sulfates or glucosiduronic acid to be excreted. These conjugate forms account for 70 and 6 percent, respectively, of the excreted melatonin derivatives. About 12 percent is excreted in a form which has been only partially characterized. It appears that the indole nucleus of this substance is altered. Trace amounts (less than 1 percent) of the melatonin are deacetylated to form 5-methoxytryptamine and then deaminated to form 5-methoxyindole acetic acid which is excreted. A portion of the serotonin is also excreted as S—methoxyindole acetic acid, rather than going to form melatonin. This pathway requires monoamine oxidase for con- version to 5-hydroxyindole acetic acid, which is then methylated to form the excretory product. The methylation step requires 5~adenosylmethionine and the enzyme HIOMT. Both of these were also used in the conversion of N- acetylserotonin to melatonin. Cohen 32 31. (1969) pointed out that melatonin satisfies many of the criteria of a hormone. It is produced by a highly specialized glandular structure; in fact, the HIOMT enzyme has been found only in the pineal gland. It is released into the circulatory system and affects a distant target organ or organs. It can also be taken up ‘I a“ ’ preferentia11y by the target organ. Additional evidence to support these statements was provided by Lerner at £1. (1959b) when they drmonstrated the presence of melatonin in the peripheral nerves of man, monkey and cow. Barchas andw Lerner (1969) provided additional evidence for the localiz- ation of melatonin in the peripheral nerves when they showed that the methylating enzyme, HIOMT, was found in the pineal, but not in other portions of the central or nervous systems. These observations indicated that the pineal must release melatonin into the circulatory system from which it is picked up by peripheral nerves. Axelrod at E1. (1963) and Wurtman 3: ii. (1969e) injected labeled melatonin to trace its uptake in body tissues of cats and rats. They found some melatonin in all tissues examined 1 hour after injection. The pineal concentrated the labeled melatonin 90 fold over the plasmah level. The ovary and iris-choroid layer of the eye concen- trated the hormone 10 fold. other endocrine glands, peripheral nerve and the sympathetic chain concentrated melatonin 3 to 5 fold. As might be expected, adipose tissue had the lowest concentration of all tisues tested. The high level of melatonin uptake cannot be accounted for by hemo- dynamic factors, indicating the presence of a selective concentration mechanism. Following the isolation and identification of melatonin as the factor reaponsible for the lightening reaction of frog skin to pineal extracts, experiments were carried out 35 to determine its function in mammals. Body weight was not affected by the daily injection of 20 micrograms of melatonin in rats starting at 27 days of age and continuing for 28 days (McIsaac g3 31.,I1969). wurtman El 31. (1963a) found no difference at the 50 microgram level. Body weight was lowered in both sexes when Tilstra and Prop (1963) gave daily injections of 100 micrograms from birth to 50 days of age. Since both the injection levels and the age of treat- ment varied, one cannot be sure which factor caused the difference in results. Gonadal stimulation was observed by Thiebolt é£_éi. (1966a) following the injection of relatively large daily doses of melatonin (100 to 1000 micrograms) for 15 days. In male rats the seminal vesicles were markedly enlarged, while the testis and prostate weights increased somewhat, but remained in the range of individual variation. Similar treatment with serotonin produced no effect. Ebels and Prop (1965) and Dappers (1962) found no changes in testis weights after injecting doeses of 30 to 150 micrograms daily. Kappers (1962) did note a possible inhibition of the seminal vesicles with a 500 microgram dosage level, but this involved only one experimental and one control animal. In females, the ovary weight of melatoninm injected rats was 90 to 50 percent larger, but the uterus showed no change in weight compared to the controls. 36 Set‘Otonin treatment produced a slight, non-significant eYl'iargementof the ovary. Kappers (1962), McIsaac 2E _a_l. ‘1965) found no change in ovarian weights after injecting 20 or 100 micrograms of melatonin daily. The 28 day test period was started with 27-day old rats. Similar injections of 5-methoxytryptophol (closely related to melatonin) sig- nificantly lowered the ovary weights, Adams Eé El. (1965), Axelrod Ei 3;. (1963), Ebels and Prop (1965) and Uurtman E: El. (1963a, 196hb) observed depression of ovary weights with melatonin injections. Dosage levels ranged from 1 to 150 micrograms per day. All rats were immature when treatments were started. Serotonin injections were without effect (Axelrod 53 $1., 1963; Hurtman 33 al., 1963a) in microgram doses. O'Steen (1965) observed significantly depressed ovarian weights in rats after injecting 10 and 25 milligrams of serotonin per kilogram of body weight. Uterine weight appears to be unaffected by melatonin (Ebels and Prop, 1965; Hurtman and Axelrod, 196“) or serotonin injections (Ebels and Prop, 1965). Sexual maturity was not affected by daily 100 micro- gram injections of melatonin given rats from birth to 50 days of age by Tilstra and Prop (1963). Adams 35 El“ (1965), Axelrod g: 3;. (1963) and Hurtman 23 El“ (1963a) delayed sexual maturity by injections of 1 to 100 micrograms of the hormone starting at 2 to h weeks of age. Serotonin in- jections were without effect (Axelrod 33 al., 1963; Hurtman \ ,"' 37 3:1 23;., 1963a) except when given at levels of 10 and 25 milligrams per kilogram body weight (O'Steen, 1965). Most reports agree that l to 20 microgram injections of melatonin given daily caused a decrease in the incidence of estrus in rats (Axelrod it 51., 1963; Chu 23 El., 196k; McIsaac if. 31., 1961s; Hurtman _e_t_ g_1_, 1963a, l96’4b). Negative results were obtained with serotonin (Axelrod gé £11., 1963; Chu g g... 1961.; Wurtman £3 11." 1963a), N- acetylserotonin (Chu :3 al., 196“), 6-hydroxyme1atonin (Chu gt $1., 196%) and 5—methoxytryptophol (McIsaac SE Ei., 196k); To test for a possible neurotransmission activity of melatonin, segments of intestine and uterine horn were observed in ZEEEE after known neurotransmitters and _ melatonin were added to the medium. Tilstra and Prop (1963) observed contractions with two neurotransmitters; acetylcholine and serotonin. Melatonin appeared completely inactive alone and did not appear to modify the action of the two active substances. These and similar results from Arutyunyan gt 51° (1963) support the view of Kappers (1962) that melatonin is not a neurotransmitter substance. However, in a recent paper, Hertz-Eshel and Bahamimoff (1965) reported that melatonin inhibited both spontaneous and serotonin-induced contractions of the rat uterus which had been sensitized during the previous 3 days by diethylstilbesterol treatments. 