.33 """" THE Ei*¥"‘§iC?S OF CONTiNUOUS LIGHT GR DARKNESS ON TH‘fRGEil AND GONAD FUNCTEON éfi‘ RATS AND MICE T213935; fer five Degree aé M. S. {w‘aiCE‘lEGAN STATE COLLEGE Guifiermo 05cm Funfiéam 1950 \ r . .. ' ' I , II\\ | \ ”5 ' . t . I .- ,. f ? I 1 I .-_' '1 l , - s . l ' I. _ I. ) e. , . , ,, a z ) :3. ‘ ‘ \ V ’ . 1 ’ .' t - 4 $ 1 ' I. ' , " I ~ ‘ , ‘ ~ ', a ' t f 3 ’ \ - e _ I . ‘k ‘ r F M ) ‘ ’ ' ‘ ‘ ~ f . ‘ ‘ ‘ ‘ l \ ‘ f ‘ Ir ‘ ' n c ' ' V ~ ‘ ' . g ’ ‘ L. a 1 I This is to certify that the Za/W/j ' ”53/234 55.23:; 2mm has been accepted towards fulfillment of the requirements fidegree in f / Date / I OZ l . ;, 7 _-.__ -- - 'fl ' __ . . ' 1 t '3. s _, f v ) 0 fl I . \ , , \W i I . ‘ \ — y 4| "5 ~ g \‘ .. ‘ \ I A. I v '. l ‘ ’ J I .‘ . l \ K 1‘ ( V . :‘ - I \. \ \ | I // _ , .- \‘ ‘ ' “ 1 7:: a \. e ‘ 'f x Y \ '\‘ f" 1 I " . l - A---._—__ l _.-..- —-‘_ . . ' , . .‘ I v f. . , ' . , , . - .. ‘ - -- . t ' . ‘ , . " ~. - --I' . . 'u ' . .' . | ' ‘ ' ‘ ’ '. . l- . .. l . . 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I. > - ': _ - uh“ - ~. . .~ 3- —:x»: . #7 v"fik 2»~$¢xfite*z - -; aw.s..~ a; - ~ laufia!” ‘ ~ ’.. ‘ t [‘7' ' _{ .v .. ' 4 - .' ""1: V. , - “f. _. . .. 1‘ L- ->.“* h . V '3 "2 .‘ _ _ “ "a” . . _ ‘ "~ I. _ ' . THE EFFECTS OF CONTINUOUS LIGHT m DARKNESS ON THYROID AND GONAD FUNCTION IN RATS AND MICE By GUILIERNO SCAR EQQJTRIANO A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER (F SCIENCE Department of Physiology and Pharmacology Year 1950 {THESlS TABLE CF CONTENTS IrmmUCTION OOOOOOOOOOOOOOOOOOOOOOOO0.00.00000000000000000. REVIEN OF. TEE LITERATIJREOOOOOQQQQOQQoooooooooooooooooooooooo Effects of Light on Thyroid Function ................. Effects of Light on the Sexual Activity of Birds...... \oqeet-a Effects of Light on the Sexual Activity of Mammalian” Evidences That Not All Birds and Mammals are Influenced by Light 01131133800000.ooooooooooooooooooooo 15 Functional Inter-relationships Between Thyroid, Gomds am Pituitm GJ-aIIdOO0.0000IOOOOOOOOOOOOOOOOOOO 16 mmmm OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0000... 18 Effects of Continuous Light or Darkness on Thyroid Flmction in MCQOOOOOOCOO...OOOOOOOOOOOOOOOO...0...... 18 Effects of Continuous Light or Darkness on Gonadal Reaction to Pregnant Mares' Sermn.-............ 20 in Rats REUL‘IS OOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOO0.0.0.000... 24 Effects of Continuous Light or Darkness on Tllyr01d Metion 0.0000COOOOOOOOOOOOOOOOO0.0.0000.00.. 24 Female Mice - Thiouracil Action .............. 24 Female Mice - Uptake of Radioactive Iodine . . . 24 Male Mice -Thiouracil Action ......:......... 32 Male Mice - Uptake of Radioactive Iodine . . . . . . 32 Effects of Continuous Light or Darkness on Gonadogen ActionOOOOOOOO.COOCO0.0000000000000000000000 38 Female Rockland Rats ......................... 38 Female Carworth Rats ......................... 38 DISCUSSION 48 SUMMARY..................................................... 53 BmLImMPIH.0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 56 no: no 7:. R ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Dr. J. Meites, Assistant Professor of Physiology, for his valuable assistance throughout the course of this work. To his generous contributions of time, energy and advice are largely due any merits this work may possess. Also to Dr. B. V. Alfred- son, Head of the Department of Physiology and Pharmacolog, for providing the facilities of the department. Thanks are also due to Drs. E. P. Reineke and L. Wolterink for their valuable suggest- ions. Finally, the author wishes to express his appreciation to Mr. John Monroe, whose careful and patient assistance in handling the animals involved contributed to the success of this research. INTRCDUCTION It has long been established that there are numerous variations in cyclic sexual activity among different species assoc- iated with environmental changes in temperature, light, food, humid- ity, altitude, rainfall, etc. The. cyclic initiation of sexual activity in some species seems to depend primarily on lengthening or shortening of days, while others are indifferent to the amount of light and re- spond to such factors as changes in temperature. Presumably these environmental factors stimulate sexual function through nervous pathways to the anterior pituitary, which is induced to increase its secretion of gonadotropic hormones and thereby initiate gonadal function. Sufficient data are available to indicate that the thyroid gland, as well as the gonads, exerts important influences on reproduct- ive function. This gland, as well as the gonads, is controlled by the anterior pituitary, and has also been shown to be influenced by envir- onmental factors, particularly changes in temperature. Inasmuch as only fragmentary and inconclusive data are available on the effects of light on thyroid function, it was the principal objective of this research to determine whether or not any conclusive relationship could be demon- strated. This was done by placing mice under continuous light or darkness for a period of twenty-eight days, and testing their level of thyroid activity by administering a constant amount of thiouracil or a tracer dose of radioactive iodine. In addition to the above, the effects of a continuous twentyheight-day'period of light or darkness on the response of the gonads to administration of a constant dose of gonadotropic hormone (pregnant mares' serum) were also studied. Although num- erous studies have already been made on the influence of light increments on normal gonadal function in birds and mammals, only one report has been found dealing with the effects of light on gon- adal reaction to administered gonadotropic hormones. Inasmuch as the gonadotropic hormone of pregnant mares' serum has received wide use in animal practice, it was considered especially pertinent to determine whether light or darkness affected its action. The data obtained in these studies indicate that changes in light increment definitely influence the thyroid secret- ion rate of mice, and also alter the gonadal response of rats to pregnant mares' serum. Inasmuch as alterations in thyroid activity have previously been shown to influence the reaction of the gonads of rats and mice to pregnant mares' serum.