THE INFLUENCE OF VARIATIONS IN ENVIRONMENTAL TEMPERATURE AND THYROID STATUS ON GROWTH AND SEXUAL [DEVELOPMENT IN THE MALE MOUSE Thesis for fhe Dogma of M. S. MICHIGAN STATE COLLEGE M Maqwcad Butt "3949 (MES: : This is to entity that the Ilwsis entitled "The Influence of Variations in Envirnnmental Temperature and Thyroid Status on the Growth and Sexual Development of Male Mice" presented by M. Maqsood Butt has bvcn awqm‘d towards fulfillment of lIu' requirements for M , S . dq‘qrc't: inlhlflifllogy Z Major professor Halo ISarch '7, 1949 THE INFLUENCE OF VARIATIONS IN ENVIRONMENTAL TWERATURE AND THYROID STATUS ON GROWTH AND SEXUAL DEVELOPMENT IN THE MALE MOUSE BY M. figqsood Butt A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in.pertia1 fulfillment of the requirements for the degree 0! MASTER OF SCIENCE Department of Physiology and Pharmacology 1949 ' TI"! 58 I S ACKNOWLEDGEMENTS The writer takes this opportunity to record his sincere appreciation to Dr. E. P. Reineke, Professor of Physiology, Michigan State College, for his encourage- ment, assistance and scholarly guidance throughout the course of this work.and for his suggestions in the preparation.of this manuscript; to Dr. B. V. Alfredson, Head of the Physiology and Pharmacology Department, for his aid in providing facilities in conducting this work; to Drs. J. Meites and L. F. Wolterink, Department of Physiology and Pharmacology, for their interest and valuable suggestions, to Dr. R. F. Langham, Department of Pathology flar the photomicrographs and to Dr. L. B. Sholl, for providinngacilities in his Pathological laboratory for the histological studies. The writer is also grateful to the Department of Education, Government of Pakistan, fer the award of a scholarship to study in the U.S.A. 218921 ,4 CONTENTS INTRODUCTION . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . Role of Thyroid on Growth , , , Influence of Seasonal Variations on Thyroxine . Secretion Rate Relationship of the Thyroid to Reproduction . . Bovines Sheep and Goats Poultry Egg Pro duction EXPERIMENTAL PROCEDURE AND RESULTS . . . . Material and Environmental Conditions . Mice Kept at 3000 Feed and Feeding Procedure Effects on Growth.and Feed Consumption . . Hyperthyroidism Experiments I and III Experiments II and IV Hypothyroidism Environmental Tanperature Therprotein and Growth of Hair Effects on Testes and Seminal Vesicles . . Hyperthyroidism Hypothyroidism Environmental Temperature Effects of Hyperthyroidism.on Male Sex Characters Histological Studies . . . . . . Testes Spermatogenesis Seminal Vesicles Technique . . . . . . . . . 13 13 15 l9 29 29 36 CONTENTS (Continued) Thyroid and Sexual Development . . Testes Seminal Vesicles Effects of Thyroprotein on Respiration Mortality Rate . . . . . DISCUSSION . . . . . . . SUMMARY . . . . . . . BIBLIOGRAPHY . . . . .e . . 51 51 53 61 6h INTRODUCTION -The scape of Endocrine Research is exceedingly broad and is interwoven with almost every phase of biol- ogy. With the recent advancements in the field of Endoc- rinology, it has been observed that compliciated physiologic mechanisms exist which effect variations among the ductless glands in the formation and release of endocrine compounds, in accordance with the needs of the body for the substances. The functional capacity of the ductless glands is influenced directly or indirectly by the hormones elaborated by the other compounds of the endocrine system. Thus a state of balance is normally maintained between the various ductless glands and between themselves and other tissues and organs of the body. It has been observed that many of the hormones are involved in a number of physiologic processes within the body instead of being concerned only with the adjustment of one particular process in the animal body. This is especially true in the case of thyroid as it plays an essential role in the physiological processes of growth, reproduction, lactation and other functions. A deficiency of thyroid hormone or an over-supply of it may reflect it- self in demonstrable changes in many organs and functional processes of the body. The thyroid is a regulator of energy metabolism. In.mild hyperthyroidism.anabolic effects maybe obtained in growing animals of some species while in severe hyperthyroidism, catabolic processes predominate. Since many tissues are dependent upon normal thyroid activity for normal functioning, it is apparent that the thyroid mechanism is one of the most important of the homeostatic mechanisms necessary for maintaining a constant internal environment in the midst of a constantly changing external environment. After growth of the domestic animals is completed, the secretion of the thyroid gland gradually decreases and they tend to fatten. The breeding animals, both male and female may become sluggish and their reproductive organs less effective. Preliminary observations indicate that by replacement thereapy with thyroid materials, the slightly hypothyroid animal can be returned to a normal condition of metabolism. Accompanying this change, there will usually be an improvement in reproductive ability in both sexes. It appears likely that the activity cf the endoc- rine glands may be modified favourably by selective breed- ing and such modifications will also result in improved productive ability among the live-stock. As the thyroid gland appears to have some practical application in the field of live stock development, the present work was undertaken to study the influence of variations in environmental temperature and thyroid status on growth.and sexual development in young male mice. The object was to apply the knowledge gained from.work on the Lu! laboratory mouse a, f to a consideration of similar prob- lems in farm.animals under different environmental condi- tions,especially in tropical countries where the thyroid secretion rate may be assumed to vary due to wide differ- ences in seasonal temperatures, influencing thereby the various physiological phenomena of the animal body. . y...) REVIEW OF LITERATURE Role of Thyroid on Growth Von Rapp (18h0) was the first to report the effects of thyroidectomy in a ruminant. Thyroid ablation depresses growth in animals and when thyroidectomy is complete, the variation is largely due, however, to the marked effect of age on the incidence of growth retardation. In younger ruminants growth depression is.marked (Simpson, 1924; Spielman et.al. l9h5 and others) while in the older animals such a depression is not so apparent. Administration of thyroid or iodinated casein to these animals completely re- stored growth processes. Engelbach (1932) states that the thyroid is responsible for the development of centers of ossification in infantile life. The pattern of growth de- pression in hypothyroidism varies considerably in various animals. Complete suppression of thyroid activity in the young ruminants is of little economic importance. Partial suppression of thyroid action in the young animal for short periods of time immediately before marketing might be of some value, if the fat deposition in the carcass could be increased, thereby improving market quality. The thyroid stimulates the growth in young;animals and if the deficiency develops after the growth has been attained, them the skel- etal.derangements are not necessarily noticeable but.myxe- dema, along with mental derangement are soon Observed in the animal 0 A study of the literature reviewed by Koger and Turner (1943) showSthat mild hyperthyroidism in young ani- mals accelerates growth but in severe hyperthyroidism, where the basal metabolism.is considerably elevated, marked losses of weight and decreased fertility occur. In mice, thyroxine in minute doses increases growth rate fairly con- sistently (Robertson, 1928; Keger et al., 19h3; Koger and Turner, l9h3 and others). Reineke and MdMillen (l9h6) reported a significant increase in the rate of gain and a slight increase in efficiency of food utilization by feed- ing small doses of thyroprotein (.005 - .006 per cent of the ration) to Berkshire pigs, starting at weaning time. Wallach et al. (1947), Beeson et al. (l9h7I, and Reineke, EcMillen and Bratzler (l9h8) reported similar results by feeding small doses of thyroprotein to different breeds of pigs. No growth stimulation was found in pigs placed on iodinated casein for a short time during the fattening period (Muhrer et al., l9h7). Parker (19h3) and Irwin, et al. (l9h3) reported that feeding iodinated casein will accelerate the growth of chicks to a limited extent provided the dosage used is low. These results have been extended by Turner, Irwin and Reineke (l9hh) who found that the accelerated growth continues only for the first six weeks of life and that prolonged