38 ‘rwo studies of the effect of melatonin injections on “\Yroid function inrats yielded contradictory results.~ Boschieri fig 51. (1963) reported that daily subcutaneous injections of 150 micrograms of melatonin for 10 days H resulted in significantly lower thyroid weights and de- creased thyroidal uptake of 131I. Histological examination showed the height of the thyroid cells to be significantly reduced. Thiebolt 55 al. (1966b) administered 100 to 1000 micrograms of melatonin daily to young prepubertal rats and demonstrated marked thyroid cell hypertrophy. Lerner it :1. (1958) had first characterized melatonin as the substance which lightens the skin color of frogs through an aggregation of melanin granules within the cells. No influence was seen on mammalian pigmentation. Additional experiments were carried out by Snell (1965) which supported this lack of effect in mammals. Large doses of melatonin were injected subcutaneously into pure red and pure black male guinea pigs daily for 30 days. He observed no gross changes in skin pigmentation and found no histological evidence that melatonin caused any positional change in melanin-granules. D. Factors Influencing Pineal Function 1. Alteration of Pineal Anatomy It is well substantiated that continuous exposure of rats to light causes a decrease in weight of the pineal gland (Fiske 33 g.” 1960; Quay, 1961; Roth §_t _a_1., 1962; 39 ‘urtman 9:1: 51., 1960b), compared to that of animals on diurnal lighting schedules. Adrenalectomy (Iiske e; 3.1., 1962; Quay, 1961), gonadectomy (Fiske it 51., 1962) or hypophysectomy (Fiske e; 21., 1962) did not prevent the effect of constant light on pineal weight. However, Quay (1961) found that bilateral intraorbital transection of the optic nerves blocked the effect of continuous light. Quay (1963b) also observed cytological and metabolic evidence of pineal inhibition in response to continuous light. Basrur and Winget (1963) compared the histology of the pineal gland of birds kept in constant darkness with that of birds kept under a diurnal light cycle. The only difference ob- served was the absence of magenta-colored granulations in the large epithelial cells of the pineal septum in birds kept in darkness. They concluded that these cells were light responsive. This report did not indicate the species of bird tested, the length of light treatment period or the age, sex and reproductive state of the birds. The human pineal undergoes extensive calcification after puberty. Kitay and Altschule (195ha) reviewed some of the early writings in which the presence of calciferous areas, called brain sand or acervulus cerebri, were observed. It was generally thought that this calcification indicated that the pineal ceased to function at or shortly after puberty. Hurtman 25 El' (196ha) found no correlation 1&0 bfitween age of the subject (3— to 70-year old humans) and ‘PiJieal enzymatic activity. All pineals from subjects over “0 years of age showed obvious calcification. They con- cluded that the pineal probably remains active throughout life. DeMartino it if. (1963) demonstrated that the number of osmOphilic granules in rat pineals increased with age. As_these granules are thought to be a product of secretory activity, they suggested a possible increase in pineal activity following puberty. Everitt and Huang (1962) showed that the pineal gland of the female rat (on a percentage of body weight basis) is larger than that of the male. Pregnancy reduced the pineal weight, an_observation also reported by Huang and Everitt (1965). The latter paper indicated that the pineal weight of pregnant rats was inversely proportional to the number of fetuses being carried. Shellabarger (1953) reported hypertrophy of the pineal in White Leghorn capons compared to intact males. Castration of rats was reported to have no effect on pineal weight (Everitt and Huang, 1962). Everitt and Huang also reported a negative effect of unilateral adrenalectomy on pineal weight (1962). Beiss 23 El. (19630) reported that the pineal shows no atrophy following hypophysectomy, as do adrenals, thyroids and gonads. In contrast to the atrophied glands, the pineal phosphorus turnover increases. bl 2. Alteration of Pineal Metabolic Activity Experiments have been conducted to determine the factors involved in control of melatonin production and/or release. Quay (196“) showed that the melatonin level in adult rat pineals follows a circadian rhythm and is cor- related with light and dark periods. Melatonin content of the pineal rises at the start of the dark and decreases during light. Wurtman 33 £1. (196Ud) also observed a reduced synthesis of melatonin in rats when exposed to light. “Hydroxyindole-O-methyl transferase (HIOMT), a melatonin-synthesizing enzyme, also shows a diurnal rhythm when rats are kept under cyclic light treatments. When lighted from 7 a.m. to 7 p.m. enzyme activity was lowest at about 6 p.m. The activity rose about 3 fold to its maximum at midnight, followed by a decrease starting shortly before the next light period started (Axelrod, éi 31., 1965). Animals kept in constant light show a decrease in HIOMT, whereas animals kept in constant or nearly constant darkness show increased HIOMT activity (Axelrod 33 21., 1965; Hurtman and Axelrod, 196b; Vurtman 33 31., 1963b). Quay (1963a, 1965a), Fiske (1961.) and Snyder 33 g. (1965a, 196hb) showed the diurnal cycle of pineal serotonin content to be approximately opposite that of melatonin; that is, it is at its maximum at about noon, drops quite quickly at the start of the dark period to reach a nocturnal low at midnight and then gradually increases toward the noon level b2 “1‘31! most of the increase occurring during the lighted Portion of the morning. Quay (1963a) suggested that the Sharp decline of pineal serotonin in the early dark period may represent release, destruction or a triggered release of melatonin synthesis from serotonin, with the latter explanation being favored. The serotonin rhythm persists in constant darkness (Snyder 33 33., 1965a, l96hb, 19653) but 13 abolished in constant light treatments (Quay, 1962; Quay and Halevy, 1962; Snyder 33 33., 1965., 196th, 19650). no rhythm is abolished by the addition of as little as h hours of light, preventing the nocturnal decline in serotonin content of the pineal (Snyder 33 33., 