(Meites and Chandrashaker, 19493 Johnson, 1949), the data reported here suggest that the gon- adal reaction to pregnant mares! serum.may be similarly affected via light-induced changes in thyroid function. These data raise two interesting questions, namely: (1) can the reports of alterations in thyroid function which have been attributed in the past solely to temperature changes also be due, in part at least, to changes in light increment? (2) to what extent may light-induced changes in thyroid function have accounted for results previously attributed only to temperature or light- induced changes in pituitary gonadotropic function? - 4 - OF THE LITERATURE m 22 Light 29. 22115229 W The possible effects of light on the thyroid gland may be important in explaining reproductive behaviour because of the intimate relation of this gland to gonadal function. However, only meager information is available in the literature on this subject. Dempsey'(l943) described the histological changes of the thyroid glands of six female rats with severed pituitary stalks and six intact female rats which.were kept under continuous light for one month. The thyroids showed a reduction in cell height, from which he assumed a reduction in secretion of thyrotrOpic hormone by the anterior pituitary. He found the same histological characteristics in the thyroids of six female rats which were exposed to heat for one month. Exposure to cold resulted in increased thyroid cell height. Kleinpeter and Mixner (1947) determined the effects of the quantity and quality'(wave length) of light on the thyroid activity of baby chicks during fourteensday periods of illumination. They showed that increased quantities of light slightly increased thyroid function 'when compared to controls kept under normal light conditions. No re- lationship between-the quality of light and thyroid function could be demonstrated. Reineke and Turner (1945) worked on the seasonal rhythm in the thyroid hormone secretion rate of the chick, and reported that in both female and male chicks thyroid secretion reached a maximum.level in the fall (OctOber and November) and declined thereafter during February and early March. During the latter part of March, thyroid secretion de— clined flu'ther in females and males, and remained at a low level until August; during October the thyroid secretion rose again towards the normal winter levels observed during the previous year. It seems possible that the thyroid secretion level my also have been influenced by seasonal variations in day-light. If so, it would be logical to assume that increasing quantities of light decrease thyroid secretion in chicks. Turner (1948) reported that the thyroid secretion rate of White Leghorn hens when two years old was decreased when compared with either the same breed or White Plymouth Rocks at six months of age. He also found that there was no seasonal decline in thyroid secretion rate between January and March. By May the secretion of thyrondne had declined as did also egg production. This decline in thyroid secretion rate during warmer weather may again be correlated with increasing day- light as well as increasing temperature. Stein and Carpenter (1943) reported that the adult green Tritm'us viridescens (salamander) exposed to normal daylight for forty days during September and October showed an increase in thyroid activity. The glands had more colloid droplets and vacuoles, higher and more vacu- olated cells, and more oval basal nuclei than those of animals kept in darkness. Measurement of cell heights corroborated the histological data and differences were significant whether based on the number of cells measured or on the number of animls observed. Exposure of _Tr_i_t— urus viridescens to artificial illmination for 150 days resulted in a greater degree of stimulation. Controls kept in darkness had less active thyroids. Low temperature also had a stimulating effect as great or greater than that of light. It was concluded that light probably plays a role in the annual thyroid cycle in Triturus viridescens as well as temperature. It will be seen in the experiments re- ported here that the thyroids of mice react just opposite to those of the salamanders when exposed to continuous light or darkness. Berliner and Warbritton (1937) observed poor fertility in rams during the summer months and suggested that it was due to a decrease in thyroid secretion rate as a result of high summer temper- atures. Bogart and Mayer (1946) also reported that high temperatures (85° - 90° F.) in the summer reduced activity of the reproductive organs of rams by inducing hypothyroidism. They