dosage is associated with growth.depression. The upper limit of the dose in such cases was .1 per cent thyroprotein in the t al. feed. Wheeler§l9t8) reported a significant increase in body weight of Rhode Island Red males but not in females at 12 weeks of age. Quisenberry and Krueger (19h8) reported in- creases in the growth rate and efficiency of feed utiliza- tion in chicks of the New Hampshire and White Plymouth Rock breeds. Influence of Seasonal Variations on Thyroxine Secretion Rate Seasonal variations of the thyroid activity in domes- tic animals is often due to differences in environmental temperatures. There are many indications that stimuli aris- ing in the external environment are capable of altering the rate of formation and discharge of secretions by the endoc- rine glands. It has been observed that there exists a reciprocal relationship between thyroid and thyrotropic hormone of the anterior pituitary. Dempsey and Astwood '(19h3) presented evidence that the rate of thyroid hormone secretion varied inversely with the environmental tempera- ture and they also suggested that the thyroid secretes its active principal more rapidly in cold than in warm.environ- meats. Cruickshank (1930) reported that thyroid weight in fowls showed marked seasonal variation, the weight being greatest from.January to March and least from mid March to mid July. Iodine content of the thyroids varied with the weight of the organs. Berliner and Warbritton (1937) observed poor fertility in rams during the summer months and they suggested that it was due to a decrease in thyroid secretion rate as a result of high summer temperature. They obtained good results by combined treatment with gonadotrophic hormon37ghyroxine. Ring (1939) states that exposure to cold causes an increase ,in metabolic rate and increases thyroid cell activity and vice versa. Reineke and Turner (l9h5) observed that the thyroid secretion ratesof male and female white Plymouth Rock chicks were highest during October to November and low- est during march to August. They further observed that with the onsetof fall the thyroid secretion rate continued to rise again toward the normal winter levels. Bogart and mayer (l9h6) state that high temperatures ' (85 - 90°F) cause a marked lowering in the activity of the reproductive organs in the ram by causing a hypothyroid con- dition. Thyroprotein or thyroxine given to rams during the period of high temperatures stimulate the reproductive or- gans and restore most of the reproductive activities to a level near that of the breeding season. Hurst and Turner (1947, 1948) reported variations in the thyroxine secretion rate in mature male mice at different temperatures. Relationship of the Thyroid to Reproduction Considerable work is still to be carried out to arrive at a definite conclusion as to the role of the thyroid status on sexual development in animals. A.number of workers hold the view that the thyroid has no direct effect on the testes and that any reproductive disturbances in the male in hypo-or hyperthyroidism is not primarily due to endocrine imbalance but to changed metabolic status (Moore, 1939) while others believe that thyroid secretion is essential for normal reproductive processes as in the absence of the thyroid gland libido is absent in the bull and the pro- duction of semen may be seriously affected. In rans ”Summer Sterility" has been observed due to decreased thyroxine secretion rate as a result of high summer temper- ature. In the domestic fowl definite stimulation of sperma- togenesis occurs by feeding Small doses of thyroprotein. In young breeding animals the thyroxine secretion rate is at its optimal level; the energy metabolism.and fertility are at a peak. With advancing age the rate of thyroxine secretion gradually slows down; the sex drive in the male declines; the time required for service increases and the fertility is also believed to decline. ‘The available literature on the subject is reviewed below. Bovines: Petersen et al. (19h1) thyroidectomized a male Jersey at four months of age and observed complete absence of libido at sexual maturity. His reaction was tested at frequent intervals with females in estrus and in no. instance could he be induced to mte. Oral adminis- tration of 25 gm. of desiccated thyroid over a period of three days restored normal activity and sexual behaviour. Brody and Frankenbach (l9h2) reported that thyroidectomized cows failed to manifest the normal physical signs of estrus. Oral administration of fresh thyroid to one cow restored normal estrual behaviour. Reineke (l9h6) reported that 1t bulls which had unsatisfactory breeding records, showed definite improvement in vigor, and improved libido was ob- served in ten, after feeding thyroprotein. The time re- quired for an observable effect to occur ranged from 7 to to days with an average of 16 days. Schultafand Davis (19h?) reported that iodinated casein increased the con- ception rate from 51.7 to 55.7 in five bulls out of seven and resulted in spermatozoa with higher motility and greater resistance to lower temperature (AOOF) storage. Sheepgand Goats: McKenzie and Berliner (1937) showed an increase in the number of abnormal sperm by keeping rams at high temperatures during the winter months. Berliner and Warbritton (1937) reported that rams produced semen of poor quality during summer months. This was be- lieved to be due to a decline in thyroid hormone secretion due to high temperatures prevailing during summer. Adminis- tration of gonadotropic hormone and thyroxine gave good results. Turner, Mixner and Reineke (19h3) observed that) a ram which had good sex drive but deficient semen was fed thyroprotein, and the semen improved in quality and after- wards settled several ewes. Bogart and Mayer (19A6) noted a decline in sperm numbers and mobility and an increase in abnormal forms, in rams made hypothyroid with thiouracil. Absence of spermatogenic activity in the testes of rams during the period of high summer temperature and also in thiouracil-treated ones was observed. They also reported that rams injected with thyroxine or fed thyroprotein during the period of high environmental temperature produced 10 more spermatozoa and had a lower percentage of abnormal cells. Warwick et a1. (19h8) reported that iodinated casein given to rams (.5 to 1.5 gms. daily) during April and May caused a deterioration in semen quality, partic- ularly in rams that lost weight. During July and August the same treatment caused improvement. Poultry: The effects of mild hyperthyroidism appears to be of practical significance in Poultry Husbandry as it stimulates growth and spermatogenesis in young birds, but the level that will be tolerated in rather narrow and semen production can be greatly impaired by giving too large an amount. Crew (1925) reported rejuvenation of seven hens 5 to 8 years of age, when they were fed desiccated thyroid daily for six months. The principle changes were the de- velopment of new plumage characteristic of younger fowls, improvement of the head furnishings and an increase in egg production. Jeep (1933) recorded that during late winter and early spring months, the testes of mallard drakes normally increase in size and spermatogenic activity. During this period an artificial increase, much greater than.that of normal drakes, is obtained by feeding daily doses of .25 to 1 gm. desiccated thyroid. The testes size ranged from 2 to 10 times that of the non-thyroid fed control. Benoit and Aron (l93h) Observed that when immature male ducks were fed with thyroid tissue or injected with ll thyroxine, they became sexually mature. They also recorded that thyroidectomy delayed the normal testicular growth in chickens and ducks. Testes of white leghorns rapidly de- creased in size. Losses were as great as 20 per cent of the normal testes size in 11 days and 90 per cent in 20 days. Benoit (1937) observed that the pelvis of ducks after ‘ thyroidectomy attains a development inferior to that attained by normal ducks with teeticles of equal size. Greenwood and Chu (1939) reported that the effects of thyroidectomy in the male Brown Leghorn were very marked and there was a regression in testes size, together with cessation of sperma- togenesis. Blivaiss and Domm (1942) recorded to to A5 per cent decrease in body weight and combs averaged 62 to 68 per cent less in thyroidectomized cockerels than the controls. Blivaiss (19h7) reported reduction in size and in degree of maturity of the gonads and sex accessary organs in thyroid- ectomized roosters. The testes remain.aspermic for as long as two years. These dysfunctions resulting from total ablation of the thyroid may be repaired by feeding desiccated thyroid or