THS- "I'HE EWECT OF ALTERAWONS EN THYRDID ACTEWTY ON THE RES?OHSE C)? THE OVARIES C‘F *ZMMATURE FEMALE RATS AND MICE TO EQUWE GONAUQTROP‘EN Theais for fine Degree 9? M; 5. [WCWGAN STATE COLLEGE Thamas N. .fiahnmn EM? This is to certify that the thesis entitled "The Effect of Alterations in Thyroid Activity on the Response of the Ovaries of Immature Female Rats and Mice to Equine Gonadotropin" presented by Thomas N. Johnson has been accepted towards fulfillment of the requirements for M:— degree in Now / Major professor Datew THE EFFECT OF ALTERATIONS IN THYROID ACTIVITY ON THE RESPONSE OF THE OVARIES OF IMMATURE FEMALE RATS AND MICE TO EQUINE GONADOTROPIN By THOMAS N. ggmsorr 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 OF SCIENCE Department of Physiology and Pharmacology Year 1949 THESIS Thomas N. Johnson ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Professor E. P. Reineke and Dr. Joseph Meites under whose careful direction this Thesis was prepared. The author is very grateful to Dr. B. V. Alfredson, Head of the Department of Physiology and Pharmac010gy, for providing the materials, equipment and laboratory space with which to carry on the experiments des- cribed herein. The author especially wishes to thank Professor B. B. Roseboom, retired Head of the Department of Physiology and Pharmacology, for his guidance and inspiration. He wishes to thank Mr. John Monroe for procuring and so dili- gently caring for the animals involved. The author also wishes to thank Mr. George Crenshaw, senior veterinary student, for his assistance in the laboratory. Thomas N. Johnson TABLE OF CONTENTS INTRODUCTION OOOOOOOOOOOOOOOOOOOOOOOO0000...... mm OF THE LITEATIIRE OOOOOOOOOOOOOOOOOOO... Effects of Hypothyroidism............. EffBCts Of Hyperthyr01disme e e e e e e e e e e 0 Effects of Altered Thyroid Status on Pituitary Gonadotropic Function.. PBOCEURE OOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOO RESULTS oeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee. 1. 2. 3. 5. Effect of Thyroxine on Gonadogen Response in Young Female Rats......... Effect of Protamone on Gonadogen Response in'Young Female Rats......... Effects of Thiouracil on Gonadogen Response in Young Female Rats......... Effect of Protamone on Gonadogen Response in Young Female Mice......... Effect of Thiouracil on Gonadogen Response in Young Female Mice......... Figure]. eeeeeeeeeeeeeeeqeeeeeeeee FigureZ eeeeeeeeeeeeeeeeoeeeeeeee Figure eeeeeeeeeeeeeeoeeeeeeeeeo 3 Figure 4 eeoeeeeeooeeeoeeeeeeeeeee 5 OOOOOOOOOOOOOOOOOOOOOOOOO Figure DISCUSSION 000......00.000.000.000...0.00...... SUMMARY AND CONCLUSIONS OOOOOOOOOOOOOOOOOOOOOO. 12 12 13 14 15 16 18 19 2O 21 22 25 26 Thomas N. Johnson TABLE OF CONTENTS (Comm) APPEIIDIX OOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOO 28 Table l .......................... 29 Table 2 .......................... 30 Tables .......................... 31 Table 4 .......................... 32 Table 5 .......................... 33 BIBLIOGRAPHY OOOOIO‘OOOOOOOO0.00.000.000.00. 34 Thomas N. Johnson INTRODUCTION It has long been established that a func- tional relationship exists between the thyroid gland and the reproductive organs. Alterations in thyroi- dal activity have a definite influence on the function of the gonads.‘ Clinical investigators have found that when reproductive disorders coexist with abnormal thyb roid function, the reproductive system often returns to normal after the abnormal thyroid condition has been corrected. Little is known regarding the physiological mechanisms of these clinical findings. Conceivably the thyroid could play a part in one or more of the following mechanisms: 1) It might have a direct effect on the repro- ductive organs. 2) It might be necessary for the elaboration of gonadotropins by the anterior pituitary. 