found that when thyh roprotein or thyroxine were given to rams during the period of high temperatures the reproductive organs and sexual activities were re- stored to a level near that of the breeding season (fall and winter). It seems possible that the hypothyroidism found in the above rams during summer may also have been induced in part by the greater quan- tities of light present at that time. This would seem to be substan- tiated by the findings of Yeates (1949) that breeding activity in sheep can be controlled at will by artificially altering light duration. This worker induced anestrus in sheep during the breeding season (winter) by putting them.under increasing periods of artificial light, and induced estrus and breeding activity during the nonpbreeding season (summer) by artificially decreasing the daily light periods. The effects of light duration on breeding activities in this species may' be more important than temperature changes. Effects 2;: Light 93 3113 §e_:g___ual Activity 2; §_i__rd_§. Since the times of Pliny and Aristotle the cyclic changes in the size of the avian gonads have been recognized to be correlated with the seasons of the year. In studies of migratory species many workers have advanced the theory that the migratory urge may be related to gonadal changes. Some of the most widely quoted investigatory efforts in this connection have been those of William Rowan (1929) whose original experiments dealt chiefly with the Junco. This worker observed that the interstitial tissue of the testis and ovary canmences its spring recrudescence prior to the initiation of migratory movement, and reaches a madman approximately at the time of the northward migration to the breeding ground. At this time the interstitial tissue and geminal elements increase and reproductive activity takes place . Towards the end of the period of northern residence the gonads exhibit a second burst of interstitial activity, and this flurry of function within the secretory portion of the gonads occurs Just before and during southward migration. Rowan (1931) utilized the above observations to formulate a definite theory, holding that the secretions of the sex glands are primarily responsible for migratory behaviour. By artificially alter- ing the duration of illumination in each twenty-four-hwr period, H he was able to control the stage of gonadal develoynent in various mig- ratory species. Thus birds could be brought into either the migratory or full breeding condition during the dead of winter. Since the inter- stitial tissue in the testis was most praninent during the periods of gonadal recrudescence and regression, Rowan (1931) concluded that in -3- the case of the Junco at least, the hormones elaborated by this cellu- lar type constituted the physiologic stimulus to migration. In a discussion of factors contributing to the migratory urge in birds, Wolfson (191.0) regarded increasing daylight and consequ- ent pituitary stimulation as important events in the chain of physiolog- ical processes which are essential to the seasonal flights. Bissonette's studies (1932) of light-induced changes in the testis of the sterling led him to believe that wave length as well as amount of illumination may also be an important factor in this respect. A study of the greenfinch by van Oordt and Date (1939) has generally confirmed the original observations of Rowan (1931) and Bissonette (1932). Birds placed in the dark at the beginning of May, when they were in full song, were killed at varying intervals. It was found that while in the dark both the testes and the ovaries decreased considerably in size and spermatogenesis came to an end. When birds were brought into the light in August, after being in the dark, their gonads increased, spermtogenesis was .re-initiated and the birds began to sing. It was also found that putting finches in the dark caused them to moult in June instead of at their usual time in August. Cole (1933), working on captive mourning doves, found that reproductive activity could be induced by increasing the duration of the daily light period. Egg laying and gonadal activity can be stimulated in ring-necked pheasants by constant illumination, according to Clark, Leonard and Bump (1939). These workers found that an increase in total illmnination produces an increase in the number eggs laid in this spec- ies. Riley and ‘11wa (1938) reported that ovarian developnent may be -9- stimulated in female sparrows during the fall and winter by increasing the light ration. Benoit (1936, 1937) made the significant mum in ducks that removal of the eyeballs and severance of the Optic nerves did not inhibit capacity to breed when artificial light was directed through a fine glass tube into the eye socket or on the pituitary. Ringoen and Kirschbaum (1939) reported that there was no gonadal re- sponse to light in sparrows if the eyes were covered. Lamoreux (1943) found that white Leghorn males exposed to twelve hours or more of light daily made significantly greater gain in semen yield than males exposed to light less than one hour. Effects _o_§ Light 93 the m Activity g_f Mammals. The first corroboration in mnmels of Rowan's (1929) work in birds was nude by Bissonette (1932) and Baker and Ransom (1932) in voles and ferrets. Since then a large amount of work on the effect of light has been done on many species of vertebrates, and this has been sulmnarized in papers by Bissonette (1936), Marshall (1936) and Rowan (1938). ' Marshall (1940) reported that in female ferrets subjected to different degrees of light intensity, as measured by putting them at different distances from a 1000 watt bulb, the acceleration of the es- trous cycle was roughly correlated with the degree of light intensity. Morgan (1949) , worldng on the female opossum during the non-breeding season, also reported that increases in the size and weight of the re- productive tract of the opossmn appeared to be directly proportional to the amount of radiant energ received through lamps with and without -10.. filters. oh the assumption that light exerts its seasonal effect by way of the pituitary, several investigators have attempted to de- termine the path of transmission of the stimulus. Le Gros Clark (1939) studied the path of transmission of the light stinnlli in ferrets. He found that when the optic nerves were sectioned, these animals did not come in heat at all, or also came in heat much later than the normal time. He also found that the normal response to visual stimulation can occur even in the absence of the visual centers of the cortex, through impulses passing either to the ventral nucleous of the lateral genicu- late body or to the mrpothalamus by the way of the accessory optic tracts . Whitaker (194.0) reported that when white-footed mice were blinded by removal of the eyes, they were not rendered sterile but exhibited no cyclic sexual activity. Those kept in continuous darkness exhibited a reduced and entirely non-cyclical reproductive activity. Furthermore , with light of the low intensity of one foot- candle power, breeding took place throughout the short portion of the year and the animals failed to go into a state of anestrus. Even at lower temperatures, when the litter could seldom be successfully rear— ed, the mice did not go into anestrus if provided with sufficient add- itional light. Hammingsen and Krarup (1937) have shown that there is a correlation between the estrous cycle and the ordinary daylight diurnal rhythm in the rat. Sexual heat and muscular activity (as recorded by activity cages) were at their maximum in the dark. All the phenomena, (mating instincts, cyclical changes in the vagina and the correlated increases in activity) were shifted 12 hours when an artificial day- night rhythm was established by exposing the animals to light at night and to darkness in the day. in eight-hour alternating rhythm of light and darkness, however, was not followed by any change in sexual rhythm. Constant light was found to stimulate vaginal corn- ification and induce heat. Gresson (1940) reported the effect of increased daily illumination and reversed day and night conditions on the estrous cycle of the mouse. Femles were kept in darkness for seven to eight hours during the day and under bright electric illumination for the remainder of the twenty-four-hour period. Controls were kept under normal conditions. It was found that ulong day“ conditions accelera- ted estrus and induced copulation in mid-winter. Reversed day and night conditions brought about daytime mating. Meyer and Meyer (1944,) stated that cotton rats which were raised in constant darkness displayed retarded developnent of the re- productive tract and delayed attailment of sexual maturity, but that constant lighting had no perceptible effect on normal development. . In apparent contradiction to the findings of most of the foregoing workers is a report by Chase (1941), who studied reproduc- tive activity in a strain of mice displaying congenital anopthalmia. It was found that females fran this eyeless strain showed vaginal in- troitus somewhat earlier than normal mice, and exhibited the first vaginal cornificaticn considerably earlier. The length of the cycle was said to have been unaffected. Anopthalmic mles matured and showed spermtogenesis at the same age as males from a normal strain. It is impossible to compare these results directly with those of the other -12.. workers who stuiied only normal animals, since it is obvious that the genetic factors responsible for congenital eyelessness may well be accompanied by other hereditary abnormalities effe cting the reproduc- tive cycle. Ievinson, welsh and Abramowitz (1941) observed that hypo- physectomized female rats displayed a marked decrease in total activity and a complete loss of activity rhythm normally associated with the sex cycle. However, females from which the pituitary had been removed con- tinued to display a diurnal rhythm of activity in which running in- creased at night and decreased during the daylight hours. This rhythm was reversed when the light-dark periods were inverted by the use of artificial lighting. Since mophysectonw results in regression of the gonads, adrenals and thyroid, it becomes apparent that diurnal activity (exercise) is independent or secretion fran any of these glands as well as fral those of the anterior pituitary itself. Apparently the normal pattern of running activity in the female rat consists of a diurnal cycle, the phasic nature of which is independent of any hormonal action. However, any quantitative increase in general activity probably depends upon secretions from the endocrine glands. Fiske (1939) observed that female rats kept on constant light showed long periods of estrus and diestrus, whereas in rats kept in constant darkness the metaestrous phase was the most protracted por- tion of the sex cycle. Bioassay revealed that