by administering thyroxine. Martinez (19h?) observed that thyroprotein when fed to fowls in doses of .01 and .02 per cent of the ration does not influence semen production or spermatogenesis and that .Oh per cent of the feed caused a definite stimulation of spermatogenesis. Both semen volume and the sperm concen- trations were increased quite markedly. Consequently the 12 total number of sperms per ejaculate was increased by approximately 65 per cent. In old roosters similar but less pronounced trends were observed. Thyroprotein when given at high levels of .08 to .16 per cent of the ration, depressed semen production and the total number of sperms decreased markedly (Wilwerth, 1948). Wheeler & Hoffmann (1948) observed in therprotein treated cockerels that the testes and pituitary weights were significantly higher and thyroid weights were significantly lower in the treated males. Egg Production: Thyroidectomy reduces egg production in poultry (Winchester, 1939). Turner and his co-workers at Missouri, U. S. A. (1945, 1946, 1947, 1948) observed that thyroprotein administration at a low level prevented the yearly egg production decline, especially during;the hot weather when a decline normally occurs in the laying hens. 13 EXPERIMENTAL PROCEDURE AND RESULTS Material and Environmental Conditions All the mice used in the following experiments were immature, male, and of about the same age with some varia- tions in size and body weight. They were bred by Rockland Farm, Garden City, New York, U.S.A. and belonged to a well established commercial strain. Before starting the experi- mental work, all the mdce were kept in cages at a controlled temperature of 24°C., with humidity which.varied from 45 to 55 per cent, for a period of one week. The object was to get them acclimatized to controlled environmental and laboratory feeding conditions. Artificial electric light was maintained during the day'throughout the experiments. Mice Kept at3000: In experiment II groups 8, 9 and 10 and in experiment IV groups 5, 6, 7, 8 and 9 were kept in the incubators with a maintained temperature of 30°C. for three to four weeks. As the mice were kept in cages in the incubators, enough artificial light was not available to them. The mice were taken out of the incu- bators every week for weighing. Feed and Feeding: The mice were fed ad libitum with finely ground standardized laboratory feed "Purina Laboratory Chow," manufactured by Purina Company, St. Louis, Missouri, U. S. A. Special feeding trays were used to prevent wastage. Water was available at all times from.in- verted bottles with a special outlet. 14 Thyroprotein (Protamone) manufactured by Cerophyl Laboratories, Inc., Kansas, Missouri, containing about .8 per cent 5 ', thyroxine and thiouracil provided by Lederle Laboratories, Inc., Pearl River, New York, were used through- out the experiments. The desired doses of thyroprotein and thiouracil were weighed on?3na1ytical balance and thoroughly .mixed with the weighed quantity of Purina Laboratory Chow in a mechanical mixer, with a view to having a uniformity of the drugs in the feeds. Procedure: All the mice were weighed and grouped, keeping the average weight of each mouse in the groups to about the desired weights. The mice in each group were numbered with picramic acid dye in a regular identification pattern. Feed and water consumption, general appearance, sex- ual behaviour and the mortality rate if any were recorded daily in the morning from 8 to 9 a.mt throughout the ex- periments. Dead mice were removed as so on as discovered to prevent being eaten up by their fellows. The mice in these experiments were killed by ether after a period of four weeks except the groups 8, 9 and 10 in experiment II which were killed at three weeks interval. Each.mouse in the experiments was dissected and its testes and seminal vesicles removed and weighed separately. In experiments I and II the seminal vesicles were weighed after gently squeezing out the seminal fluid, while in experiment III and IV these were weighed intact with.the seminal fluid. 15 Effects on Growth and Feed Consumption Hyperthyroidism: Experiments I and II at 24°C. The daily average feed and water'consumption per mouse in the control group 1 (Expt. I), was 3.9 and 8.4 gms., re- spectively. Tables IV and V show that the feed and water consumption of the thyroprotein treated groups increased with the increase in thyroprotein levels. The mice in group 3, receiving .025 per cent thyroprotein in the feed,on an average consumed 4.7 gms. of feed and 10.7 gms. of water per day per mouse. The feed and water consumption at the start of the experiment was 3.4 and 7.1 gms. per mouse, with a continuous increase fromLthe fifth day onward and on the last day of the experiment the feed and water con- sumption was 5.5 and 12.8 gms. Each mouse in this group consumed 19.4 and 27.3 per cent more feed and water than the controls. Each mouse in group 4, having .05 per cent thyroprotein in the feed,oonsumed on a daily average 5.4 and 11.4 gms. of feed and.water respectively. In this group each mouse consumed 3.4 gms. of feed and 7.0 gms. of water on the first day; with a continuous increase from the 4th day onward, to 6.3 gms. of feed and 14.1 gms. of water on the last day of the experiment. Each.mouse in this group consumed daily 30.2 and 35.7 per cent more feed and'water respectively, when compared with the control. Groups 5, 6 and 7 consumed 5.8, 6.1 and 4.2 gms. of feed and 11.8, 12.4 and 9.2 gms. of water per day per mouse. The average body weight of each mouse in Group I TABLE I GENERAL INFORMATION ABOUT THE EXPERIMENTS 16 No.0f Mice Average wt.gms. Expt. Group Duration At At At At No. No. Dosage of expt. Start End Start End Maintained at 2490 I 1 Control 4 weeks 10 10 18.0 28.0 n 2 .2% TH " 10 10 . 17.4 25.9 n 3 .025% TP** " 10 6 17.3 28.6 n 4 .05% TP " 10 10 17.4 29.8 a 5 .1% TP " 10 9 18.0 28.1 n 6 .2% TP " 10 8 18.1 27.0 " 7 .2% TH-+.05TP " 10 10 17.9 26.5 Maintained at 30°C I1 8 Control 3 weeks 10 7 14.2 19.6 9 .025% TP " 10 3 14.2 16.6 10 .2% TH " lO 8 14.4 18.0 maintained at 2420 III 1 Control 4 weeks 10 10 18.1 28.4 2 .025% TP " 10 10 18.4 30.2 ' 3 .05% TP " 10 10 18.4 31.5 " 4 Control " 7 5 16.9 25.8 Maintained at30°C IV 5 Control 4 weeks 7 6 17.0- 24.3 n 6 .005% TP 7 7 17.3 26.9 " 7 .01% TP " 10 10 17.2 24.6 " 8 .02% TP " 10 8 16.7 23.4 " 9 .1% TH " 7 7 16.8 22.9 * Thiouracil ** Thyroprotein 17 was 18.0 gms. at the start of the experiment and 28.0 gms. at the end. Each mouse made an average gain of 10 gms. or 55.5 per cent of the body weight in the four-week period. The gains made by groups 3, 4, 5, 6 and 7 were 63.4, 66.4, 49.1 and 51.8 per cent respectively (Table II). Comparing the feed and water consumption and the gain in body weight of all the groups in Table II, it is clear that group 4 receiving .05 per cent thyroprotein in the feed consumed 30.2 per cent more feed and 35.7 per cent more water per day than the control group and made a total gain of 12.1 per cent in body weight. ‘With higher dosages a decrease in body weight gains was observed (Fig. 2). In experiment III, groups fed .025 and .05 per cent thyroprotein at 24°C for a period of four weeks gained 7.4 and 13.8 per cent more body weight when compared with the control group. The increase in body weight gains‘was fairly constant in the groups fed low levels of thyroprotein. Experiments II and IV’atBOOC. Various levels of thyroprotein were fed to the mice in these experiments (Table I). Group 6 (Expt. IV) fed .005 per cent thyro- protein in the ration made a gain of 3.4 and 12.5 per cent more when compared with.the control.groups 4 and 5 at 24 and 300 respectively. The feed consumption per gram body weight gain was 10.2 gms. and that of the controls 11.1 and 12.2 gms. respectively (Table VI). This means that very small doses of thyroprotein even at high.temperature caused better utilization of feed than the controls. 18 Decreases in body weight gains were observed in groups given .02 and .025 per cent thyroprotein at 30°C for 3 to 4 weeks. Hypothyroidism: Goitrogens when given at an early age result in greatly reduced gains accompanied by symptoms of » cretinism in animals, (Hughes, 1944). IMixner, Tower and Upp (1946) reported an increase in the rate of gain ahd a decrease in the feed required per unit of gain in older cockerels fed thiouracil. Reineke and McMillan (1946) and Vander Noot et a1. (1947) reported an increase in body weight gain.and efficiency of food utilization in swine. Kempster and Turner (1945) and Glazener and Jull (1946) state that thiouracil caused a decrease in the weight of the chicken and increased the amount of feed required to produce a pound of gain when compared with the control. In the experiment I, group 2 fed .2% thiouracil for a period of four weeks, the daily average feed and water consumption per mouse was 3.3 and 7.5 gms. respectively. There was a decrease of 19.5 per cent in feed consumed per day per mouse when compared with the control group because the goitrogen causes a decrease of 20 to 30 per cent in B.M.R. (Reineke et a1. 