3) It might act on the gonads indirectly through changing the metabolic rate of the body as a WhOle o It was thought that a good experimental approach to one aspect of the problem would be to determine the influence of experimental alterations 1 trim Thomas N. Johnson of thyroid function in immature female rats and mice on the response of the gonads to a constant dose of exogenous gonadotropic hormone. Hyperthyroidism.was induced by administering either thyroxine or Prota- mone, and hypothyroidism.was induced by feeding thiouracil. The gonadotrophin used was pregnant mare's serum, which is predominantly a follicle stimulating hormone. REVIEW OF THE LITERATURE Functional Relationships between the Anterior Pituitary, Thyroid and Ovary Thyroid-Ovarian Relations Effects of Hypothyroidismg' Menstrual irregularities have been associated with the symptoms of both hyper- thyroidism.and hypothyroidism. Foster gt_gl. (1939) treated fifty patients who had dysmenorrhea, oligo-' menorrhea, amenorrhea, metrorrhagia or menorrhagia with desiccated thyroid. The average BMR preceding treatment was -15 per cent and all patients were other- wise healthy with normal pelvic findings. As a result of the thyroid treatment, 89 per cent of these patients recovered. 2 Thomas N. Johnson Lerman.g§_gl. (1942) reported that patients with myxedema due to primary dysfunction of the thy- roid had amenorrhea that was not due to the men0pause. The ovarian cycle returned to normal after the admin- istration of thyroid. Krohn (1947) observed that the normal estrous rhythm.in mice was disturbed by daily subcutaneous injections of 0.3 mg. propyl-thiouracil. The cycles were lengthened and irregular or disappeared. Estrous rhythm.returned 14 days after the cessation of injec- tions. Single injections of 5 I.U. of chorionic gonad- otropin into six mice showing prolonged anestrous dur- ing thiouracil treatment were followed in each case by a single estrus period. Vaginal sensitivity of spayed mice to estrogen was unaffected by propyl-thiouracil treatment. Aranow, Engle, and Sperry (1946) administered increasing amounts of thiouracil up to 0.8 gm. daily for 14 months to 4 adult female Rhesus monkeys and observed menstrual irregularities with frequent periods of amenorrhea. Scow and Simpson (1945) thyroidectomdzed 440 rats on the first day of life and reported that sexual infantilism.occurred in these animals. 5 m. Thomas N. Johnson Jones, Delifs and Foote (1946) reported that hypothyroidism induced by thiouracil had no effect on the reproductive system in adult male rats as Judged by their ability to sire litters. Sterility was not induced by thiouracil in females, but gestation was abnormal when the treatment was prolonged. Resorp- tion of the fetus resulted in 100 per cent of the cases which received prolonged doses. When the treat- ment was administered during a period of less than 100 days, some of the females delivered litters that were normal in growth, development and reproduction. Effects of Hyperthyroidism: Reineke and Turner (1945) used synthetic thyroprotein to influence some of the body processes of farm animals. It was pointed out that replacement therapy with thyroid material could restore the normal metabolic rate in the slightly hypo- thyroid animal and improve the reproductive system markedly. In males it was pointed out that normal sex life can be restored, and the quality of the semen improved, while in females the ovaries become active and normal estrous cycles ensue. Ershoff (1945) found that female rats raised to maturity on diets containing 0.5 per cent and 1.0 per cent desiccated thyroid showed marked diminution in ovarian development. The ovaries remained infantile 4 was Thomas N. Johnson in both.weight and histological appearance. Stein g£_gl. (1947) noted that single thy- roxine injections into mice invariably caused retarda- tion of proliferation in the germinal epithelium. Retardation was greater in immature than in mature mice. It was also greater during diestrus than during estrus. Langham and Gustavson (1947) administered d, l-thyroxine daily by subcutaneous injections at levels of l, 2, and 5 Ug. per gram of body weight for 3, 6, and 10 days and thereby decreased the estrus response of ovariectomized rats to 1.5 gamma of estrone as deter- mined by vaginal smears. Twenty-eight days after the 10-day period of thyroxine dosage the response to estrone returned to normal. The hormone secretion rate of rats is equivalent to approximately 3.5 micrograms of thy- roxine per 100 grams body weight (Monroe and Turner, 1946). Since Langham‘g§_al. (1947) administered thy- roxine at the rate of 100 micrograms or more per 100 grams body weight, their rats were probably stimulated to the thyrotoxic level. Hill (1948) administered thyroxine within the physiologic range and reported that the