pituitaries of constant- light animals contained high amounts of follicle-stimulating hormone, and those in constant-darkness were characterized by high quantities of luteinizing hormone. Adult males kept in constant darkness had larger pituitaries and more highly developed testes and seminal vesicles than -13.. did males in constant light. Female rats raised under constant light- ing reached sexual maturity earlier than did females in normal light- ing conditions, whereas animals maintained in constant darkness were the last to attain maturity. In a later paper, Fiske (1941) showed that injections of the gonadotrOpic hormone of pregnancy urine into male and female rats kept under continuous light for fifteen to twenty days gave better ovarian and seminal vesicle development than in animals living in dark- ness. Injections of pituitary follicle stimulating hormone (FSH) into inmature females kept under continuous light for fifteen to twenty days gave less ovarian growth than in females kept in darkness. Male rats similarly injected with FSH had heavier seminal vesicle when kept in the light than in the dark. These are the only data which the writer has found dealing with the effects of light on the reaction of the go- nads to administered gonadotropins. They are difficult to interpret, however, because of the apparent contradiction in the results. Truscott (1941.) reported that the attainment of sexual maturity in rats is accelerated by constant lighting. Furthermore, he found that when the optic nerve was severed, maturation was delayed beyond the normal period despite constant lighting conditions. Pomerat (191.2) claimed that the. ‘pituitaries of rats kapt in continuous darkness for one and one-half months resembled those of young castrated females. The acidophils were increased, the basophils were doubled in nmnber, and degranulated basophils and castrate cells were present. Maw of these changes persisted after three, six and twelve months of darkness but were not as pronounced. He also found that the ovaries of rats kept under continuous light for one and one- 5'14- half, three or twelve months were consistently smaller than those in control rats of corresponding age, and contained fewer corpora lutea. The ovaries of rats kept in continuous darkness showed an even greater reduction in size and in the number of corpora lutea, especially after one and one-half months. Rice (1942) and Hammond (1938) have summarized the ob- servations pertaining to the stimulating effects of light upon gonadal function in domestic animals. The breeding season in the mare occurs during the spring and summer when the amount of light per twenty-four hours is increasing. Transfer 0f mares from the northern to the south- ern hemisphere results in a change of the breeding period to fit the new seasons. Domestic mammals such as sheep, deer and goats breed during short or shortening days. Bissonette (1941) has observed that although goats usually show their last heat period not later than middle of March, fertile mating may be induced in July if the day length is art- ificially shortened during the preceding two months. Comparable out- of-season sexual behaviour and reproduction has been produced in sheep by Sykes and Cole (1944), and Yeates (1949) as a result of experimental modification of the amount of light per twentybfour hours. Yeates (1949), working with grade sheep and Suffolk ewes, found that the natural sexual season (which embraces the autumn and winter'months) may be modified or even reversed by suitable alteration of the daily- light ration. In grade Suffolk ewes he noted that the onset of the sexual season was a response to a decreasing daily amount of light which occurred thirteen to sixteen weeks after the change from increas- ing to decreasing length of day. These responses occurred irrespective -15.. of the level at which the changeover in trend of daily lighting occurred and were unrelated to specific "threshold" amounts of light. In the latitude of Cambridge, Massachussets, domestic cats usually breed from the middle of January until the middle of July. Dawson (1941) has elicited estrus in this species during November and December by increasing the amount of illumination for each twenty-four hours. Evidences M go_t_ Al_._1 m g m; Q Influenced .131 Light Changes. In the preceding review, examples have been cited of birds and mammals which respond to changes in light. However, there are exceptions among both. Thus sex activity in the guinea pig is little affected by changes in the amount of light (Dempsey, 1934.) and the same appears to be true of the spermophile (Johnson and Cm, 1933). Guinea pigs are tropical. 