1945). Each mouse in group 9 (Expt. IV) fed .1 per cent thiouracil.consumed 2.7 gms. of feed and 7.5 gms. of water daily (Tables IV and V). It was observed that the feed requirement per gram.gain in body weight in the thiouracil treated groups for a period of four weeks at 240 and 30°C was higher than in the contro ls . 19 In these treated animals, group 2 (Expt. 1), group 10 (Expt. II) and group 9 (EXpt. IV) there was a decrease of 6.7, 13.1 and 15.7 per cent in body weight gains when compared with the controls (Table II and III), and the de- crease was more marked at 30°C than at 24°C. Group 9 in experiment IV was kept for three weeks fiend the others for four weeks. Environmental Tenperature: A decrease of 9.1 per cent in the body weight gains of mice in group 5 (Expt. IV) kept at 30°C, was observed when compared with the control at 24°C (Table III) and there was also a decrease in feed and water consumption (Tables IV and V). Thyroprotein and Growth of Hair: It was noticed that the thyroprotein treated mice exhibited a fine sleek coat but groups given .05 and .1 per cent at 24°C and .01 per 0 cent thyroprotein.at 30 0 did better than other treated ones. Egéects on Testes and Seminal Vesicles Hyperthyroidism: Administration of iodinated casein (thyroprotein) in normal animals produces hyperthyroidism. In experiment I, thyroprotein in doses of .025, .05, .1 and .2 per cent was fed to mice for a period of four weeks at an environmental temperature of 24°C. Table VII shows that the weights of the testes and seminal vesicles in group 3 given .025 per cent therprotein were 181.9 and 66.1 gms. and those of group 4 were 187.8 and 71.7 gms. When compared with the control group there was a significant increase in the weights of the testes and seminal vesicles 20 TABLE II AVERAGE BODY WEIGHTS OF CONTROI.AND VARIOUS TREATED GROUPS OF MICE AT 24°C Average Weekly Body_Wt.in gms. Total %'Wt.* Group At wt. gained No. Start lst 2nd 3rd 4th gained or lost Experiment I 1** 18.0 22.4 24.3 27.3 28.0 10.0 -- 2 17.1} 21.5 2207 214,09 2509 805 -607 3 17.3 22.7 24.8 27.6 28.6 11.3 +7.9 4 17.4 23.1 25.9 28.2 29.8 12.4 +12.1 5 ' 18.0 22.1 24.7 27.4 28.1 10.1 +. 6 -l8.l 21.3 23.7 26.6 27.0 8.9 ..6.4 7 17.9 21.0 23.1 26.2 27.2 9.3 —-3.7 gaperiment III 1** 18.1 2302 21403 2601 28.1} 1003 -" 2 18.4 23.3 26.0 2713 30.2 11.8 4'7.4 3 18.4 24.4 27.4 30.2 31.5 13.1 -+13.8 * Weight gained or lost when compared with the weight gained by the control groups. ** Control groups. TABLE III 21 AVERAGE BODY WEIGHT OF CONTROLS AND VARIOUS TREATED GROUPS OF MICE AT 30°C Average Weekly Body Wt. in Gms. Total % Weight* Group At Weight Gained No. Start lst 2nd 3rd 4th Gained or Lost Experiment II 8** 114‘02 16.6 1808 1906 ." 501+ -" 9 11402 lheh 1609 1606 "- 20‘; “2101 JD like“ 1603 1705 1800 "- 306 -1300 Egriment IV 4** 16.9 19.2 20.7 22.5 25.8 8.9 -- 5** 17.0 17.9 20.0 22.1 24.3 7.3 - 9.1 6 17.3 21.3 23.5 25.3 26.9 9.6 (+ 3.4 (+12.5*** 7 1702 1901 1909 2203 2406 70‘} (" 808 (+ .3**3F 8 16.7 18.5 19.5 20.7 23.4 6.7 (-11.9 (" 2e8*** 9 16.8 17.2 19.4 20.9 22.9 6.1 (-15.7 (_ 6.6*** * Weight gained or lost when compared with the weight gained by the control groups. ** Control groups in the two experiments and group No. 4 was kept at 24°C. *** Gain or 1033 in Body Weight when compared with Group N00 50 5)? capture}— an mar at 2 tummy /5* (Journal. are: 025.719 ,//5 .0051 rpwmnon /4- 13.... /'/,4- a. . . J .. . . ,/ 02 . - .2... /,,.-/' J .721. ,3 .2 -TU. /-/ / 'V,’ d 205-713 ' ~ ,./' /3 (O . ,/ / 5]? / ,/'/ "xx // ‘ . / '- / Lu ”W / .I/"Iv- /// E g/o ' Ct Lg 9 3 a 3. g 7 0L 1 A A £xpr]4r24% £1097. yAraooc 0) KlVAS‘ 490v: «0/ 454mm: 38* K5 .7. 3*: a g 6 85‘ Lk 4 f, . 'E 3 Q J 000ka The influence of thyroid status on feed and water consumption of male mice at two different Fig. 1 envi ronment a1 t emper at ures . 22 23 TABLE IV WEEKLY AVERAGE FEED CONSUMPTION PER DAY BY THE CONTROLS AND VARIOUS TREATED GROUPS OF MICE Daily Daily*‘ Feed intake Average per cent Expt. Group Food increase or No. No. lst 2nd 3rd 4th Consumption decrease Maintained at 21L°C I 1* 3.7 3.9 4.1 4.1 3.9 -- n 2 3.4 3.3 3.3 3.4 3.3 -19.5 n 3 4.2 4.8 4.9 5.2 4.7 '19.4 . 4 4.3 5.5 6.0 6.2 5.4 ' 30.2 n 5 4.5 5.6 6.3 7.0 5.8 44.1 n 6 4.6 6.0 6.6 7.2 6.1 51.2 8 7 3.9 4.1 4.3 4.2 4.2 2.4 IV 4 3.2 3.4 3.5 3.9 3.5 -- Maintained at 30°C IV 5** 2.8 3.1 3.2 3.6 3.2 -- . 6 3.3 3.5 3.6 3.7 3.5 9.3 n 7 3.4 3.5 3.7 3.9 3.6 12.5 . 8 3.5 3.7 3.9 4.2 3.8 18.7 * 9 2.6 2.5 2.7 2.9 2.7 ~15.6 * Calculated by comparing with the controls ** Cont rol gro ups 24 TABLE V WEEKLY AVERAGE WATER CONSUMPTION PER DAY BY THE CONTROLS AND VARIOUS TREATED GROUPS OF MICE - Daily Daily* water intake Average per cent Expt. Group Water increase or No. No. 1st 2nd 3rd 4th Consumption decrease Maintained at 24°C I 1** 7.4 8.1 8.9 9.2 8.4 -- " 2 7.2 7.4 7.6 7.7 7.5 10.7 " 3 8.3 10.4 11.3 12.7 10.7 27.3 " 4 8.7 10.9 12.1 13.9 11.4 35.7 h 5 8.9 11.2 12.9 14.3 11.8 40.4 " 6 9.1 12.2 13.4 14.8 12.4 47.6 " 7 8.3 9.0 9.6 10.1 9.2 9.5 Iv 4 7.3 7.9 8.2 8.8 8.0 -- Maintained at 30°C IV 5** 7.5 8.0 8.2 8.4 8.0 -- " 6 7.5 8.4 9.1 9.5 8.6 7.5 . 7 7.8 8.6 8.9 10.2 8.9 9.8 " 8 8.0 8.7 9.5 10.5 9.2 15.0 E 9 7.1 7.0 7.3 7.9 7.5 -6.2 * Calculated by comparing with the controls ** Control Groups ' i 25 EXP! 1472‘ ”(0 TU/NRWN 17p: . 8,. r A ‘ : 4L k: xprlarao? mrgArao‘E cavrna covrml. 4r 2 0257. mwnar/ov . . g3 . ru. . 8.0661 mm RANOV r a ' e l. o 1 a .l ' rU .. -. ' y/’6 20 g /8 Q) a? /6 - i g M “ 0 a 2 3 2 3 4 WEEKS WEEKS Fig. 2 The effects of thyroid status on body growth of the male mouse at two different environmental temperatures. 26 TABLE VI FEED INTAKE IN GRAMS PER GRAM BODY WEIGHT GAIN IN CONTROL AND TREATED MICE AT DIFFERENT ENVIRONMENTAL TEMPERATURE Group Total Feed Total Gain Feed intake Dosage No. Consumption Body Weight Per gm.gain J 0 Experiment I maintained at 24 C Control 1 110.2 10.0 11.0' .2% TH 2 90.7 8.0 11.3 .025% TP 3 131.6 11.3 11.5 .05% TP 4 153.3 12.3 12.4 .1% TP 5 160.3 10.1 15.8 .2% TP ‘6 167.3 8.9 18.8 .2% TH +.%g% 7 113.5 9.3 12.2 Control 4* 98.0 8.9 11.2 Experiment IV maintained at 30°C Control 5 89.6. 7.3 12.2 .005% TP 6 98.1 9.6 10.2 .01% TP 7 100.8 7.4 13.6 .02% TP 8 106.4 6.7 15.8 .1% TE 9 75.6 6.1 12.3 * Control from.Experiment It’kept at 24°C 27 was observed (Figs. 3 and 4). This shows that small doses of thyroprotein caused a significant increase in the weights of the testes and seminal vesicles while high doses (0.1, 0.2 per cent) caused a decrease in the weights of these organs. Significant increase in the weights of the testes and seminal vesicles was also observed in the mice fed .025 and .05 per cent levels of thyroprotein in experiment III. In experiments II and IV an attempt was made to study the effects of both high and low dosages of thyr0protein on sexual development at 30°C. In experiment II, group 9, fed .025 per cent thyroprotein for a period of three weeks, a decrease in the weights of the testes and seminal vesicles and high.morta1ity rate was observed (Tables VIII and X). Keeping in mind these adverse effects produced by .025 per cent thyr0protein, groups 6, 7 and 8 (Expt. IV) were given .005, .01 and .02 per cent levels of thyroprotein for a period of 4 weeks. Significant increases in the weights of the testes and seminal vesicles were observed in group 6 given .005 per cent thyroprotein when compared with the control group 5)kept at 30°C (Table VIII). In groups given .01 and .02 per cent thyroprotein, a slight increase in the weights of the testes and seminal vesicles was observed in comparison with that of the control (GrOUP 5) at 30°C. Figs. 3 and 4 show that there were no marked differences among groups 4, 7 and 8. In these experiments the weights of the testes and seminal vesicles of the controls and treated groups, when 28 calculated per gram body weight, were highest in groups given .05 and .005 per cent thyroprotein at temperatures of 24° and 300 respectively (Table IX). Hypothyroidism: Goitrogens in general appear to exert a depressive influence on the reproductive system in birds and mammals (Andrews and Schnetzler, 1946; Leathem, 1946; Bogart and Mayer, 1946; Shaffner and Andrews, 1948 and others). In the present experiments administration of .1 and .2 per cent thiouracil to young male mice at both 240 and 30°C caused a decrease in the weights of the testes and semdnal vesicles. The decrease in the weights