estrous response of immature rats decreased with high doses of thyroxine; and conversely, increased with the 5 if“! Thomas N. Johnson administration of thiouracil. Young rats given toler- able doses of thyroxine (7 mg. per 100 gm. rat), over a period of ten days, showed a decrease in response to estrone. Senescent rats given similar dosages showed a slightly increased response. Hyperthyroidism appears to exert either a favorable or negative influence on reproductive pro- cesses in the rat but this does not appear to be true in other species. Martinez (1947) fed thiouracil to the domes- tic fowl at a dosage of 0.1 per cent of the ration for a period of four weeks and reported little change in the semen volume, motility, or sperm concentration. Thyroprotein fed as 0.01 and 0.02 per cent of the ration did not influence semen production, but when fed at 0.04 per cent the total number of sperm.per ejaculation increased by 65 per cent. Meites and Chandrashaker (1949) reported that thyroprotein partially or completely inhibited the response of the seminal vesicles and coagulating glands of male rats when pregnant mares' serum was injected, while therprotein increased the gonadotro- pic response in the male mouse. These investigators also found that thiouracil increased the gonadotropic 6 ta; Thomas N. Johnson 7 response to pregnant mares' serum in rats and reduced the response in mice. Effects of Altered Thyroid Status on Pituitary Gonado- tropic Function: It is evident that there are complex interrelationships between the pituitary, thyroid and ovary, and it appears that a balance must be maintained between these glands for efficient reproductive func- tion. P'an (1940) reported that the gonadotropic potency of the anterior lobe of the pituitary was decreased following thyroidectomy in normal and cas- trate rats and normal rabbits. Evans and Simpson (1929) reported that the gonad-stimulating properties of the anterior pituitaries from.hyperthyroid rats were increased, while the glands from hypothyroid rats were less effective than normal. Chu (1944) found that in thyroidectomized rabbits the ovaries contained many more large follicles than normal controls, but ovulation did not take place after coitus. The animals that were operated on readily ovulated after the injection of pregnancy urine extract, and the ruptured follicles were more numerous than in normal estrus animals. Fresh saline extracts were prepared from the pituitaries of the normal and thyroidectomized m Thomas N. Johnson rabbits for the assay of their relative amounts of ovulating hormone. The pituitary extract from.the normal animals caused 60 per cent ovulation in estrous rabbits, whereas pituitary extract from the thyroidectomized rabbits caused no ovulation and only induced growth of follicles. Reineke, Bergman, and Turner (1941) reported on the effect of thyroidectomy in eight male kids. They were thyroidectomized between the ages of five and twenty-four days and killed at the end of four months, after growth stasis had appeared. Their pituitaries were assayed for gonadotropic hormone and compared with groups of normal controls of the same weight and age. The gonadotr0pic hormone was found to be present in low concentration and the testes showed lack of stimulation, weighing less than in the young- est group of normal controls. It appears from.the review cited that hyper- thyroidism.generally exerts unfavorable influences on reproductive processes in rats, while in other species a slight degree of hyperthyroidism.is favorable to repro- ductive action. It was thought that further light might be thrown on some of the endocrine interrelationships 8 sis Thomas N. Johnson involved by determining (l) the extent to which the response of the rat and mouse ovary to gonadotropin could be modified by alterations of thyroid function, and (2) whether there was any species or sex differ- ence in the gonadal reactivity of the rat and mouse to these hormonal stimuli. PROCEDURE A total of 22 groups of immature female albino rats (averaging 6 animals per group) of the Michigan State College strain, and 14 groups of imma- ture female albino mice of the Rockland strain were used for these studies. Various degrees of hyperthy- roidism were induced in the first series of rats by the injection of thyroxine subcutaneously for a ten day period in dosages equivalent to 1,5 to.12.0 ,‘ times the approximate normal thyroid secretion rate. Hyperthyroidism was induced in the second‘series of rats by incorporating Protamone” in the ration (Purina Laboratory Chow) in concentrations of .04 to 1.28 per * An iodinated casein product containing approximately 3 per cent thyroxine as determined by chemical assay. ”Protamone was made available through the courtesy of the Cerophyl Laboratories, Inc., Kansas City, Mo. 9 Thomas N. Johnson. 