1965a,_1965c)3 Snyder and Axelrod (4965) presented evidence to show that serotonin is released from a bound form during darkness, allowing it to be acted on by enzymes for melatonin production or excretion as needed by the body. The binding of serotonin during the light hours accounts for its increasing levels during the day. The enzyme, 5-hydroxytroptophan decarboxylase (5-HTPD), is involved in the synthesis of serotonin; and monoamine oxidase (MAO) in serotonin metabolism. Effects of light on these enzymes would influence the pineal serotonin levels. Snyder and Axelrod (1965) found no cyclic rhythm in pineal levels of 5-HTPD or HAO in rats in cyclic light. Snyder and Axelrod (l96ha) and Snyder 33 33. (l96ha, 1965b) 43 a“hm-ted that constant light increased pineal 5-HTPD and constant darkness decreased its level in comparison to an intermediate level in rats under cyclic lighting treatments. No change was effected in the pineal MAO content by con- tinuous darkness (Unrtman and Axelrod, 1965; Uurtman 33 33., 1953b). The recognition of light as an important controlling factor of pineal function suggests involvement of the eyes as a receptor. Hurtman 33 33. (l96bd) showed that bilateral enucleation (complete removal of both eyes) resulted in a loss of the capacity of the pineal to respond to altered illumination. Blinding abolsihes the RIGHT response to light changes (Axelrod 33333., 1965; Hurtman and Axelrod, 196k). The circadian rhythm in pineal serotonin content persistedgafter enucleati3n (Snyder 33 33., 196kb, 1965a). The elevation in pineal 5-HTPD with constant light is eliminated by removal of the eyes. Removal of the pituitary (Snyder 33 33., 1965a, 1965c; Wurtman and Axelrod, 196“; Hurtman 33 33., 196bd), gonads (Snyder 33 33., 1965c; Whrtman and Axelrod, 196a; Wurtman et a1., 196ud, 1965), thyroid (Snyder 33 33., 1965a, 1965c), or adrenals (Snyder 33 33., 1965a, 1965c) has not been shown to exert any effect on the pineal metabolic activity. wurtman 33 33., (1965) reported that the HIOMT activity increases during diestrus and decreases during proestrus and estrus phases of the estrous cycle in rats. an 13‘13 ‘Hould appear to indicate‘a relationshipretween estrogen outqnnt and HIOMT activity. However, injections of estradiol produced no change in HIOMT activity in immature rats and only a slight decrease in mature animals. Thus, the authors concluded that the RIGHT activity changes during the estrus cycle were probably not a consequence of variations in the level of circulating estrogens. There is no definite evidence for a hormonal control of the pineal. Anatomically it was shown that sympathetic innervation is the primary, if not the only, functional nervous supply to the pineal. This innervation has been interrupted by surgical and pharmacological means in order to determine its function in the control of pineal metabolism. Surgical denervation of the pineal has usually been effected by bilateral superior cervical ganglionectomy. Previous ob- servations showed that this operation effectively destroys the main pineal nervous supply, as evidenced by the degen- eration of the two nerve conarii and the loss of intrapineal innervation (Kappers, 1960). Vurtman 23 21. (l96hd) showed that sympathetic denervation of the pineal interrupts the effect of light on melatonin synthesis. The pineal HIOMT cycle in diurnal light is abolished (Axelrod g: 31., 1965), as is its response to constant light or darkness (Unrtman and Axelrod, 1961»). Following bilateral ganglionectomy, the serotonin level of the pineal is reduced (Pellegrino de Traldi E: 31., 1963) and the cyclic variation in serotonin level is abolished (Fiske, 196“; Snyder and Axelrod, 1965; U5 Snyder 3531;, 1965a, 1964b, 1965c). According to Quay and Balevy (1962), transection of the optic tracts also reduced the pineal content of serotonin and related amines. Constant light and superior cervical ganglionectomy both increase the pineal S-BTPD level (Pellegrino de Iraldi and Rodriguez de Lores Arnaiz, 196%; Snyder and Axelrod, l96ba; Snyder fig éi., 196ha, 1965b). fi ” Boura it ii. (1959) and Boura and Green (1959) des- cribed bretylium, a compound which blocks the peripheral sympathetic nervous system without impairing the para- sympathetic or central nervous system functions. No effect on the pineal serotonin rhythm was observed by Snyder and Axelrod (1965) following bretylium treatment of rats. This result was unexpected since sympathetic denervation abolishes the serotonin rhythm._ Snyder E: 51. (1965b) found that “ bretylium-treated rats failed toushow an alteration of 5-HTPD enzymatic activity, agreeing with the denervation experiment results. The negative results with modifications in hypophyseal, gonadal, thyroidal and adrenal endocrines would indicate that the pineal gland is not directly controlled by hormonal factors, although some modifying actions of hormones may be present. The general reduction in melatonin production accompanying bilateral enucleation and interruption of the sympathetic nervous system suggests that the primary control mechanism involves light induced impulses from the retina. #6 These impulses probably pass through the optic tracts, through unknown central nervous system pathways, through pre- and postsympathetic ganglionic fibers and to the pineal via the nervi conarii. In the pineal gland, neurotransmitters are probably released to influence the activity of various enzymes, especially S-HTPD and HIOMT, controlling serotonin and melatonin metabolism. Snyder and Zweig (1966) reported evidence of a non- retinal pathway for light influence on the pineal of very young rats. Extending the light period given blinded, 12- day old rats partially prevented the nocturnal fall in pineal serotonin. Covering the skull of these young rats or the use of 27-day old blinded rats produced the normal serotonin rhythm. These results suggested that light might penetrate the thin skull structure of very young rats and exert an effect directly on some portion of the brain. The subsequent ossification of the skull and growth of the hair covering prevent any appreciable direct influence of light on the brain of older animals. If the normal pathway of light influence on the gonads involves the retina as a receptor, blinding would be expected to interrupt the results usually obtained. Hoffman and Reiter (1965b) performed bilateral enucleations which re- sulted in significant atrophy of the testes of hampsters kept in a stimulatory light regime (16 hours light and 8 hours darkness, daily). Whrtman gt El° (l96bc) prevented the constant light induced hypertrophy of the ovaries and uterus