of the testes and seminal vesicles was more marked at the .2 per cent level for three weeks at 30°C than at7°f per cent level for a period of four weeks (Tables VII and VIII). There was a significant decrease in the weights of the testes and seminal vesicles of group 10 in experiment II. Thiou- racil (.2 per cent) when fed to mice in group 2 (Expt. I) for'a period of four weeks, caused a decrease in the weight of the testes and seminal vesicles which was significant when compared with the control. It was observed that the decrease in the weight of the seminal vesicles was more marked than the decrease in the weight of the testes in thiouracil-treated groups. Environmental Temperature: It has been observed that high environmental temperature causes a decrease in thyroid secretion rate. The mice in group 8 in experiment 29 II and in group 5 in experiment IV, which were kept at 30°C for a period of 3 to 4 weeks, showed afdecrease in the weight of the testes and seminal vesicles when compared with the controls (Tables VII and VIII). Effects of Hyperthyroidism.on Male Sex Characters The mice in groups 4 and 5 in experiment“ I and group 3 in experiment III,respectively,showed a tendency to mount each other during the third week of the experi- ments. Some degree of excitement was also observed in mice given high doses of thyroprotein. Histological Studies Histology of Testes: The substance of the testis is divided into lobules by fan-like extension of septa from.the mediastinum testis and within each lobe are a number of tortuous canals called the seminiferous tubules. The interpasses between the seminiferous tubules are occupied by blood and lymph vessels, nerves, Leydig's inter- stitial cells, connective tissue and several types of cells (Maximow and Bloom, 1943). Leydig's cells are modified connective tissue cells and these cells are re- sponsible for the secretion of male sex hormone. In the adult the seminiferous tubules are lined by the complex seminiferous epithelium with its two kind of cells. The Cells of Sertodi which are the supporting and nutrient elements and the spermatogenic cells which) through their proliferation and complex transformation, furnish 30 TABLE VII AVERAGE ORGAN WEIGHTS OF CONTROLS AND TREATED GROUPS OF MICE IN EXPERIMENTS I AND II Group No.0f Testes Seminal Vesicles No. Mice Dosage Weight EV Weignt** -§w Maintained at 24°C . l 10 Control 164.3 -- 60.4 -- $6.2 16.6 2 10 .2% TH 151.8 2.73 52.3 2.62 _ 16.9 15.3 3 6 .025 TP 181.9 2.94 66.1 2.51 1‘803 £602 4 10 .05% TP 187.8 4.21 71.7 3.18 £903 1' 07 5 9 .1% TP 169.8 1.13 65.9 2.03 1707 1502 6 8 .2% TP 160.9 .92 56.0 1.34 3:902 £600 ,_ 7 10 .2% TH 161.4 .71 55.4 1.46 +.05 T? 17.6 17.3 Maintained at30°C 8 7 Control lh60h “ #508 -- i507 3.7600 9 3 .025% TP 139.8 2.64 37.6 2.73 1‘60“ 1507 10 8 .2% TH 135.2 2.83 32.3 3.01 -:7.7 15.2 i.Standard deviation ‘ "t" value obtained by comparing the groups with _ their respective control groups. ** The seminal vesicles were weighed after gently squeezing the seminal fluid. 3 TABLE VIII AVERAGE ORGAN WEIGHTS OF CONTROLS AND TREATED GROUPS OF MICE IN EXPERIMENTS III AND IV 1 Group No.0f Testes Seminal VesicIes No. Mice Dosage Weight 8*7 Wt.‘ f Maintained at 24°C 1 10 ContrOl ' 17106 "" 7508 -" 110.8 19.7 2 10 .025% TP 186.6 3.13 84.2 2.94 110.5 $8.8 3 10 .05% TP 194.4 4.22 89.4 3.65 17.9 $9.6 ‘ A: 5 CODtl‘Ol 16208 -" ‘ 700A "" 110.4 $10.0 Maintained at 30°C 5 6 Control 151.3 1.12 60.5 1.94 111.6 ** ‘i7.0 ** 6 7 .005% TP 179.0 2.41 5.23 79.6 2.51 4.14 1706 1502 7 10 .01% TP 158.4 .34 .44 67.5 .43 .72 111.7 110.1 8 8 .02% TP 150.6 1.36 .21 58.4 1.42 .45 9 7 .1% TH 136.2 3.02 2.63 49.3 3.13 2.74 :10.5 19.2 1' Standard deviati on * fl value obtained by comparing groups 2 and 3 with group 1 and groups 5, 6, 7, 8 and 9 with group No. 4, reSpectively. ** fl‘value obtained by comparing group Nos. 6, 7, 8 and 9 with group 5. *** The seminal vesicles were weighed with the seminal fluid. 32 . 4 a a w E S. _( E «me. e on To. 1 a: $8. _ ohm. awooESo T ”Nata degree _( E a m. 7 6558. 7 some 28 22%} a: «no. 7 ac ammo. _ 00328 93m. _) Qk .\. \. 0K .\. m6. _( 0a a 8:3 aw. fl _ naswmo. 4 Sum. 7 003 20,0 200- [80 [60 - I40 20 Fig. 3 The influence of variations in environmental temperature and thyroid status on the weigit of testes of mice. 33 0 0 am' 3a: /00’ 4 r—' ‘80? _. r. 8 ~ 1 t ‘ k. 50 fl *4 i g T- P '5‘ ii m 40 FT )8 a r - ~ . _ and M Q ‘5‘ - 0 a 0.0. 1‘ KQ k ,3 $20» $282338 gr: (gm? ggw‘ige. 2swwngfi ON 8 “ ‘38“ a =Bw99\ w u 3 99“ Boeeqej G (mmpm1/234587 l23 890 456789 EmprAmz I I" .I 1? Fig. 4 The influence of variations in environmental temperature and thyroid status on the weight of seminal vesicle of the mouse. The seminal vesicles were weighed with the fluid removed in Expts. I am II and intact with contained fluid in Expts. III and IV. 34 TABLE IX WEIGHTS 0F TESTES AND SEMINAL VESICLES PER GM. BODY WEIGHT IN CONTROLS AND TREATED GROUPS AFTER 4 WEEKS Expt. Group Testes S.Vesicle No. No. Dosage nmmh mgm. maintained at 24°C 1* 1 Control 5.9 2.1 n 2 .2% TH 5.8 2.0 " 3 .025% TP 6.4 . 2.3 n 4 .05% TP 6.9 2.4 " 5 .1% TP 6.0 2.3 " 6 .2% TP 5.9 2.2 n 7 .2% TH .+.05% TP 5.9 2.0 III** 1 Control 6.0 2.6 " 2 .025% TP 6.2 2.7 " 3 .05%TP 6.2 2.8 IV** 4 Control 6.1 2.6 Maintained at 30°C " 6 Control 6.0 2.4 n 6 .005% TP . 6.6 2.9 " 7 .01% TP 6.4 2.7 " 8 .02% TP 6.4 2.5 " 9 .1% TH 5.9 2.2 *Seminal vesicles weighed after gently squeezing seminal fluid. **Seminal vesicles weighed with seminal fluid. 33 0 0 am we /00- 4 60 r F g rrL F F - k. 50 — fl _, g .1 r _1 - In .0. F we E -— «m klx . (g at Q Q Q Q Q g Q g _gS&KEES Sgt E e Egret gsmhufia CNS 3““ 558“ : fow99E N no. new UUQ§Q\% G GROUPNO./2.34567 I23 499/0 45678.9 EXPr/vo. I I I 1.17 Fig. 4 The influence of variations in environmental temperature and thyroid status on the weight of seminal vesicle of the mouse. The seminal vesicles were weigied with the fluid removed in Expts. I am II and intact with contained fluid in Expts. III and IV. 34 TABLE IX WEIGHTS OF TESTES AND SEMINAL VESICLES PER GM. BODY WEIGHT IN CONTROLS AND TREATED GROUPS AFTER 4 WEEKS Expt. Group Testes S.Vesicle No. No. Dosage mgm. mgm. maintained at 24°C I* 1 Control 5.9 2.1 " 2 .2% TH 5.8 2.0 " 3 .025% TP 6.4 . 2.3 n 4 .05% TP 6.9 2.4 . 5 .1% TP 6.0 2.3 n 6 .2% TP 5.9 2.2 n 7 .2% TH .+.05% TP 5.9 2.0 III** 1 Control 6.0 2.6 n 2 .025% TP 6.2 2.7 " 3 .05% TP 6.2 2.8 IV** 4 Control 6.1 2.6 Maintained at 30°C ' 6 Control 6.0 2.4 " 6 .005% TP , 6.6 2.9 " 7 .01% TP 6.4 2.7 " 8 .02% TP 6.4 2.5 " 9 .1% TH 5.9 2.2 *Seminal vesicles weighed after gently squeezing seminal fluid. **Seminal vesicles weighed with seminal fluid. 35 mature spermatozoa. In a tubule with active spermatogenesis the Sertoli cells are slender, pillar-like elements, per- pendicular to the basement membrane to which they are attached. They are separated from one another at somewhat regular intervals by the densely crowded spermatogenic cells. The outlines of the sertoli cells can not be seen distinctly and the nuclei are oval shaped, with a compound nucleolus. Under normal conditioning the sertoli cells are never seen to divide either mitotically or amitotically. Spermatogenesis: The following are the three phases in the process of spermatogenesis: 1. The germ.cells undergo repeated mitosis and certain structural changes and ultimately give rise to new cells called Spermatids. 2. Reduction in the number of chromosomes by meiosis. 3. The Spermatids undergo a series or complex transformations which result in mature sperm. As a rule the earliest generations of spermatogenic Cells are near the basement membrane of the seniniferous tubules while the mature forms line the lumen. The period of growth starts with the completion of the last sperma- togonial division and each spermatogonium gradually in- creases in size and its nucleus undergoes marked trans- formations. This growth causes a further shifting of the cells towards the lumen of the tubules. The growing cell is known as a primary spermatocyte and when this cell reaches its full development, the period of maturation begins and it divides into two new cells called secondary TABLE VII 30 AVERAGE ORGAN WEIGHTS 0F CONTROLS AND TREATED GROUPS OF MICE IN EXPERIMENTS I AND II Group No.of Testes Seminal Vesicles No. Mice Dosage Weight T7 Weight** ETD Maintained at 24°C _ l 10 Control 164.3 -- 60.4 -- t6.2 26.6 2 10 .2% TH 151.8 2.73 52.3 2.62 - $6.9 15.3 3 6 .025 TP 181.9 2.94 66.1 2.51 1:803 £602 4 10 .05% TP 187.8 4.21 71.7 3.18 1903 1:707 5 9 .1% T? 169.8 1.13 65.9 2.03 1707 52502 6 8 .2% T? 160.9 .92 56.0 1.34 3:902 £600 .- 7 10 .2% TH 161.4 .71 55.4 1.46 +.05 T? :7.6 :7.3 Maintained atA3O°C 8 7 Control 146.4 -- 45.8 -- 1.5.? 