9.111sz and live under comparatively uniform daily conditions as regard light and temperature. They prob- ably do not possess the capacity to respond to those seasonal con- ditions which are the main factors in fixing the periods of breeding among animals living away from the equator. Among birds too, some species do not respond to increase in light, such as the guinea fowl (Scott and Payne, 1937) which likewise inhabits the tropics. It has been shown that some animals inhabiting tropical lands are as unvarying in their reproductive cycles as others living in temperate climates. The bats described by Baker and Bird (1936) are examples fran among maummls. Among birds, the garden whistler inhabiting the northern New Hebrides is as seasonal in its reproduct- -15... ion as are the birds of a temperate climate (Baker, 1940). There is said to be no seasonal change in diet and no other environmental changes are known to control the breeding season. The outstanding fact remains, however, that in nearly all animals showing sexual periodicity, breeding phenomena occur in response to seasonal changes and in the majority of these animals, as shown by observations under both natural and experimental conditions, the principal stimuli are changes in light duration or changes in tem- perature. The anterior pituitary is recognized as the organ activated by the light stimulus. Light impulses are received by the eye from which they are probably passed to the Impothalamus and to the pituit- ary along neural pathways . meeting; Islam-Wm 292mg lazing. tease and Pituitary _G_1_a_r_1d. Since this thesis is concerned with the effects of light on the function of the gonads and thyroid gland, it is per- tinent to review some of the functional inter-relationships between the thyroid, (gonads and pituitary gland. There is ample proof that alterations in thyroid activity may alter gonadal function. Thus Meites and Chandrashaker (1949) reported that in young male rats thy- roprotein partially or canpletely inhibited the response of the seminal vesicles and coagulating glands to a constant dose of pregnant mares' serum, while in young male mice thy-roprotein increased the gonada- tropic response. These investigators also found that thiouracil in- creased the gonadal response to pregnant mares' serum in male rats and reduced the response in male mice. Similar findings were reported by Johnson (1949) in innnatureefemale rats and mice. The response of the -17.. ovaries of immature female rats to pregnant mares' serum when given thryroxine or thyroprotein was reduced, but was increased when given thiouracil. The response of the ovaries of inmature female mice to pregnant mares' serum given thy-reprotein was increased, but there was no significant change when given thiouracil. Reineke, Bergnan and Turner (1941) reported that thyroid- ectomy of male goat kids resulted in a reduction in the gonadotropic content of the pituitary. Pan (1940) likewise found that the gonado- ‘ trepic potency of the anterior pituitary was decreased following thy- roidectomy of nomal and castrate rats and normal rabbits. Evans and Simpson (1929) reported that the gonad-stiflating properties of the anterior pituitary fran hyperthroid rats were increased, while the glands from hypothyroid rats were less effective than normal. Chu (1944) noted that in thyroidectcmized rabbits the ovaries contained many more large follicles than nomal controls, but ovulation did not take place after coitus. The animals that were Opera- ted on readily ovulated after the inJection of pregnancy urine extract, and the ruptured follicles were more numerous than in normal estrous animals. He also prepared fresh-saline extracts fran the pituiteriee of nomal and thyroidectomized rabbits and assayed their relative amounts of ovulating hormone . The pituitaries fran the normal animals induced sixty per cent ovulation in estrous rabbits, whereas the pit- uitary extracts from the thyroidectanised rabbits caused no ovulation and only induced growth of follicles. These results indicate that alterations in thyroid activity may not only affect the gonads directly, but may also change gonadotropic secretion in the anterior pituitary. -18- PROCEDURE Effects_2f Continuous Light gr Darkness 3? Thyroid Function $932.99.: These experiments were performed on male and female albino mice (Rockland strain). The animals were fed a balanced stock diet and drinking water was available at all times. In each experiment uniform groups (by weight) of ten animals each were placed in ample-sized screen cages. For those groups which were to be maintained under continuous light, a fifteen.watt elec- tric light bulb was placed about four inches in front of each cage. The cages of the animals kept under continuous darkness were com- pletely'covered, except that the covers were not tight enough to exclude air circulation. Control groups of animals were kept under the normal dayanight conditions prevailing in the animal room, or on about nine hours of light and fifteen hours of darkness daily. All experiments were conducted for twentyheight days and the animals were sacrificed on the last day; These experiments (as well as those dealing with gonad function) were all conducted in an air conditioned animal room at a constant temperature of 75°F. Several temperature checks made with a chemical thermometer in both the continuous-light and continuous- dark cages showed that there was not more than one-half degree varia- tion from room.temperature. It is believed, therefore, that tempera- ture changes can be excluded as a factor in these experiments. The procedure used in the thiouracil-treated animals is given in Table 1. A constant dose of ten mg. of thiouracil sus- pended in 0.1 normal sodium.hydroxide was injected subcutaneously _19.. TABLE 1 PRWEDURE IN STUDYING EFFECTS G" LIGHT 0R DARKNESS ON THE REACTION (r THE remains TO THIOURACIL No. per Group group Treatment I 10 Controls Normal day and night for 28 days II 10 Controls Normal day and night for 28 days Thiouracil injected during last 10 days III 10 Continuous light for 28 days Thiouracil.injected during last 10 days IV’ 10 Continuous darkness for 28 days Thiouracil injected during last lOdayB -20- daily in 0.2 cc. volume to each animal for ten days. On the day of sacrifice, body weights were recorded and the thyroids were care- fully removed and weighed on a Roller-Smith balance. The procedure used in the radioactive-iodine treated animals is given in Table 2. 