by removal of both eyes and by bilateral cervical sympa- thetic ganglionectomy of rats. The associated decreases in pineal weight and melatonin synthesis were also prevented. VHUrtman gt El. (196he) observed a decrease in the melatonin concentration in the ovary when mature rats were placed in constant light. Since no change was observed in h the amount of the hormone present in the heart, a light con- trolled mechanism may influence the concentration of melatonin by the ovary. ,There is indirect evidence of a species variation in ‘ melatonin synthesizing ability. Snyder and Axelrod (l96hb) showed that the rat pineal contained approximately twice the 5-HTPD activity of bovine pineals. The level in quail pineals was slightly less than that of bovine pineals. The enzyme is involved in serotonin production. HIOMT, the melatonin—forming enzyme, is even more variable, as shown by Axelrod and Weissbach (1960) and Axelrod gt fil' (196k). Monkeys have about 500 times as much pineal HIOMT as rats and birds had 3 to h times the activity of monkeys. It is not known whether these enzyme levels reflect a difference in amounts of serotonin or melatonin actually produced. E. Summary of Literature 1. Bodily gorwth Growth is not inhibited by removal of the pineal gland. Papers reporting acceleration of bodily gorwth or lack of change in growth have appeared in nearly equal numbers for l #8 both fowl and mammals. Most of the researchers who employed statistical analyses reported the lack of a significant difference between pinealectomized, sham-operated and control animals. The injection of pineal extracts and the implantation of pineal glands has also produced inconsistent results in birds and mammals. Reports of growth stimulation, growth in- hibition or lack of effect have occurred with nearly equal frequency. Only three papers have reported the effect of melatonin injections on body weights of rats. Two of the papers reported no significant difference in body weights, whereas the other reported a decrease in both sexes. There is no consistent evidence that the pineal gland has any im- portant influence on bodily gorwth. 2. Reproductive Functiou There is sufficient evidence in the literature to support a proposed inhibitory function of the pineal gland on reproductive function. Extirpation of the pineal gland of immature chicks and mammals has generally resulted in gonadal hypertrophy. In cases where no change or gonadal inhibition were observed, the animals were usually sacrificed after reaching full maturity. While the results were somea what more variable, there is also good evidence.that injections of pineal extract will prevent gonadal hypertrophy in pinealectomized animals and cause some gonadal inhibition in intact animals. Melatonin, a pineal elaboration, appears 1+9 to be the factor in pineal extracts which is responsible for the inhibitory action on gonadal development. The relationship of the pineal gland to sexual maturity of rats is less clearly shown. Pinealectomy caused earlier sexual maturity in some experiments, while producing no change in others. Those finding advanced sexual maturity usually observed a difference of only a few days. However, pineal extract and melatonin injections were reported to delay sexual maturity of rats, with only a few exceptions. The literature contains strong evidence of a relation- ship between pineal function and photoperiods.w_The pineal gland increases in size and produces more melatonin when an animal is housed with photOperiods of short light and long dark periods or total darkness, compared to normal labora- tory photOperiods or constant light. Thus, pinealectomy produced more striking results as the length of the dark period was increased. The results were negative with constant light treatment. Evidence for any pineal influence on other organs of the body is inconsistent and nonconclusive. Adrenal function has been most closely associated with the pineal gland. Further investigation is required to clarify pineal function, if any, on other body organs. OBJECTIVES 'the overall objective of this study was to determine the function of the avian pineal gland as it relates to the control of reproductive function by phot0periods through pinealectomy and melatonin injection experiments. A. B. Pinealectomy experiments: 1 .‘ 2. To determine whether pinealectomy will permit full or partial gonadal develOpment of immature quail reared under inhibitory phot0periods.i To determine whether pinealectomy will alter gonadal deve10pment of quail reared under stimula- tory phot0periods. To determine whether pinealectomy will prevent or reduce gonadal atrophy in mature quail following a change from stimulatory to inhibitory photoperiods. Melatonineinjection experiments} 1. To determine whether melatonin injections will prevent or reduce the gonadal develOpment of immature quail reared under stimulatory photOperiods. To determine whether melatonin injections will cause atrOphy or reduced function in gonads of adult quail housed under stimulatory photOperiods. 50 EXPERIMENTAL PROCEDURE A. General Management Japanese quail (Coturnix coturnix japonica) from mis- cellaneous genetic lines were used as the test animal in all experiments. All quail chicks were grown in a battery brooder to the age of 5 weeks. Birds remaining on experiment past 5 weeks of age were housed in individual cages, h to 5 inches wide, 7 to 8 inches deep (front to back) and 5% to 7 inches high, with the floors sloping toward the front. Several groups of quail were transferred to a windowless test chamber at 3 weeks or more of age. The chamber is 9} feet deep by 8% feet wide. A temperature control unit kept the room temperature between 64 and 700 F. Quail between the ages offl3 and 5 weeks were put in a battery broader which had non-glowing, encased heating elements and lacked pilot lights. Thus, complete light control was possible. Birds over 5 weeks of age needed no additional heat and were placed in individual cages U inches wide, 7 inches deep (front to back) and 5% to 7 inches high. A 25 percent protein quail breeder ration and water were provided 22 libitum. The composition of the ration was as follows: 51 52 Ground yellow corn “12.5 (lbs) Soybean oil meal, dehulled, 50% 370.0 Alfalfa leaf meal, dehyd., 17% 50.0 Dried whey 25.0 Meat a bone scraps, 50% 25.0 Fishmeal, menhaden, 60% 25.0 Ground limestone 50.0 Dicalcium phosphate 15.0 salt, iodized 5.0 My“, Vit. premix (Nepcosol) 2.5 (Fat 20.0 All quail were wing banded for identification purposes at 1‘ to 2 days of age. B. Pinealectomy Operation All quail to be pinealectomized were operated on during