2:600 9 3 .025% TP 139.8 2.64 37.6 2.73 :60“ 1507 10 8 .2% TH 135.2 2.83 32.3 3.01 i707 i502 t.Standard deviation ‘ "t" value obtained by comparing the groups with . their respective control groups. ** The seminal vesicles were weighed after gently squeezing the seminal fluid. AVERAGE ORGAN WEIGHTS OF CONTROLS AND TREATED GROUPS OF TABLE VIII MICE IN EXPERIMENTS III AND IV 31 Seminal VesicIes Group No.0f Testes No. Mice Dosage Weight EII' Wt.* f Maintained at 24°C 1 10 CODLrOJ. ' 17106 -- 75 08 '- ilO.8 19.7 2 10 .025% TP 186.6 3.13 84.2 2.94 110.5 $8.8 3 10 .05% TP 194.4 4.22 89.4 3.65 17.9 $9.6 4 5 Control 162.8 -- ' 70.4 -- $10.4 t10.0 Maintained at 30°C 5 6 Control 151.3 1.12 60.5 1.94 111.6 ** '17.0 ** 6 7 .005% TP 179.0 2.41 5.23 79.6 2.51 4.14 1706 1502 7 10 .01% TP 158.4 .34 .44 67.5 .43 .72 111.7 110.1 8 8 .02% TP 150.6 1.36 .21 58.4 1.42 .45 9 7 .1% TH 136.2 3.02 2.63 49.3 3.13 2.74 110.5 19.2 ‘* Standard deviation - * t value obtained by comparing groups 2 and 3 with group 1 and groups 5, 6, 7, 8 and 9 with group No. 4, respectively. ** d value obtained by comparing group Nos. 6, 7, 8 *** The seminal vesicles were weighed with the seminal £11116. 0 and 9 with group 5. 32 ,_A_. F E 83/0 456767 246 I23 I I 200 r l80 M WAG/234567 [MOI/tn Fig. 3 The influence of variations in environmental tenperature and thyroid status on the weight of testes of m ice. 33 a SEMNAL VESCLE [It-MT.“- Fig. 4 The influence of variations in environmental temperature and thyroid status on the weight of seminal vesicle of the mouse. The seminal vesicles were weigied with the fluid removed in Expts. I and II and intact with contained fluid in Expts. III and IV. WEIGHTS OF TESTES AND SEMINAL VESICIES PER GM. TABLE IX 34 BODY WEIGHT IN CONTROLS AND TREATED GROUPS AFTER 4 WEEKS Expt. Group Testes S.Vesicle No. No. Dosage Emma mgm. maintained at 24°C I* 1 Control 5.9 2.1 n 2 .2% TH 5.8 2.0 " 3 .025% TE 6.4 . 2.3 a 4 .05% TE 6.9 2.4 ' 5 .1% TE 6.0 2.3 . 6 .25 TP 5.9 2.2 fl 7 .2% TH .t.05% TE 5.9 2.0 III** 1 Control 6.0 2.6 n 2 .025% TE 6.2 2.7 " 3 .05% TE 6.2 2.8 17** 4 Control 6.1 2.6 Maintained at 30°C ' 5 Control 6.0 2.4 n 6 .005% TE 6.6 2.9 N 7 .01% TE 6.4 2.7 n 8 .02% TE 6.4 2.5 u 9 .1% TH 5.9 2.2 *Seminal vesicles weighed after gently squeezing seminal fluid. **Seminal vesicles weighed with seminal fluid. 35 mature spermatozoa. In a tubule with active spermatogenesis the Sertoli cells are slender, pillar-like elements, per- pendicular to the basement membrane to which they are attached. They are separated from.one another at somewhat regular intervals by the densely crowded spermatogenic cells. The outlines of the sertoli cells can not be seen distinctly and the nuclei are oval shaped, with a compound nucleolus. Under normal conditioning the sertoli cells are never seen to divide either mitotically or amitotically. Spermatogenesis: The following are the three phases in the process of spermatogenesis: 1. The germ.cells undergo repeated mitosis and certain structural changes and ultimately give rise to new cells called Spermatids. 2. Reduction in the number of chromosomes by meiosis. 3. The Spermatids undergo a series of complex transformations which result in mature sperm. As a rule the earliest generations of spermatogenic Cells are near the basement membrane of the seminiferous tubules while the mature forms line the lumen. The period of growth starts with the completion of the last sperma- togonial division and each spermatogonium gradually in- creases in size and its nucleus undergoes marked trans- formations. This growth causes a.further shifting of the cells towards the lumen of the tubules. The growing cell is known as a primary spermatocyte and when this cell reaches its full develOpment, the period of maturation begins and it divides into two new cells called secondary ‘9 36 spermatocytes. The spermatides are the last generation of spermatogenic cells and they do not divide. Crystalloids are present in Sertoli cells and spermatogonia. Degenerated spermatogenic cells which finally disintegrate into granular and fatty debris are sometimes seen in the lumina of the seminiferous tubules. This is not a pathological phenomenniprovided it does not exceed certain limits (Maximow and Bloom, 1943). The de- generated cells are usually seen close to stretches of active seminiferous epithelium with normal spermatogenesis in full progress. Seminal Vesicles: The seminal vesicles are tortuous elonated, hollow bodies with a very irregular branched lumen and numerous out-pocketings. Their wall consists of an external connective tissue sheet with elastic nets, of e.nuddle layer of smooth muscle and of a mucous membrane resting upon a thin submucous layer. The mucous membrane forms an intricate system.of thin.and high primary folds which branch into the lumen and anastomose very frequently with one another. The epithelium shows great individual variations which probably depend on age and on functional influences. Technique: The testes and seminal vesicles were fixed in Bouin's fluid. All the tissues were dehydrated in a series of graded alcohols for varying intervals, depending upon the stage of dehydration. Lithium.Carbon- ate was used during the dehydration process to bleach the 37 picric acid residue in the fixed tissues. Picric acid is one of the reagents used in the preparation of Bouin's f1uid.~ Cedar Oil was used as a clearing agent. The stand- ard histological technique was used for cutting paraffin sections which were .7 u in thickness. The slides were stained by Harris hematoxylin and an eosin counterstain. Thyroid and Sexual Development In this note a brief general description is given of the histological studies of the testes and seminal vesicles of the control and treated mice while the de- tailed studies are in progress. Testes: Histological examinations of the testes of thyroprotein treated mice (.025 and .05 per cent at 24°C and .005 per cent at 30°C) showed an increased spermato- genic activity in the seminiferous tubules when compared with that of the control groups (Figs. 5, 6, 7, 9). Out of these groups, the one fed .05 per cent thyroprotein gave the best results. Most of the lumina of the tubules contained numerous mature sperm; the spermatogonia showed active pro- liferation.and the cells appeared well arranged (Fig.:9). Decreased spermatogenic activity and some degenerative changes were observed in groups given .02 per cent thyro- protein at 30°C (Fig. 11). Testes of Thiouracil treated mice at 30°C for a period of four weeks, showed atrophic and degenerative changes in some of the seminiferous tubules and also 38 cellular disorganization (Fig. 15). In others limited spermatogenesis was observed. The testes of the mice kept at 30°C also showed limited spermatogenesis and some de- gree of degenerative changes in some of the seminiferous tubules (Fig. 11). Seminal Vesicles: The seminal vesicles of the mice fed .05 per cent thyroprotein at 24°C and .005 per cent at 30°C, showed an increased proliferation of the epithelial cells lining the mucosa when compared with that of the controls (Figs. 8, 10, 14). The epithelial cells showed glandlike structures containing numerous granules and there was an increase in cell height. Slight desquamation of the epithelial cells lining the seminal vesicles was observed in some sections of the thiouracil-treated mice (Fig 16). Accumulation of these desquamated cells with seminal fluid was seen in the Alumina of the seminal vesicles. The epithelial cells showed decreased proliferative changes and a comparative decrease in cell height. Somewhat decreased proliferative activity and degenerative changes were seen in the seninal vesicles of mice kept at 30°C for four weeks (Fig. 12). 39 Fig. 5 Testis section of a normal mouse kept at 24°C. showing the degree of spermatogenic development. H. and E. Stain (x 116). Fig. 6 o Testis section of a mouse kept at 24 C and fed .05% thyroprotein. Note the increased spermato- genic activity in the hypertrophied seminiferous tubules (x 116). I A a——-g.-——._,h.__ 4O Fig. 7 Testis section of a normal mouse kept at 24°C, showing the degree of spermatogenic develop- ment (x 590). Fig. 8 Section of a seminal vesicle of a normal mouse showing the presence of a few granules. (x 590). 41 42 Fig. 9 Higher Magnification of Fig. 6., Testis section of a mouse fed .05% Thyroprotein at 24°C. Numerous mature sperm are seen in the lumen of the seminiferous tubule . The spermatogenic cells show active proliferative changes and are well organized (x 590). Fig. 10 Section of seminal vesicle of a mouse fed .05% thyroprotein (24°C), showing numerous secretion granules and increase in L;cell height (x 590). 