‘1. 1“ H. I' 1 m "1. 1 \ \‘ L '1 \ i “ii W l f if - _ THE EFFE"? OF Raga mm “Eqé‘é‘i'P-QED STATUS: SN TfiE RESPGNSE OF OVAREEQT‘GMSZED RAYS "Fail ESTRONE Thesis far the Degree of M. S. MICHIGAN STATE CCLLEGE Marion Han!ey HE" 1948 Thisistooertlfgthatthe thesis entitled The Effect of Age, and Thyroid Status on the Response of Ovariectomized Rate to Estrone presented by Marion Henley Hill has been accepted towards fulfillment of the requirements for Jain—degree mm 037 Major professor Dme_lnlz_21,;UEflim. 3 1" mm or AGE, AND macro sums on THE RESPONSE OF OVARIEClOMIZED RAB 10 BSIBONE MARION my A THESIS Submitted to the School of Graduate Studies of Hichigen Stete College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of “BER OF SCIENCE Department of Physiology end Phemeoology Year 1948 .THESJS Inrion Henley 3111 DEDICATIONS To my husband, Professor John D. Hill, this Thesis is affectionately dedicated. His constant understanding end co- operation in caring for our children, Joan Patricia, John Dillard and fiery Elisabeth have made this work possible. It is also dedicated to my parents , Kr. and Mrs. Edmund C. Henley, who have provided a continual source of inspiration. Marion Henley Hill Harion Henley Hill WW3 The author wishes to express her sincere gratitude to Professor E. P. Reineke, under whose careful direction this lhesis was prepared. To his generous contributions of time, energy and advice are largely due any merits this work may possess. She also wishes to express her thanks to Professor B. B. Roscbocm, Head of the Department of Physiology and Pharmacology, for providing the materials , equipment and laboratory space with which to carry on the expert-outs des- cribed herein. Finally, the author wishes to express her appreciation to Mr. John Monroe, whose careful and patient handling of the animals involved, contributed in no small neasure to the success of the experinents. Marion Henley Hill TABLE OF CONTENTS INEQDUCTION ......COOOOOOOOOOOO0.0....00..00.0.0000... MOP MLITERAWRE ..OOOOOOOOOOOOOOO0.0.00.0.0... 1. 2. 3. 4. 5. Functional Relationship between the Therid Gland and thO Ovaries eeeeeeeeeeeeo Response to Estrogen as Observed by thinal SMOQTB eeeeeeeeeeeeeeeeeeeeeeeeeeee Relation Of Thyroid t0 Pregnancy eeeeeeeeee Relation of lhyroid to Sperm Production ... Gonad, Thyroid, Anterior Hypophysis ROICtiODShiP eeeeeeeeeeeeeeeeeeeeeeeeeeeeee mmEDm .....OOOOCOOOOOOOOOO............OOIOCOOOOO... REsm’m .........OOOOOOOCOCOOOOOO.....OOOOOOOOOOOOOOOOO 1. 2. 3. 4. DISCUSSION Response of Old Rats to Estrone and Effect Of Thyroxine eeeeeeeeeeeeeeeeeeeeeee Response of Young Rats to Estrone . . . . . . . . . Effect of Thyroxine on Estrone Response in Young Rats eeeeeeeeeeeeeeeeeeeeeeeeeeeee Effect of Estrone, Thyroxine and Age, on weight Of Old Rats eeeeeeeeeeeeeeeeeeeee Figure 1 eeeeeeeeeeeeeeeeeeeeeeeeeeee Figure 2 eeeeeeeeeeeeeeeeeeeeeeeeeeee WY AND CONCLUSIONS ......OOOOOOOOO....OOOOOOOOOOOO mmn 0......0.0.000.........OOOOOOOOOOOOOO000...... T‘blGI 1 - 8, Viginfll SMSGT Ratings eeeeeeeeeee Table 9, Procedure for Making up Solutions of Estrone and d,l-thyroxine . . . . . . . . . 10 13 15 16 17 18 18 20 21 22 24 25 26 Marion Henley Hill TABLE OF CONTENTS (Con't.) Table 10, Vaginal Smear Classifications ....... 36 BIHIIOGRHHY ......OOOOO0.00000000.00.0000...0000...... 