the day of hatching or on the following day, under light to moderate ether anesthesia. Iris scissors were used to make a longitudinal out about 10 to 12 millimeters long through the skin on tOp of the head. The edges of the skin were separated to expose the soft, developing skull bones. The cartalaginous suture line between the frontals was pierced transversely, about 2 millimeters anterior to the frontal fontanelle, using a No. 12 Bard~Parker surgical blade. Care was exercised in this and the following steps in order to avoid cutting or injuring the cerebrum. Fine iris scissors were used to extend the cut transversely to either side, into the frontal bones. The final opening through the skull was about 6 millimeters long. The dura which adheres to the skull was cut at the same time. In a blind operation, fine Vatchmaker's forceps were inserted through the opening, just urider the dura in a caudad direction, and past the frontal "A 53 fontanelle to grasp the pineal body and remove it. The stalk remained attached to the pineal body and was removed at the same time. The occurrence of some bleeding could not be prevented, but the amount was not excessive. As soon as the active bleeding had stopped and the blood was removed with a cotton ball dampened with physiological saline, the skin was closed over the skull with three or four sutures. In preliminary pinealectomy trials, attempts were made to expose the pineal before removal by extending the cut described above caudally on both sides, across the junction of the frontal and parietal bones. The flap of develOping bone could then be turned back, exposing the pineal. However, this method markedly increased the amount of bleeding and resulted in excessive mortality. Most of the mortality observed in the test birds during pinealectomy was thought to be due to an overdose of ether. Any birds in which the operation appeared to have resulted in incomplete pineal removal or injury to brain tissue were destroyed immediately. The success of all pinealectomy operations was further checked at the end of each trial. Any suspic- iOus tissue was sectioned and stained for histological observation. Those birds which showed any remaining pineal tissue were eliminated from the experiment. The successfully operated birds were usually alert and active within 1 or 2 hours following surgery. Mortality in the battery broader was no greater in the pinealectomized 5t quail chicks than in their controls. ‘Pinealectomized and control birds of the same age were reared together in the battery brooders. The time required for each Operation precluded the use of larger numbers of experimental animals in each test. 0. Melatonin Injections Melatonin (Nutritional Biochemical Corporation, lot No. 8231) was dissolved in chloroform with subsequent 1020 dilution in sesame oil (5 percent chloroform solution). An 0.2 milliter dose contained 500 micrograms of melatonin. There was some tendency for the melatonin to come out of solution when stored at temperatures below 2&0 C. (75° F.). Subsequent solutions_were made in 5 percent ethanol. Levels up to 2000 micrograms melatonin in O.“ milliliters of solution remained in solution through the experiments. It was necessary to stir and heat this highest concentration to 50° c. on a Temco Stir-Plate (A. s. Aloe Co.) to get .11 of the melatonin into solution. All injections were subcutaneous; placed in the area of the lateral body apterium, just anterior to the femoral feather tract. Daily injections were given, alternately on the right and left sides. Melatonin levels and volumes of injections are specified under the respective experiment description. Injections were given with l milliliter hypo- dermic syringes using a 1% inch, 22 guage needle. D. Description Of Experiments Experiment 1 Eighteen (18) to 25 pinealectomized and a like number Of control quail chicks were started in each of four groups. MOrtality and the elimination Of unsuccessfully Operated birds lowered the numbers of birds completing the experiment. They were grown in broader batteries with an L:DII 16:8 (light period : dark period 3 16 hours : 8 hours, daily) light cycle. At 3 weeks of age prior to puberty, they were moved to a brooding battery located in a windowless chamber. From 3 weeks Of age their light schedule was L:D - 2:22. The two groups remaining on experiment past 5 weeks of age were moved to individual cages within the same windowless chamber. Groups were sacrificed at b, 5, 6 and 7 weeks Of age. The body, adrenal, thyroid, pituitary (6 and 7 weeks only), spleen, bursa, testes, ovary and oviduct weights were recorded. Organs to be weighed were carefully removed and placed on wet paper towels. Excess tissue was dissected away before weighing. Roller-Smith balances were used to weigh all tissues. All birds that had been Operated on were macro- and microscOpically examined to determine whether the pineal had been completely removed. -§§g§riment 2 Twenty (20) to 25 pinealectomized and control quail were started in each Of 3 groups. They were kept in L:D 8 16:8 light cycles throughout the eXperiment. At the age of 5 weeks, the females were moved to individual cages, while the males remained in the battery brooders until sacrificed. At the ages of 5, 6 and 7 weeks, the groups were sacri- ficed, with the exception of one group of females which were kept in individual cages until sexually mature. Thus, only males were sacrificed in the 6 week age group. Body, pituitary, thyroid, adrenal, spleen, bursa, testes, ovaryw and oviduct weights were recorded as in Experiment 1. Be- cause Of size several of the 7 week Old quail ovaries and oviducts were weighed on a triple beam balance (Ohaus Scale Corp.). Sexual maturity was assumed when the first egg was laid by each bird. All pinealectomized birds were examined to determine whether the Operation was successful. Experiment 3 Twenty (20) pinealectomized and 25 control Japanese quail were started in the battery brooder under an L:Dés 16:8 light schedule. At 26 days Of age they were trans- ferred to a battery brooder in a windowless chamber with L;D I32822 light cycles. At 38 days Of age they were placed in individual cages within the same test chamber. The light period was increased to provide an L:D - lh:lO schedule at the age of 56 days and egg production was recorded to 57 determine age Of sexual maturity. After 28 days Of increased light treatment, the photoperiod was abruptly reduced to 1:Da- 6:18 and gradually lowered (by 15 minutes per day) until a light schedule of L:D I “:20 was Obtained. Egg production records were continued for an 18 day period to determine time Of cessation of egg production. The males were raised under the same lighting conditions, but were sacrificed after 16 days Of the final photOperiod. Testes weights and body weights were recorded. Presence or absence Of the pineal was determined for all Operated birds at the end Of the experimental period. sgj.