43 I... 44 45 Fig. 11 . Testis section of a mouse kept at 30°C for four weeks, showing limited spermatogenesis and de- generative changes in the tubules (x 590). Fig. 12 Section of seminal vesicle of a mouse kept at 30°C for four weeks, showing some degenerative changes (x 590). 46 47 Fig. 13 Testig section of a mouse fed .005% Thyroprotein at 30 C, showing some spermatogenic activity and organized spermatogenic cells. Compare with Figs. 7 and 11. (x 590). Fig0 11+ Section of seminalovesicle of a mouse fed .005% thyroprotein at 30 0, showing increased cellular activity. Compare with Fig. 12. (X 590). 49 Fig. 15 Tegtis section of a mouse fed .1% thiouracil at 30 C for four weeks. Note the atrophic and degenerative changes and limited spermatogenesis. (x 590). Fig. 16 Section of eminal vesicle of thiouracil-treated mouse at 30 0, showing desquamated epithelial cells, and decreased cellular activity (x 590). a : ‘73-‘33???" *v-ffi“ . .. fig 50 51 Effects of Thyroprotein on Respiration At a temperature of 30°C it was observed that the mice in group 9 (Expt. II) and group 8 (EXpt. IV) fed .025 and .02 per cent thyr0protein reapectively showed an in- crease in the respiration rate when compared with the con- trol groups or the mice fed thiouracil. This increase in respiration was more marked within the first 10 days of the experiment. It may be that the high dosages of thyro- protein at 30°C caused some degree of excitation of the respiratory centers in the medulla and thereby increased the respiration rate or maybe due to increased metabolic rate and interference with the homeostatic mechanism. Mbrtality Rate Mortality rate among the control and various treated groups of mice was variable. However it was observed that the trend of the percentage of mortality was high in mice given high doses of thyroprotein at 30°C (Table X). High metabolic rate due to large doses of thyroprotein admin- istration at high environmental temperature, interferes with the normal homeostatic mechanism of the mouse and thereby causes extra stress on the organism.which ultimately succumbs. 52 TABLE X MORTALITY RATE AMONG CONTROLS AND TREATED GROUPS OF MICE Experiment Group Mortality No. No. Dosage % Maintained at 24°C I 1 Control 0 " 2 .2% TH O " 3 .025% TE 30 n 4 .05% TE 0 " 5 .1% TE 10 n 6 .294 TE 20 '3 7 .2% TH + .057: TE 0 Maintained at 30°C I1 8 Control 30 " 9 .025% TE 70 " 10 .2% TH 20 Maintained at 24°C Ill 1 Control 0 " 2 .025% TP 0 " 3 .0575 TE 0 IV 4 Control 28.4 Maintained at 30°C " 5 Control 14.2 n 6 .005% TE 0 " 7 .0193 TP 0 " 8 .02% TP 20 " 9 .194 TH 0 53 DISCUSSION During recent years considerable work.has been done in the field of thyroid physiology, but the role of the thyroid in male fertility needs further clarification. Some workers point out that the thyroid has no definite effect on the testes and it is probable that many repro- ductive disturbances in the male in hypo- or hyperthyroidism are due not primarily to endocrine imbalance but to changed metabolic status (Moore, 1939) or to a complex inter- relation between the endocrine system and the body metabol- ism as a whole (Cameron, 1945). Berliner and warbritton (1937); Bogart and beer (1945); Reineke (1946) and others believe that the thyroid plays an important part in male fertility. 'With this work in view, an attempt has been made to study the influence of variations in environmental temperature and thyroid status on growth and sexual de- velopment in the growing male mouse. Varying degrees of hypo- or hyperthyroidism were produced by the administra- tion of thyroprotein or thiouracil. In the present experi- ments it was observed that .05 per cent thyroprotein when fed to male mice for a period of four weeks at 24°C stimulated body growth and the weights of the testes and seminal vesicles were significantly increased. Hurst and Turner (1948) state that stimulation of the growing mouse by 80 times its own thyroid secretion rate is detrimental 54 to growth, but stimulating the growing mouse by 20 to 60 times its own thyroid secretion rate is beneficial to growth. This being so, administration of small doses of thyroxine to growing animals, will maintain an optimal thyroxine level in the body and will thus improve the .metabolic rate within the physiological limits. Moreover, Evans et al. (1939) and Scow and Marx (1945) state that there are some indications that the thyroid hormone is necessary for the normal elaboration of the hypophyseal growth hormone. Large doses of thyroprotein (.2 per cent) in the feed markedly increased the feed and water consumption due to increased metabolic rate but there was a decrease in body weight gains and also of the testes and seminal vesicles. It may be pointed out here that increased feed con- sumption on high doses of thyroprotein treatment is in it— self no criterion as to the increase in body weight. It has been observed that the mice with the highest feed con- sumption did not experience the maximum increase in weight. This suggests that there is a point of diminishing returns, wherein the increase in metabolism induced by the thyropro- tein exhibitSpredominantly a’tatabolic" effect, in relation to the relative "anabolic" effects which are produced by small doses of thyroprotein treatment in growing animals. There was an increased feed consumption with the increase in dosages of iodinated casein, but there was no relation 55 as regards the increase in body weights. In experiment IV, Table VIII it is shown that when .005 per cent of thyroprotein was fed to growing mice at 30°C, there was also a significant increase in the weight of the testes and seminal vesicles when compared with the control kept at an environmental temperature of 30°C. In this group a fairly constant increase in the body weight and feed consumption was also observed. The daily feed consumption per gram body weight gain was slightly less than that of the controls at 24° and 30°C. There was a decrease in body weight gain, feed consumption and in the weights of the testes and seminal vesicles of the control group (Expt. IV) kept at 30°C, when compared with the control group 4)at 24°C. This shows that there occurs a decrease in the thyroid secretion rate at high temperature and the growing animals with their changing rate of thyroid hormone secretion, could not maintain their normal thyroxine level in the body while the groups given small doses of thyroprotein did better. Hurst and Turner (1948) observed a decrease in the thyroxine secretion rate in male mice at 87°F when compared with those kept at 80°F. The decrease in thyroid secretion caused a decrease in metabolic rate and further upset the pituitary-thyroid relationship. As has been previously pointed out, administration of small doses of thyroxine will maintain the thyroxine level at its Optimum and will increase the metabolic rate to about normal. At 30°C, the mice on .01 5 ‘ ... per cent 56 level of thyr0protein gained more body weight, consumed more feed and also there was an increase in the weights of the testes and seminal vesicles in comparison with the control group kept at the same temperature. Thyroprotein (.025 per cent) when fed to the mice at 30°C for a period of three weeks caused a decrease in body weight gains and tie decrease in the weights of the testes and seminal vesicles was more marked than by feeding ..2 l L .02 per cent thyroprotein in experiment IV. It appears that high doses of thyroprotein at high environmental temperatures are detrimental to the animal body, because at high temperature there is a decrease in thyroxine secretion rate and, further, administration of large doses of thyroxine does hot maintain the thyroid secretion rate within physiological limits. The explanation is that large doses of thyroxine in the blood causes the anterior pituitary to cease secretion of thyrotropic hor— mone and the thyroid steadily decreases in size. The re- sult is that the complex Pituitary-Thyroid relationship is upset. In experiments I and IV, the mice fed .05 and .005 per cent thyroprotein at environmental temperatures of 24° and 30°C showed greater beneficial results with re- gard. to growth and sexual development than that of the other thyroprotein—treated groups of mice at the same temperatures. This indicates that an increase of only 6°C it 240 to 30°C in environmental temperature caused a ten times reduction in the optimal thyroprotein dosage, 57 which means that the demand for thyroxine at high temper- ature is less than that at low temperature. Administration of .1 and .2 per cent thiouracil at 24° and 30°C caused hypothyrordism in the mice, and this would result in a considerable decrease in B.M.R. The .feed consumption decreased and there was a decrease in body weight gains and in the weights of the testes and seminal vesicles. The decrease in the weight of the