0n the day prior to sacrifice each animal was injected intraperitoneally with a tracer dose of 1131 con- taining approximately 0.1 microcurie of activity. On the day of sacrifice, the thyroids were removed, weighed, air dried and then placed in small copper discs for counting under a Geiger-Muller tube. All important data were treated statistically. The standard error of the mean was determined by the following formula: €012 n(n - l) S.E. = Significant differences between means were determined by the following formula: In1 " m2 S.D. = 2 2 V131 + E2 Effects 2; Continuous Light 2;; Darkness _o_1_1 gonadal Reaction to Pregwt Mares' Serum. These experiments were conduc- ted only in rats (Rockland and Carworth strains) according to the procedure given in Table 3. These animals were kept under continu- ous light or darkness as in the previous experiments, except that - 21 - TABLE 2 PROCEDURE IN STUDYING EFFECTS OF LIGHT CR.DARKNESS ON THE UPTAKE OF RADIOACTIVE IGDINE BI'THE THYROID No. per Group Group Treatment I 10 Controls Normal day and night for 28 days Radioactive iodine injected 16 hours before sacrifice II 10 Continuous light for 28 days Radioactive iodine injected 16 hours before sacrifice III 10 Continuous darkness for 28 days Radioactive iodine injected 16 hours before sacrifice TABLE 3 PROCEDURE IN STUDYING EFFECTS (I? LIGHT 0R DARKNESS ON GWADAL REACTION T0 PREGNANT MARIS' SERUM NO. pr Group group Treatment I 10 Controls Normal day and night for 28 days II 10 Controls Normal day and night for 28 days P.M.S. injected during last 4 days III 10 Continuous light for 28 days P.M.S. injected during last 4 days IV 10 Continuous darkness for 28 days P.M.S. injected during last 4 days - 23 _ a constant dose of pregnant mares' serum.(one Cartland-Nelson unit) was injected subcutaneously into each animal during the last four days of the twentyaeight day period. On the day of sacrifice, the ovaries and uterus were dissected out and weighed. -24- RESULTS Effects of Continuous Li ht or arkness on oi ction M 11133 - Ihiouracil 49.11.1- The results of two preliminary experiments in female mice are given in Table 4 and Fig. 1. It can be seen that the thyroids of the animals which were under continuous light plus thiouracil weighed significantly less than the thy-raids of the animals kept under normal day-night lighting plus thiouracil. The next experiment (Table 5) also shows that continuous light (group IV) reduced the effectiveness of thiouracil when compared to the controls (group II). In this experiment, continuous darkness did not significantly alter the action of thiouracil (group II). A similar experiment (Table 6, Fig. 2) showed again that continuous light decreased thiouracil action (group IV) while continuous darkness sig- nificantly increased thiouracil action (group III) when compared to the controls (group II). M flag - m 9;: Radioactive M. The results of this experiment (Table 7, Fig. 3) corroborate the data obtained in the thiouracil-treated mice. It can be seen that the thyroids of the animals under continuous light (group III) weighed less, while the thy- roids of the mice under continuous darkness (group II) weighed more than the controls (group I). The uptake of radioactive iodine by the thy- roids of the continuous-light group was significantly less than by the thyroids of the control group, whether calculated on a thyroid or body weight basis. On the other hand, the thyroids of the mice which had been in continuous darkness took up a significantly greater amount of - 25 _ some no sense unsusopm © ub.m a none .388 pgamanmfim HHosHsOHmp msHm sum u 2.3 $6 u 2.8 made 8.3. seen” 353380 m H e x e \ Hfiosnsowmp $4 .. 813 8.0 .. one 8.3 8.3 33 3.580 S H e x e x . mQIN " OOQQ 188% pgamanmam . 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[NB-1 l54 ’0' '— 5.4 Con’rrols Conlrols Dink L13“ Thiouracll Thlouracil Th'iouracil L___.V_H -_r0_ ,¥__- ,W,, , _- - _" Effect of Continuous Light or Darkness on Thiouracil Action in Female Mice -30- . 14.1.3341 EFFECT or CONTINUOIB LIGHT 0R mamss ON THE UPTAKE or RADIOACTIVE 1mm: IN FEMAIE MICE Ave Av. Ave Ave No. orig. final thyroid tugoid wt. per body body wt. 100 gn. body wt. Group group Treatment wt. gm. wt. gm. mg. mg. I 0 / o I 10 Controls 23.70 28.88 2.53 - 0.11 8.76 - 0.36 ’l o 2‘ @ ous dark- ness ,1 o ,1 o III 10 Continu- 24.00 27.53 2.1!. - 0.14 7.77 - 0.25 ous light Group Radioactivity in counts per second Av. no. counts Av. no. counts Av. no. cotmts per per thyroid per mg. thyroid twoid 100 at. body wt. ~ 1‘ o ,1 o ,1 o I . 21057 " 1.72 8.74 "' 0038 75044 " 6059 ,l o ,1 o / @ II 28023 "' 2052 9089 " 0042 99076 " 6095 0 0 - 0 III 12.39 {- 1.45 6.01 f 0.30 44.21 ,5 4.15 Significant Significant Sipificant _ differences : differences. differences 3 Groups Groups Groups Infill-2.18 IanlII=2004 IandII=2.54 1 and III-4.08 I and III-'- 5.65 I and III- 4.01 0 Standard error of mean -31.. Female Mice - AV. 30‘ 1 Counts I , per 30‘ Thyrond. ’0 ‘ fl 0 AV. /2 ‘ Counts [0‘ “-1 per I * m3. of 6 ‘ . 