10 cent and feeding each mixture for a ten-day period. Hyperthyroidism was induced in the mice by incorporat- ing Protamone in the ration in concentrations of 0.2 to .52 per cent for a ten day period. Varying degrees of hypothyroidism were induced in the rats by incorporating 0.1 per cent thiouracil in the ration and feeding for four to twenty day periods. Hypothyroidism was induced in the mice by including 0.1 per cent thiouracil in the drinking water for periods of from.seven to twenty days 0 Those groups of rats and mice which were to receive thiouracilfor the longer periods were started at an earlier age in order to reach approximately the same body weights at the end of the experiment as the groups treated for shorter periods. A constant dose of pregnant mare's serum (Gonadogen), one Cartland-Nelson unit, was always injected during the last four days of thyroxine, thy- roprotein, or thiouracil administration in the rats. Kindly supplied by the Upjohn Company, Kalamazoo, Michigan. Thomas N. Johnson 11 One-half Cartland-Nelson unit was similarly injected into the mice. Two control groups of rats, one untreated and the other injected with Gonadogen only, were used in the series of rat eXperiments. Two control groups of mice were similarly used in the thyroprotein-treated mouse series. A control group of mice, treated with- Gonadogen only, was run with each group of mice which received thiouracil. All animals were fed a balanced stock diet (Purina Laboratory Chow) and drinking water was avail- able at all times. The animal groups were kept in individual cages, at a constant temperature of 75 degrees F. Standard errors of the mean were determined for each group of animals. The significance of the differences between averages was determined by the "t“ test as described by Snedecor (1946). Thomas N. Johnson. 12 RESULTS 1. Effect of Thyroxine on Gonadogen Response in Young Female Rats. For the purpose of estimating the thyroxine dosage,the thyroid secretion rate was assumed to be equivalent to 4.65 micrograms of d, l-thyroxine daily as reported by Monroe and Turner (1946). The first series of rats received thyroxine injections subcutaneously in dosages ranging from.l.5 to 12 times their estimated normal secretion rate dur- ing a 10 day period, plus one Cartland-Nelson unit of pregnant mare's serum (Gonadogen) injected subcutan- eously during the last four days. In all the thyroxine treated groups, the response of the ovaries to the Gonadogen was reduced ‘when compared to the controls which received Gonadogen only (Table 1, Fig. l). The rats which received the lowest thyroxine dose (1.5 times the normal) had ovaries weighing 71.56 mgs. per 100 gm. body weight, which represents a sig- nificant decrease below the control value of 99.0 mgs. Thomas N. Johnson 15 At thyroxine doses of 4 times normal and above, ovarian weight was depressed still further below that observed at 1.5 times the normal level. A statistical check (t test) shows these differences to be highly significant. 2. Effect of Protamone on Gonadogen Response in‘Young Female Rate. The second series of rats were given Prota- mone in their feed in doses ranging from.0.04 to 1.28 per cent of the total diet during a period of ten days, plus one Cartland-Nelson unit of Gonadogen injected during the last four days (Table 2, Fig. 2). The group of animals which received 0.04 per cent Protamone (lowest dose) had ovaries weighing 66.58 mg. per 100 gm. body weight, a highly signifi- cant decrease below the control value of 99.0 mg. There was a progressive decrease in the ovarian weights of all groups of rats which received doses of .08 to .52 per cent Protamone. However, the ovary weights of the rats that received 0.64 and 1.28 per cent Protamone were slightly above those that received 0.52 per cent Protamone. Thomas N. Johnson 14 A statistical check (t test) showed no significant differences between the groups which received .52, .64, and 1.28 per cent Protamone. 