‘.1n.nt”'u Sixteen (16) male and 16 female quail were moved from the battery broader to individual cages at 36 days of age. These were all unoperated birds. Starting at 37 days of age, prior to the puberal phase Of gonadal development, half of each sex were injected daily with 0.2 milliliters Of carrier solution (chloroform in sesame 011) containing 500 micrograms of melatonin. The control birds were shamwinjected with an equal volume of the carrier only. The quail were sacrificed after 1 week of injections. The body, pituitary, thyroid, adrenal, spleen, bursa, testes, ovary and oviducts were weighed, following the procedure outlined for Experiment 1. Experimeht.5 Twentwaour (2“) male quail were randomly divided into three groups at 6 weeks of age. They were individually caged in the test chamber with a light schedule of L:D - 1&310. Daily melatonin injections were given subcutaneously at levels Of 0 (control), 100 and 500 micrograms, in 0.2 milliliters of carrier (aqueous ethanol) for 7 days. The birds were sacri- ficed on the 8th day (age - 50 days, during the puberal phase of gonadal growth), and body, pituitary, thyroid, adrenal, spleen, bursa and testes weights were recorded as in Experiment 1. Experiment 6 Twelve (12) male and 12 female quail were placed in individual cages at 5 weeks Of age. Their light schedule remained at L:D a 16:8. Both sexes were randomly divided into three groups which were given daily injections of 0 (control), 10 and 50 micrograms Of melatonin in 0.1 milliliters Of carrier solution. The males were injected for 2 weeks, covering both prepuberal and early puberal phases Of gonadal growth, and sacrificed the day following the last injection (at 50 days Of age). Body, pituitary, thyroid, adrenal, spleen, bursa and testes weights were recorded. The inn jections were continued in the females until all birds had reached sexual maturity. The age at first egg was recorded. §3periment 7 Six grOUps of young mature (in egg production) female Coturnix quail were housed in individual cages. Their photo- period remained at L:D - 16:8. Daily injections were given to provide dosages of 0 (control), 50, 100, 500, 1000 and 2000 micrograms of melatonin. The levels between 0 and 500 micrograms daily were given in 0.2 milliliters of carrier solution. The two higher doses (1000 and 2000 micrograms daily) were given in a volume Of 0.“ milliliters, also in a 5 percent ethanol carrier. Individual egg production records were kept from h days before the start of injections to the end of the test period. The injections were given for 12 days, except for the 50 and 1000 microgram levels, which were continued for 2“ days. E. Statistical Treatment Organ weights were statistically analyzed on a whole organ weight basis and on a milligrams per 100 grams Of body weight basis. The F-test was used to detect significant differences between standard deviations. When such dif- ferences were found, the approximate t-test was used. All other data were analyzed by the two sample t-test. Standard errors were computed from the pooled standard deviation (3) except in cases where the F-test was significant in which case the error was computed from the individual standard deviations (sx and sy) (Gill, 1966: Adler and Roessler, 1958). 60 All tables of results were constructed on a similar basis. The number Of quail (n) is listed for each group. Any exceptions in the number Of Observations, occurring as a result Of the loss or incomplete removal of body organs, are noted in the footnotes. The values listed for each measured parameter give the mean value I the standard error of the mean. Body organ weights were calculated on both the whole weight basis and on the adjusted weight (milligrams per 100 grams Of body weight) basis. The symbols used to indicate statistical results are: + = Significant difference between standard deviations Of experimental and control results; P‘( 0.10 (10% level). if Significant difference between standard deviations Of experimental and control results; P<( 0.05 (5% level). ' iii = Significant difference between standard deviations Of experimental and control results; P <:0.02 (22% level). * = Significant difference between means Of experimental and control results; P < 0.05 (5% level). ** = Significant difference between means Of experimental and control results; P < 0.01 (1% level). Significant differences between standard deviations are indicated, since the statistical treatment of these Obser- vations differed as described above. Except in the case 61 where significant differences between standard deviations were found, the standard errors were calculated from the pooled standard deviations. This statistical procedure gives a more accurate estimate of the expected range, but eliminates its value as an indicator of the variation in the actual data being tested. RESULTS ,Among all quail on which pinealectomy was performed, about 70 percent survived for more than #8 hours. The 30 percent mortality was divided into the following causes: (1) Five (5) percent due to an overdose Of ether, (2) Ten (10) percent due to excessive blood loss and (3) fifteen (15) percent sacrificed due to suspected brain damage or incomplete removal of the pineal gland. In the three pinealectomy experiments there were 137 pinealectomized“ quail which survived to the planned autOpsy age. Macro- sc0pic and microscOpic examinations showed complete removal of the pineal in 97 Of these birds. The remaining ho quail had tissue varying in appearance from a small, atypical to a definite pineal structure. Experiment 1 ,The body and organ weights of the U-week Old quail in Experiment 1 are summarized in Table 1. These birds had been moved abruptly from L:D = 16:8 (light period : dark period = 16 hours : 8 hours, daily) to L:D = 2:22 photo- periods. A general unthrifty appearance Of the birds indicated a partial failure to adapt to this new light cycle. Two females (one pinealectomized and one control) appeared Obviously weak and emaciated and were eliminated from the experiment. The only statistically significant difference Observed was between adrenal weights of the male pinealecto- mized and control groups at the 5 percent level. 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"Amev mucmwoz cameo 0.0:: is t 3.3 :.m h 2.3 m~.m « 2.3 9.: n. 8.3 153. 