4 . Th'rmd Z . ~ 0 Av. [30‘ Coun+s w '——‘ per 80‘ 100 gm. 50‘ M Body Wt 30 I l 0 Controls Dark Lighf Fg.2 Effect of Continuous Light or Darkness on the Uptake of Radioactive Iodine by the Thyroids of Female Mice -32.. radioactive iodine than the controls. __Ma_lg gig - Thiouracil m. Continuous light or darkness in male mice elicited the same reaction to thiouracil as in female mice. The first experiment (Table 8) was performed on an initially mture group of male mice. A younger group of male mice were used in the second experiment (Table 9, Fig. 1.). In both, the effect of continuous light (group IV in each experiment) was to sig- nificantly reduce the action of thiouracil, while continuous darkness (group III in each experiment) significantly increased the action of thiouracil when compared to the controls (group II in each experiment). Mg Q32 - £31152 9; Radioactive Lodi__n_e_. The data ob— tained in this experiment (Table 10, Fig. 5) corroborate the results . obtained with thiouracil. Continuous light produced a significant decrease in thyroid weight (group III) and continuous darkness a sig- nificant increase in thyroid weight when compared to the controls (group I). The uptake of radioactive iodine was significantly decreas- ed in the thyroids of the mice under continuous light (group III) and significantly increased in the thyroids of the mice under continuous darkness (group II), when compared to the controls (group I). In summary, the preceding experiments in female and male mice conclusively demonstrate that continuous light decreases thyroid function, as evidenced by (1) a reduced thyroid reaction to a constant dose of thiouracil (2) a decrease in thyroid weight and (3) a reduced uptake by the thyroids of a tracer dose of radioactive iodine. Con- tinuous darkness, on the other hand, definitely increases tkvroid activity, as indicated by (1) an increase in thyroid reaction to a -33- 58. 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[sz m .. \ r—-‘ \ > 5‘ l Canfrols Conjmls Dirk LIB M I Thiouracil Thiouracil Thiouracll i Fig, 4 Effect of Continuous Light or Darkness on Thiouracil Action in Male Mice -36- TABLE 10 EFFECT OF CONTINUOIB LIGHT 0R DARKNESS ON THE UPTAKE CF RADIOACTIVE ICDIEE IN MAIE MICE AV. Av. Av. Av. I No. orig. final thyroid tgygoid 1Q. per body body Ht. 100 gm. “" wt. Group poup Treatment wt. an. wt. 9n. mg. mg. 0 l e " @ II 10 Continu- 21.40 31.76 2.62 - 0.21 8.25 - 0.61 ous dark- nesa l o " @ III 9 Continu- 21.80 31.67 1.93 - 0.12 6.09 - 0.36 ous light Group Radioactivity in counts per second Av. no. counts Av. no. counts Av. no. counts per per tlvroid per mg. ttwroid tmoid 100 m. body wt. ,1 c _ o o I 22060 "’ 2097 10019 " 0046 74059 " 10048 y‘ c ,l o l o II 31003 " 300° 11.51 - 0047 117043 "‘ 12035 c - o ,l o III 13.70 " 2.26 60% - 0.78 41.77 "' 6.06 Significant Significant Significant differences: differences: differences: Groups Groups Groups I and III - 203° landfill-3.64 0 Standard error of mean ‘ Av. Coun+5 per Thyroid Av. Counts per m3. of Thyroid. Av. Coun+s per [00 gm. Body Wt -37.. Mefle Mice IZ‘ /0‘ <>N"'°‘°° IZO- [00‘ °$$$$ "—1 (“I Conirols Dark U8h+ Fig. é Effect of Continuous Li ght or Darkness on the U tak of Radioactive Iodine by the Thyroids of p 6 Male Mice -33.. constant dose of thiouracil (2) an increase in thyroid weight and (3) an increase in thyroid uptake of radioactive iodine. Effects of Continuous Light or Darkness on Gonadogenigction. Female Rockland Rats. The data given in Table 11, Fig. 6 show that continuous light for twentyheight days (group III) augmented the action of pregnant mares' serum on the ovaries and uterus of Rockland female rats when compared to the controls which received pregnant mares' serum (group II). Continuous darkness for twenty-eight days (group IV) did not change the activity of the equine gonadotropin. The results were essentially similar in a repeat exper- iment (Table 12, Fig. 7). Continuous light again augmented the effects of the gonadotropin on ovarian and uterine weights, while continuous darkness produced no change in ovarian weight. The latter condition, however, did appear to increase the weight of the uterus. - A third repeat experiment (Table 13, Fig. 8) corrobora- ted the results obtained in the previous two experiments. It can be concluded, therefore, that continuous light for twentyaeight days enhances the effects of a constant dose of gonadogen on the ovaries and uterus of Rockland female rats, whereas continuous darkness for twentyheight days appears to exert no effect on the action of gonad- ogen. Female Carworth Rats. The results of two repeat experi- ments in Carworth rats (Tables 14 and 15) indicate that neither continuous light nor continuous darkness for twentyaeight days in- fluenced the action of a constant dose of equine gonadotropin in some .«0 909.8 gunman 0 -39.. 005. u HHH 0m.~ u HHH and HH modem 05 HH museum » $053.33 u mooomnommwv pneoaadmwm #5303an 5000980 no.3 30803.0 3.04 ... $.34 mm.m ... N93. OWNS 13.8 mm.m.n.n 00.3 98.23980 m B e x e \ ammoggom 83 Ema Awméw I 40234 mm...” I 44.04 w4.mmm $.00 mwommfi 8.3 9532.3qu 0 EH 0 x 0 \ nomouugm meg. m 00.43” $.N M H93 flung” 31mm 0m.mNH 00.3 no.3 adorned—co 9” HH 0 0 No.3 I magma R...” I mimm 00.3.” Sewn 3.03.. 00.3. 3.6.550 m H e x e \ .ma .ma .3 .03 .am .5 engage 059% mocha 3.: .33 .3 8H :3 38 .8 8H :5 ..t. :3 38. ...E .38 non 3t» was»: .03 a names 50 a.“ Hug—”mane .02 $4 $4 $4 $4 $4 . $4 2.4m 58m ESE zH chao4 E8300 20 gm mo E05 MDOPZHHZOO .8 90% HH mags -40.. FemaIe Rats M. of w+, or Ovaries u+erus or > I"r IOOPBm. 3/0 I00 31:. 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