0n the whole, these results agree with the thyroxine injected series, since both thyroidal substances reduced the ovarian response to Gonadogen. 5. Effect of Thiouracil on Gonadogpn.Response in 'Young Female Rats. The groups of rats which received 0.1 per cent thiouracil in their diet for periods of four and seven days, plus Gonadogen, showed a highly signifi- cant increase in their ovarian weights over the non- thiouracil treated controls. The rats which received the thiouracil for the shortest period of time (4 days) had ovaries weigh, ing 155.98 mg. per 100 gm. body weight (first group) and 161.74 mg. per 100 gm. body weight (second group), a highly significant increase above the control value of 99.0 mg. The two groups which received thiouracil for seven days showed a still greater increase in ovarian weight (Table 5, Fig. 5). The rats which received thiouracil for 10 and 15 days had ovaries weighing more than the controls but tr] Thomas N. Johnson 15 less than the groups that received the drug for 4 and 7 days. The rats which received thiouracil for 20 days had ovaries weighing only 65.5 mg. per 100 gm. body weight (first group) and 66.19 mg. per 100 gm. body weight (second group), which represent signifi- cant decreases below the control value of 99.0 mg. Thus thiouracil increased the ovarian response to Gonadogen in all groups except when given for a 20 day period. 4. Effect of Protamone on Gonadogen Response in YOung Female Mice. Six groups of mice, consisting of seven animals each, received doses of Protamone varying from .02 to .16 per cent of their diet during a period of 10 days, plus one-half Cartland-Nelson unit of Gonadogen injected subcutaneously during the last four days. The group of animals which received .02 per cent Protamone (lowest dose) had ovaries weighing 51.07 mg. per 100 gm. body'weight, a highly signifi- cant increase above the control value of 41.14 mg. Thomas N. Johnson 16 In all the Protamone groups, the response of the ovaries to Gonadogen was increased when comp pared to the controls which received Gonadogen only (Table 4, Fig. 4). There was a progressive increase in the weights of the ovaries in the groups that received .02 to .08 per cent Protamone. Although the group that received .16 per cent Protamone showed an increase in weights of the ovaries above the control value of 41.14 mg. per 100 gm. body weight, the ovaries of these mice weighed less than the ovaries of the mice which received the lowest dosage of Protamone. A statistical check (t test) shows signifi- cant differences between the untreated control group of mice and the control group which received Gonado- gen only. Highly significant differences were also found between the control group which received Gonado- gen only and all the groups which received Protamone plus Gonadogen. 5. Effect of Thiouracil on Gonadogen Response in‘Young Female Mice. It can be seen that the ovarian weights of Thomas N. Johnson 17 the four groups of mice which received thiouracil in their drinking water for periods of 7 to 20 days were not significantly different from the ovary weights of their corresponding control groups (Table 5, Fig. 5). The group which received thiouracil for the shortest period of time (7 days), had ovaries weighing 68.94 g 5.99 mg. per 100 gm. body weight, while the 7 day control group had ovaries weighing 62.54 g 1.27. The 10, 15 and 20 day groups also did not differ sig- nificantly from.their corresponding controls. /00 90 80 3 70 a- g 60 k S x § 50 i ; 40 o s E, 30 20 /0' Thomas N. Johnson I WDOGEN ONL y 7. CON 7340’. 6. 