23.: tom 2 s m m 3 :2... .6 6: .Onucou u0~HEOu00flmmcma .Ocucou n0mfl60u00_00cma uc0suooth no.050m no.0: xom 4.5.2.0 nuance: a" 2303 505 55300—033 >4 “03:03.2; no 0mm *0 3.003 m an 0.0.050 arm: «Nam u 9.. tops: pomso; :30 xmchsuou 30 0.0031: me 3:30: cameo use 2303 recon.— 39E. 2.. 6h observation was significant only on the adjusted weight basis, which showed the control quail to have larger adrenals (9'38.E 1.10 vs. 5.86 i 0.28 milligrams per 100 grams of body weight). Tables 2, 3 and b summarize the body and organ weights of 5, 6 and 7 week old quail, respectively. The quail seem F to be in much better condition than those at h weeks of age, as evidenced by appearance and body weights. The only sig- . nificantly different whole organ weights found were the i pituitaries of the 7-week old males (5 percent level). The pinealectomized quail had lower pituitary weights. 4 The reproductive organs showed little, if any, develOp- ment in any of the groups tested. In several cases, the F-test showed a significant difference in standard deviations between the weights of pinealectomized groups and those of the controls. However, there was no consistent trend seen in the weights of the testes, ovary or oviduct. Emperiment 2 Coturnix quail in EXperiment 2 were raised under L:D = 16:8 light cycles from hatching to the end of the experiment. The results are summarized on Tables 5, 6 and 7 for quail at 5, 6 and 7 weeks of age, respectively. Table 6 contains data for males only, as the females were kept to determine age of sexual maturity. file __ u c N . nu C — .o_.o v m .mcomumm>ou uhmucmum coozuon oucogo*wmv ucquchmmm + 86 «3.2 :m; «8.2 u: u..- 3:35 8.~ Mmmén m5: a 8.3 .3: .3: >33 .3: In: «93.. 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I: uoavm>o W 8.3.133. «3.2.338... --- 5 tea --- R- 36248.33 m..~.~« 8.33 «82; Rd « 6.9... om... «flim .3...«mo.$ ~52 «3.3 99:5 36 «ifi $.25 «8.8 m5:«2.$ rm“... «3.3 :8...“ 5.. «mmé. o... «and 3.. «8.2 8.. «8.: 22.9.2 2.. «fl.» R. «an; a“. «i... m... «8.0 «22%: 2... «m... Bo... «2.. £6.19. 2... «mm; 53.3... om.~ M .m.o~. o_.: H Nm.ou_ "A E 3; mo: cm .5 0.95 .Ké Mega S.~ «85m .8. 2.32. 3.8 w J m AcV —mm:c.wo .02 _o.. :8 non 2.300. .0: E .9. econ. ton meson—mo: Z 2253.... 3—25..— m0. at . . xum ‘I‘ ‘11 4t [Toto .8223: an .2903 c025 >Eouu£aoca >3 tacos—v.5 on 3—93 ”Em: m. m. I o. ._ .525 tonnes .22... x2253 Bo xoo3un mo 3:303 .590 new 3303 zuOLuK 32h 71 At 5 weeks of age, the pinealectomized females Weighed 31enificantly less than their control group (5% level). The males were not significantly different at this age, but the pinealectomized males were slightly heavier. The 6e_ week old control males slightly outweighed their counter- parts (nonsignificant). By 7 weeks of age, in both sexes, the Operated and control birds showed no difference in body weight 0 The pituitary glands and spleen showed no significant or consistent differences in any of the age groups, At 5 weeks of age the thyroids of both sexes were larger in the pinealectomized groups, but the difference was not sig- nificant. The 6-week old pinealectomized males had non- significantly lower thyroid weights. The adrenals of pinealectomized males at 7 weeks of age were nonsignificantly larger. The other groups had no significant differences and showed no consistent trend. While the bursa weights were not significantly different, there appears to be a trend toward slightly larger bursas in the pinealectomized birds. The testes weights of male pinealectomized quail were not significantly different from those of their controls at 5, 6 or 7 weeks of age° There was, however, a difference between the standard deviations of the experimental and control groups at 5 weeks of age. The original data show a range in testes weights of “69.0 to 1082.0 milligrams in 72 the pinealectomized birds versus 89.2 to 1309.8 milligrams in the controls. Six of the individual control obser- vations were less than the minimum experimental observations and three control observations were greater than the maximum experimental observation. The individual testicular weights and the standard deviations of the 6- and 7-week old groups were much less variable. The mean ovarian weights of pinealectomized and intact control quail were almost equal at 5 weeks of age (66.53 vs. 70.6? milligrams). A significant difference between the standard deviations (P < 0.02) resulted from the wide range of individual control observations (32.5 to 133.0 milligrams). At 7 weeks of age the mean ovary weight of the pinealect- omized group was significantly higher than that of the control group at the 5 percent level (5025 vs. 1651 milligrams). A part of these quail henswas sexually mature, causing an extremely wide range in individual observations within a treatment group. Individual ovary weights in the pinealectomized group varied from 1800 to 7500 milligrams, while the control group varied from 119.6 to 5100 mil— ligrams. Only two of the 8 control birds had ovarian weights as high as 1800 milligrams, the lowest weight in the experimental group. No significant differences were found in the mean oviduct weights at 5 or 7 weeks, although the latter age had differences approaching borderline significance. At 5 weeks of age the mean oviduct weight of the pinealect- omized.quail was appreciably lower than that of the control group (72-77 V5. 236.1h milligrams), but extreme variation in the individual data (26.25 to 107.2 and 12.3 to 915.6 milligrams, respectively, prevented statistical differences. The 7-week mean oviduct weights gave contradictory results. The pinealectomized quail had the larger oviducts (#950 vs. 3121 milligrams average). Although their individual variation was reduced (ranges of 3500 to 6700 compared to 300 to 6500 milligrams for the controls) the difference between means was not significant. Seven pinealectomized and 10 control female quail made up the groups being tested to determine the effect of pinealectomy on age at sexual maturity. The average age of sexual maturity in the pinealectomized quail was 52.1 days, with a range from #8 to 57 days. The control mean age at sexual maturity was “8.9 days and the range was from #3 to 58 days of age. The difference between these means was not significant. Experiment 3 The test groups in Experiment 3 consisted of 6 pinealectomized males, 12 control males, 6 pinealectomized females and 8 control females. At the end of the stimula- tory light treatment (L:D = 10:10) period, all of the quail appeared to be sexually mature as evidenced by the presence of an active cloacal gland in the males and by egg production 74 in the females.1 No significant difference was found in the day of first egg for the females. The average time for the beginning of egg production was 18.17 days after being given the stimulatory photoperiod in pinealectomized birds, compared to 18.75 days for the controls. The respective ranges of individual observations were 16 to 20 and 13 to 22 days. A After 28 days of stimulatory light treatment, an in- hibitory light cycle was imposed. The males were sacrificed after 16 days of the inhibitory light treatment. There was no significant difference in the mean body weights of pinealectomized and control males (117.73 vs. 115.63 grams, respectively). However, the heavier mean weight of the testes of pinealectomized males approached borderline sig- nificance. The mean weight of testes in pinealectomized birds was 868.30 milligrams compared to 616.98 milligrams in the controls. 