seminal vesicle was more marked than the decrease in the weight of the testes. Histological studies of the testes and seminal vesicles showed that mild hyperthyroidism stimulated spermatogenic activity in the testes and epithelial pro- liferation of the mucosa of the seminal vesicles when com- pared with the control or thiouracil-treated mouse. Some atrophic changes and limited spermatogenesis were seen in the testes; desquamation of the epithelial cells lining the mucosa of the seminal vesicles of the mouse given 0.1 per cent thiouracil for a period of four weeks at 30°C, was also notiéed. It may be pointed out here that the increase in the weight of the testes and seminal vesicles and the in- creased spermatogenesis in the testes of low level thyro- protein-treated groups, appeared to be due not simply to increased metabolism or increased body weight gains, as the weights of the testes and seminal vesicles of the control and treated groups when calculated per gram body 58 weight, were highest in the groups fed .05 and .005 per cent thyroprotein at 24° and 30°C respectively. The mechanism whereby the thyroid influences gon- adal function has not been fully elucidated. Reineke et al. (1941) observed that the gonadotropin secretion is reduced in.thyroidectomized young male goats. Meites and Chandrashaker (1948) further observed that in male mice the response to gonadotropic hormone is reduced by thiouracil and augmented by feeding optimal levels of thyroprotein. The directly opposite results obtained in rats is undoubtedly due to differences in hormone balance in the two species. Van Dyke and Chen (1933) found a decrease in the concentration of the ovulation producing factor for rabbits,in thyroidectomized rabbit pituitary in comparison with pituitaries from.1itter-mate controls. Pan (1940) reported that the gonadotropin content of the pituitary of normal and castrate rats and of rabbits was decreased_after thyroidectomy. Castration lowered the thyrotroprm contents of the A. P. gland in cattle (Reece and Turner, 1937), in rats (Turner and Cupps, 1940) and also lowered the thyroxine secretion rate in the chicken (Schultze and Turner, 1945). Stein and Lisle (1942) observed a decrease in the gonad stimulating potency of young male rats following thyroidectomy. Chu and You (1945) gave thyroid to rabbits and found that the pituitary F.S.H. was lowered and the L.H. increased. It has also been observed that thyroidectomy decreases 59 reproductive processes in animals (McKenzie and Berliner, 1937; Smelser, 1934, 37, 39; Bhrliner and warbritton, 1937; Benoit, 1937; Turner et al. 1943; Bogart and Mayer, 1946 and others). Grumbrecht (1939) reported that thyroid increases the weight of the ovaries of infantile rats receiving a constant dose of gonadotropic substance, the increase in the weight being proportional to the dose of thyroid. Schultze and Davis (1947. 1948) reported that thyroid hormone is capable of influencing directly the metabolism of semen. Addition of thyroxine to bull's semen at a critical concentration range caused an increase in 02 consumption and increased the conception rate when compared with that of the controls. Pén (1948) reported a significant decrease in testes weight of young rats fed .2% Sulfamethazine and absence of Spermatogenic activities. The epithelial cells of the seminal vesicles showed a definite decrease in height. In these experiments the increase in the weights of the testes and seminal vesicles and the increased sperm- atogenesis of the young male mouse, observed in mild hyperthyroidism, may probably be due to the increased output of hypophyseal gonadotropic hormones which are responsible for the growth of the testes. The follicle stimulating hormone of the anterior pituitary directly stimulatesthe growth of seminiferous tubules and the lutenizing hormone stimulates the secretion of male sex so hormone by the Ieydig's interstitial cells of the testes. The male sex hormone in turn is responsible for the growth of the seminal vesicles and other secondary sex.organs (Fraenkel et a1. 1940 and Simpson et a1. 1942). The de- crease in the weights of the testes and seminal vesicles and the decreased spermatogenesis in the testes of mice fed thiouracil or kept at 30°C, was probably due to decreased output of gonadotropic hormones by the anterior pituitary as a result of decreased thyroid secretion rate. In the light of the present knowledge on the subject, it may be suggested that the thyroid secretion may facilitate the utilization of hypophyseal gonadotropic and gonadal hormones by the organism, or the increased output of gonadotropin by the anterior pituitary in mild hyperthyroidism stimulates the growth of male sex organs. Furthermore, the thyroid hormone may stimulate sexual development directly by metabolic conditioning of the cells involved. From the results obtained with young male mice, it seems clear that a mild degree of hyperthyroidism has no deliterious effects on growth or sexual development and in fact would be conducive to optimal reproductive per- formance. This suggests interesting possibilities for therapy in live-stock, particularly under conditions where the thyroid functions may be subnormal. However, the exact role of the thyroid in reproduction will need to be estab- lished for each species of domestic animals before such therapy can be used under field conditions. 61 SUMMARY The results obtained in.the present experimentsin- dicate that mild hyperthyroidism stimulates while hypo- thyroidism depresses body growth and sexual development in the growing male mouse at environmental temperatures of 24° and 30°C. The comparatively stimulated sexual development and spermatogenesis may occur through inter- relationships between the pituitary, thyroid and gonads. 1. There was a significant increase in the weights of the testes and seminal vesicles of the mice given .05 per cent thyroprotein in the feed, for a period of four weeks, at 24°C while higher doses caused a decrease in their weights. Thyroprotein (.005 per cent) when fed to mice, kept at 30°C, caused a significant increase in the weights of these organs when compared with the control at 30°C. High environmental temperature of 30°C alone caused a decrease in the weights of testes and seminal vesicles. The weights of testes and seminal vesicles of controls and treated groups, when calculated per gram body weight, were highest in groups fed .05 and .005 per cent thyro- protein at environmental temperatures of 24° and 30°C, respectively. 2. Administration of thiouracil (.1 and .2 per cent) in the feed caused a decrease in the weights of the testes and seminal vesicles which was.more marked at high environmental tenperature. The decrease in the weight of a, .) 62 the seminal vesicles was more marked than that in the testes. 3. Histological studies of the testes and seminal vesicles showed that mild hyperthyroidism stimulated the spermatogenic activity in the testes and epithelial pro- liferation of the mucosa of the seninal vesicles with numerous granules, when.compared with the control or hypothyroid male mouse. Hypothyroidism.produced by the administration of thiouracil or by keeping the mice at 30°C caused some atrophic and degenerative changes,with limited spermatogenesis in the seminiferous tubules. In thiouracil-treated mice some degree of desquamation and inactivity of the epithelial cells lining the mucosa of seminal vesicles was also observed. 4. Thyroprotein when fed at .025 and .05 per cent levels to growing male mice for a period of four weeks, at 24°C caused an increase of 7.9 and 12.1 per cent more in the body weight gains when.compared with the control group. Thyroprotein when given.at the .2 per cent level in the ration caused a decrease of 6.4 per cent in body weight gain at 24°C. At 30°C a decrease in body weight gain was observed in mice fed .02 and .025 per cent thyroprotein and also in the controls. 5. The daily feed and water consumption of the thyroprotein-treated mice was higher than that of the controls and thiouracil—treated ones. The increase in feed and water Consumption increased with the increase 173 63 in dosages of thyroprotein. The.mice given .005 per cent thyroprotein for a period of four weeks at 30°C, consumed .8 gm. less feed per gram.gain in body weight and gained 2.8 and 18.4 per cent more weight when compared with the control groups at 24° and 30°C respectively. There was an increase of 19.5 and 30.2 per cent in feed consumption per mouse per day in groups fed .025 and .05 per cent thyroprotein at 24°C, while thiouracil treatment or high environmental temperature caused a decrease in feed and water consumption. 6. 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