973» L NV‘QN soaks 7HVRO Y/NE DOSAGE" Figure 1 Effect of Thyroxine on Gonadogen Response in Young Female Hats. 18 Thomas N. Johnson 1 /00 f 90- 80' 3. * ,3 70- a; x - 60' 2 ~ a [ § 3 _ Lu ——T X 5 50 g E § 40' o Q (g 30_ Q) Q. 7' f3: Egfisawssas ékdddgdggg PER GEN 7 mmopno new /A/ Ea)“ Figure 2 Effect of Protamone on Gonadogen Response in YOung Female Rate. 19 Thomas N. Johnson & /80 - I60 - L90 ' /00 ‘ — 0W W7. l 60 20" .1: 646. Pm l00 CM. 300/ wfl v v '\ Q \ ‘0 \ Q N Q a. eCON ”a :covma - am” (My L , 5 Effect of Thiouracil on Gonadogen Response in‘Young Female Rats. DAKS‘ FED. 0./ FER CENT TH/OURAC/l. a“ 20 ts] Thomas N. Johnson 21 E K g 60*- E x g 50- 0: E a 40% Q U € 30- ] 20- * /0 - -:%?82 A W? CENT THVPOPPOTE/N /N ( FEED ICOVTRO’. :mvm-Gcwaoocm ONLY L _ a Figure 4 Effect of Protamone on Gonadogen Response in'Young Female Mice. Thomas N. Johnson 22 /00 90- 80- g: 70- K. g 60- ’— 3 3' 50. E 3 45. § § 0 g 30* “20 /0b °°“Mo°o DAYS FED 0./ PER CENT TH/OURAC/L . coma: - comboCEN our Figure 5 Effect of Thiouracil on Gonadogen Response in Young Female Mice. Thomas N. Johnson 23 DISCUSSION The results of this work indicate that when thyroxine or Protamone is administered with the object of modifying gonadotropic function, both the species of animal and the hormone dosage levels are very impor- tant. The conflicting results of some of the earlier investigations were probably due either to a failure to take into account the very high dosage of thyrox- ine used, or to recognize that different species may react differently to the same hormonal stimuli. Meites and Chandrashaker (1949) found that the administration of thiouracil or thyroprotein alone in male rats and mice had no effect on the weight of the seminal vesicles and coagulating glands. When pregnant mares' serum.was injected, the growth res- ponses of the seminal vesicles and coagulating glands were partially to completely inhibited by all except Thomas N. Johnson 24 the lowest levels of thyroprotein in the rat, while in the mouse all except the highest concentration of thyro- protein increased the gonadotropic response by 72 to 140 per cent. Thiouracil, particularly when fed for the longer periods, increased the gonadotropic response in rats by as much as 300 per cent, while the response in mice was reduced by 40 to 73 per cent. The results reported in this thesis on female rats and mice are in agreement with the findings of Meites and Chandrashaker (1949) in male rats and mice, with the exception of the female mice which received thiouracil. A reduction in response to Gonadogen.when given with thiouracil was not found in the female mice. A sex difference in the response of male and female mice to thiouracil plus Gonadogen is therefore indicated in these findings. Species differences are evident in the fact that the female rats showed a decreased response to Gonadogen when thyroxine or Protamone was administered, while the female mice showed an increased response to Thomas N. Johnson I25 Gonadogen when Protamone was administered. Thiouracil increased the response to Gonadogen in the female rats, but had no effect on the response in female mice. The question remains to be answered as to how alterations in thyroid function influenced the ovarian response to pregnant mare's serum in these ani- mals. The possibility exists that altering thyroid function may change the secretion of gonadotropic hor- mone by the animal's own pituitary. However, previous work indicates that immature rats and mice of the age used in these experiments do not secrete gonadotropic hormones (Moore, 1939). Furthermore, it has been shown, (Meites and Chandrashaker, l949),that in male immature rats and mice, the administration of Protamone or thiouracil does not alter the weights of the reproduc— tive organs of these animals during short term treat- ments such as were used in these experiments. It is possible that the thyroid hormone may exert a direct effect on the gonads, altering their reactivity to gonadotropic hormones. This would seem to be indicated from the recent observation of Warner and Meyer (1949) that the administration of thyroxine to ovariectomized female -- intact female parabiotic rats prevented ovarian hypertrophy in most of the intact partners. Thomas N. Johnson 26 The general metabolic changes induced in the body by altering thyroid function must also be taken into consideration, since changes in metabolic rate alone may affect gonadal function. However, no con- clusive information is available on this point. 1. 2. 