'One individual in the control group had relatively large testes (131%.2 milligrams) which probably kept the difference in means from being significant. Seven of the 12 control males had testes weights falling below 1Externally, the cloacal gland appears as a swelling dorsal and posterior to the cloaca of sexually mature Coturnix males. It produces a foamy substance of unknown function, which is excreted with the feces. Wolfson (1952) suggests that activity of this structure is a dependable indicator of sexual function. . 2‘ .7. ..-—.—__-—--I:. I.” __ w'f”_wgmo‘._£j .. ‘ , 75 that_of the smallest weight in the pinealectomized group. The cloacal gland appeared to be inactive in the pineal- ectomized and control males. The females remained under the inhibitory photoperiod for 21 days and egg production records were kept to determine the time of cessation of egg production. The mean number of days required to inhibit egg production under these conditions was 13.0 days for pinealectomized birds (range of 11 to 17 days) and 12.62 days for the unoperated controls (range of 10 to 16 days). The difference was not significant. The mean number of eggs produced per bird was alSo not significantly dif- ferent between the pinealectomized and control quail (20.17 vs. 19.13 eggs, respectively). Eight male and 8 female Coturnix quail were given 500 micrograms of melatonin daily from 37 to #3 days of age, in 0.2 milliliters of a chloroform and sesame oil carrier. Similar groups were given the carrier alone as a shamminjected control. Two of the melatonin-injected females died early in the experiment, leaving 6 birds in that group. Table 8 summarizes the results of Experiment b. The difference between mean body weights in the melatoninmtreated and control groups of males was insig- nificant. In the females, body weights of the melatoninu injected group were lower than those of the controls at the 1 percent level of significance. 0f the endocrine organs, Evmmfimnr\ I! u ‘. __, . ." fl J ..e.o v_a .eeeoee...e eeee.m.em.m ee .me.o v_. .ooeoto...e ecee...em.m a .No.o v.a .mcomuow>op paupcoum cooxpon oocouommmp accommmcmmm +++ .mo.o v_m .ncomuo.>op paupcoua coozpon ooceuowmmp acmomwmcmmm ++ _ .o..o v_a .acomus.>op puopceum coosuon oocouommmp peoomm.cmmm _ h u c . 3&3 a $33.... £39m « 2.2 a-.. an... .332. 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S... a 8.8 a... m 9.3 Res. 2...... 2.8 m - o w m Agv .mssu mo .02 a>.co Lomaueov cm:0uo_ox a>pco somaaouv echuepo: escapmouk .Ouucou as com _0uucoo . an cam xom nopofium mO—Qz mcomuuomc. succuo.oe M ‘ - 6...; pastes» « Emmet zoos. one .msp >3 pauses—me. as .mosu chusuou w we gov +3 3 R 30..» o mugmmoz cameo was 23.: 2.8-... 39... 77 Pituitary, thyroids and adrenals, the only difference approaching significance was observed when comparing the mean pituitary weights in females. The experimental group had lower pituitary weights than the controls. When the mean pituitary weights are adjusted to the body weight, the difference was reduced. The mean bursa weights were only slightly smaller in the experimental males and females, compared to the respective sham-injected controls. The average spleen weights of females treated with melatonin were nonsignificantly larger than those of the controls. When adjusted to body weights (milligrams per 100 grams of body weight) the difference was significant at the 5 percent level. The testes of the melatonin-injected birds were lighter in weight, averaging h65.83 milligrams compared with 838.36 milligrams in the controls; however, the wide range of individual weights in both groups (28.0 to 921.0 milligrams in melatoninutreated birds and 19.5 to 1771.8 milligrams in controls) eliminated any statistical sign nificance. Similar results were found for both ovary and oviduct weights. The mean ovary weights for quail treated with melatonin and the controls were 56.99 vs. 1690.17, respectively, with ranges of 39.5 to 8h.5 milligrams and 02.“ to 6700 milligrams. The average oviduct weights were 26.73 (melatonin-injected) and 1889.6“ (control). A large range in the individual results (16.0 to 48.9 and 18.25 to 6000 milligrams, reapectively), again prevented statistical significance. Experiment 5 Levels of 0, 100 and 500 micrograms of melatonin were injected in 0.2 milliliters of aqueous ethanol solution daily, from #2 to U9 days of age. The results are sum- marized in Table 9. Body weights were not significantly affected. The birds receiving 500 micrograms of melatonin had larger adrenals which were significantly different from the controls at the 1 percent level. The birds on the 100 microgram melatonin dosage level had significantly smaller bursae (5 percent level). fifiii‘fignl’é ' Four male quail in each of three groups were givenw 0.1 milliliter injections containing 0, 10 and 50 micro- grams of melatonin in aqueous ethanol carrier daily from 35 to h9 days of age." The results are summarized in Table 10. Body weights and organ weights except for the spleen showed no significant differences. The spleen of the group receiving the 10 microgram dosage was larger than their controls (significant at the 5 percent level). The testes weights were slightly reduced at the 50 micro= gram dosage level. EXperiment 7 Twelve daily injections of 0, 100, 500 or 2000 micrograms of melatonin in aqueous ethanol solution did not cause sexually mature Coturnix hens to cease laying eggs. No difference was seen in the number of eggs 2.3.9 Olen- .3... v .. .338. .8... agent... 323.2%; 3. .mo... v .. .338. S... 28...... 3.53:3... .. « mafia: .845 a 8.32 $33 a £52 .3»... mad. a. 3.3 3m.m a. 36m . 5.2 a" «Qua 09:5 :3... n... S. 3 8... a 8.2 a"... a 3.3 =8...” .33... « mm... .. a... « as. i... u." 35 £2.22 9.... a m... «.8... a 8.. 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