3. SUMMARY AND CONCLUSIONS Immature female rats given tolerable doses of thy- roxine (1.5 to 12 times the approximate thyroid secretion rate), over a period of ten days, showed a decrease in response to the gonadotropic hormone of pregnant mares' serum (Gonadogen). Immature female rats given tolerable doses of Protamone (.04 to .52 per cent in the diet), over a period of ten days, showed a decrease in response to the gonadotropic hormone of pregnant mares' serum. Higher dosages of Protamone (.64 and 1.28 per cent in the diet), did not decrease the response of the ovaries more than .32 per cent Protamone. Immature female rats given 0.1 per cent thiouracil for varying periods of time (4 to 15 days), showed an increase in response to the gonadotropic hor- mone of pregnant mares' serum. However, when these 5. Thomas N. Johnson 27 ratswere given thiouracil for 20 days, the ovaries showed a decrease in response to Gonadogen. Immature female mice given tolerable doses of Protamone (.02 to .08 per cent in the diet) over a period of ten days showed an increase in their response to Gonadogen. This response indicates a species thyroid difference between rats and mice. However, when the mice were given a higher dosage of Protamone (.64 per cent of the diet), the response of the ovaries to Gonadogen was less than with low dosages of Protamone. Immature female mice given 0.1 per cent thiouracil for varying periods of time (4 to 20 days in the diet or drinking water), showed no significant changes in their ovarian response to Gonadogen when compared with controls which.received Gonadogen only. This again indicates a species difference between the rat and mouse, since in the former thiouracil increases the response to Gonadogen while in the latter it decreases the response to Gonadogen. Thomas N. Johnson 28 APPENDIX Thomas N. Johnson 29 APPENDIX Table 1. Effect of Thyroxine on Gonadogen Response in Young Female Rats. -__—-_ Treatment Ave. Ave. Ave. Ave. Wt. No. Gamma XNa Orig. Final Wt. of Ovaries Group of Thy- Thy- Body Body of per 100 gm. Rats roxine roxine Wt. Wt. Ovar- Body‘Wt. gm, gm. ies mg. h 1118- 1 6 Controls 45 69.7 17.55 25.17 2. 6 Controls / Gonadogen only 40 77.1 76.68 99.0 - 4.564% 5q 6 6.9 1.5 47 96.66 68.98 71.52 i 2.71 4.. 6 9.2 2 45 88.5 61.7 69.61 g 1.75 5. 6 18.4 4 50 89.5 51.6 57.71 if 0.82. 6? 6 27.6 6 47 96.4 57.66 57.00 i 0.87 7. 6 - 1.51 55.2 12 47 103.0 41.03 41.74 a XN = Times normal thyroid secretion rate. 21‘ «(m-I) * These groups received Gonadogen. #4 Standard error of mean = \ Thomas N. APPENDIX Johnson Table 2. Effect of Protamone on Gonadogen Response in Young Female Rats. Treatment Ave. Ave. Ave. Ave. Wt. No. Orig. Final Wt. of Ovaries Group of Protamone Body Body of per 100 gm. Rats % Wt. Wt. Ovar- Body Wt. 'gm. gm. ies mg. mg. 1 Con- 6 No Treat- 45 69.7 17.55 25.17 trols ment 2 .- % Coné‘ 6 Gonadogen 40 77.1 76.68 99.0 4.56% trols Only 5*. 5 .04 45-50 90.2 59.86 66.58 g 1.48 ‘4L' 6 .08 45-50 85.41 54.6 65.9 5 1.25 5% 6 .16 40 76.66 45.8 59.72 5 1.57 6*. 6 .16 45-50102.85 55.58 56.55 {2.06 7".” 5 .52 50 107.8 54.48 50.56 £5.12 8; 6 .52 45 91.0 45.0 49.68 g 1.66 94 ‘6 .64 45 95.6 50.2 52.49 i 1.54 101; 6 1.28 40-45 85.9 44.04 55.55 5 1.19 a Standard error of mean 55!]- 71(71‘1) \(7t These groups received Gonadogen. 30 \‘ . '1 APPENDIX Thomas N. Johnson 51 Table 3. Effect of Thiouracil on Gonadogen Response in Young Female Rats. Ave. Ave. Ave. Ave. Wt. No. Days Orig. Final Wt. of Ovaries Group of on Body Body of per 100 gm. Rats Trial Wt. Wt. Ovar- Body'Wt. gm. gm. ies mg. mg- 1 . Con- 6 10 45 69.76 17.55 25.17 trols 2.x / Con- 6 10 40 77.1 76.68 99.0 - 4.564 trols 5h. 6 4 40 72.5 111.5 155.98 5 5.12 4h 6 4 40-45 65.0 101.7 161.74 5 2.57 5.’ 6 7 40-45 78.16 145.28 185.51 5 2.12 6i 6 7 50 80.0 151.40 189.42 £ 2.58 71' 10 10 45 86.5 97.2 112.41 £ 1.65 81: 6 15 45 85.75 87.7 104.71 £ 1.85 95 6 20 45 95.0 60.6 65.5 i 1.29 101; 4 20 45 79.2 52.5 66.19 5 2.5 a Standard error of mean 5.6" ”(n-t) -y1These groups received Gonadogen. \H/ APPENDIX Thomas N. Johnson Table 4. Effect of Protamone on Gonadogen Response in‘Young Female Mice. Treatment Ave. Ave. Ave. AveTNNt. No. Orig. Final Wt. of Ovaries Group of Protamone Body Body of per 100 gm. Mite Wt. Wt. Ovar- Body Wt. gm. gm. 188 mg. mg. 1 Con- 7 NO Treat- 8'10 16.4 4.5 27.8 trols ment 24' / Con- 7 Gonadogen 8-10 15.55 7.2 27.8 - .654 trols Only 51: 7 .02 10 17.5 7.84 41.14 1! 1.27 4{ 7 .04 10 16.5 8.71 51.07 i 1.51 5? 7 .08 10-11 16.5 9.26 52.27 i 2.82 6% 7 .16 11-12 15.6 7.54 55.74 5 2.47 73*1‘ 8 032 10 "" "“'- 47049 f 1080 «a Standard error of mean /2 d1 710H-0 as The animals in this group began to lose weight after the third day and died between the third and seventh days, possibly due to the toxicity of the high con- centration of the drug. .3' 1’These groups received Gonadogen. 32 Table 50 APPENDIX Thomas N. Johnson 55 Effect of Thiouracil on Gonadogen Response in Young Female Mice. Ave. Ave. Ave. Ave. Wt. Days Orig. Final Wt. of Ovaries Group on Body Body of per 100 gm. Mice Trial Wt. Wt. Ovar- Body Wt. gm. gm. ies mg. mg. Con- / trol 7 11-12 15.64 9.8 62.54 - 1.29% l 7 11-12 17.55 11.7 68.94 i 5.99 Con- / trol 10 10-11 21.2 12.55 58.25 - .60 2 10 10-11 17.5 10.5 61.55 Z 8.50 Con- / trol 15 8-10 21.7 12.1 55.42 - .68 5 15 8-10 18.0 11.4 60.29 a 4.11 Con— / trol 20 8-10 20.8 16.74 80.57 - 1.55 4 20 8-10 21.4 17.1 79.81 g 5.56 4 Standard error of mean 2 d1 «(n-I) One-half Cartland-Nelson unit Gonadogen injected during last 4 days of each experiment. Thomas N. Johnson 54 BIBLIOGRAPHY Aranow, Chu, J. Thomas N. Johnson 55 BIBLIOGRAPHY- H., Engle, E. T., and Sperry, W. M. 1946. Some effects of the administration of thiou- racil to monkeys. Endocrinology, Vol. 58, p. 551. P. 1944. Influence of the thyroid gland on pituitary gonadotropic activity in the rabbit. Endocrinology, Vol. 54, p. 90. Ershoff, B. H. 1945. Effects of thyroid feeding on ovarian development in the rat. Endocrinology, V01. 57, p. 2180 Evans, H. M., and Simpson, G. 1929. A comparison of anterior hypophyseal implants from normal and gonadectomized animals with reference to their capacity to stimulate the immature ovary. .Am. J. Physiol., Vol. 89, p. 571. Foster, R. 0., and Thornton, M. J. 1959. Thyroid in the treatment of menstrual irregularities. Endocrinology, Vol. 24, p. 585. Hill, M. H. 1948. The effect of age, and thyroid status on the response of ovariectomized rats to estrone. Thesis for degree of M.S., M.S.C. Thomas N. Johnson 56 Jones, G. E. S., Delifs, E., and Foote, E. C. 1946. The effect of thiouracil hypothyroidism on reproduction in the rat. Endocrinology, Vol. 58, p. 557. Krohn, P. L. 1947. The effect of propyl-thiouracil on the estrous cycle in mdce. Journal of Endocrinology, Vol. 5, p. 55. Langham, W. H., and Gustavson, R. G. 1947. Effect of level of thyroid activity on response of ovari- ectomized rats to estrone. Am. J. Physiol., Vol. 150, p. 760. Lerman, J. 1942. Glandular physiology and therapy. Am. Med. Assoc., Chicago, p. 406. Martinez, C. 1947. The influence of varying levels of thyroid activity on semen production in the domestic fowl. Thesis for degree of M.S., M.S.C. Meites, J., and Chandrashaker, B. 1949. The effects of induced hyper- and hypo-thyroidism on the response to a constant dose of pregnant mares' serum in immature male rats and mice. Endocrin- ology, Vol. 44, p. 568. Thomas N. Johnson 57 Monroe, R. A., and Turner, C. W. 1946. Thyroid Secre- tion Rate of Albino Rats During Growth, Preg- nancy and Lactation. University of Missouri Agricultural Experiment Station Research Bul- 1etin 403. Moore, C. R. 1959. Sex and Internal Secretions. Williams and Wilkins Co., Baltimore, Chapt. 7. P'an, S. Y. 1940. The gonadotropic potency of the anterior lobe of the pituitary of thyroidec- tomized rats and rabbits. Chinese Jour. of Physiol., Vol. 15, p. 189. (Biological Abstracts, Vol. 14, p. 1217). Reineke, E. P., Bergman, A. J., and Turner, C. W. 1941. Effect of thyroidectomy of young male goats upon certain anterior pituitary hormones. Endocrinology, Vol. 29, p. 506., Reineke, E. P., and Turner, C. W. 1945. Synthetic thyroprotein, a new drug available in veter- inary practice. J. Am. Vet. Med. Assoc., V01. 21, p. 102. Scow, R. C., and Simpson, M. E. 1945. Thyroidectomy in the newborn rat. Anat. Rec., V01. 91, p. 209. Thomas N. Johnson 58 Snedecor, G. W. 1946. Statistical Methods. The Iowa State College Press, Ames, Iowa. Stein, K. P., Quimby, J., and Moeller, A. 1947. R88- ponse of germinal epithelium to thyroid tissue or thyroxine in the ovarian capsule of the mouse. Anat. Rec., V01. 99, p. 249. Warner, E. D., and Meyer, R. K. 1949. The effect of thyroxine on the female reproductive system in parabiotic rats. Endocrinology, Vol. 45, p. 55. ‘Williams, R. H., Weinglass, A. H., Bissell, G. W., and Peters, J. B. 1944. Anatomical effects of thiouracil. Endocrinology, Vol. 54, p. 517. . lh44... xjnlcdmnll‘lbiiu .....}I|I’..Ill.'n.!l7t[n..1r|i. I‘l- - v)... .. . .I It. :5 .. . , o. . ROOM USE ONLY MICHI AN STATE UNIVERSITY LIBRARIES llIIIII IIIII IIIIII I 3 1293 062 3023