37 Marion Henley Hill mmggsc no; The number of investigations providing quantitative data on the effects of both hyper- and hypothyroidism on the response of spayed rats to estronc is rather limited. In general, it is thought that thyroid at low dosage exerts an anabolic effect in contrast to the catabolic action of medication with larger quan- tities. llcyer end Hertz (1938) used desiccated thyroid powder, thyroid globulin and d,1-thyroxine, and found that treatment with these materials at moderate doses for three days did not interfere markedly with the action of estrous; however, the de- mand for estrogen rises when thyroid is administered for more than a five day period. lhese experiments were carried on with eighty~day~old rats. Much of the literature indicates that results obtained are negative, with doses outside the physiological range. By physiological range, is meant a dosage level the suns as, or very close to the normal secretion rate of the animal 3 a rate at which no undesirable side effects can be determined by obser- vation. It is believed that young rats are usually hyperthyroid. Experiments have shown that their estrus response decreases with high doses of thyroxine; and conversely, increases with the admin- 1 Earion Henley Hill 2 istration of thiouracil, . thyroid-inhibiting drug. Normally, with increasing age, thyroid secretion declines. It is the purpose of this paper to describe experiments showing that the estrus response is in- creased in old, presumably hypothyroid rats, iiich have been adminis- tered a tolerable dosage of thyrozine for a period of time. It will also be observed that some-here between twelve and fourteen nonths, the sensitivity to estrogen decreases and the sensitivity to thyrozine increases. mm or m mummy: here is evidence that thyroxine, in some way, plays a role in reproduction in the female. The evidence of a specific relation- ship between the thyroid, ovaries or testes, and the anterior hypophy- sis is conflicting. As Francois Rabelais so appropriately put it, (Pantagruel, Boo]: III, Chapter 4) “he hierarchy (of theorgans) is such that incessantly one borrows from another, one lends to the other, one is the other's debtor.... each member prepares itself and strives anew to purify and refine this treasure.... to preserve and perpetuate the Human Face. All this is done by Loans and Debts one to the other." It is well to recall then, while considering the following experiments, tint one primary function of an organism is to maintain a normal endocrine balance. llarion Hanley all 3 1. Functional Relationship between the Thyroid Gland and the Ovaries lhe inter-relation between the thyroid and the ovary has been appreciated clinically for centuries, on accomt of the increased size of the thyroid at menstruation and pregnancy. Clinical cases have been cited by Lerman (1942), in which patients with myxedem. due to primary dysfunction of the thyroid had ancnorrhea not attributable to the menopause. 0n administration of thyroid the ovarian cycles of these patients became normal. This suggests that the thyroid, by stimlating'the ovary - either directly or indirectly, increases the production of estrogen and/er progestin, and the noraal cycle returns. This conception fits with the observations of Grumbreoht (1939) that thyroid increases the weight of the ovaries of infantile rats receiving a constant dose of gcnadotropic substance, the increase in the weight being proportional to the dose of the thyroid. Eagle (1945) reported that the hypothyroid monkey 1. usenorrheic to the extent of about one or two periods a'year. hat when a hypothyroid. monkey is put on a very low thyroid dosage for a period of ten days, the menstrual function always returns. a single treataent will give either two or three successive normal menstrual cycles. In the hypothyroid monkey the amount of estrogen needed to in- duce estrogen withdrawal bleeding is greatly increased. In monkeys treated with thieuracil, the menstrual cycles will again become very irregular. Amenorrhea or extremely prolonged cycles of over fifty days are the rule. Marion Henley Hill 2. Response to Betm a; Observed bllginal Smears Lee (1925), in his studies on the estrus cycle in the rat, found that following thyroidectomy there was, on the average, a one day lengthening of the cycle. His results also indicate that pro-pubertal thyroidectomy did not materially change the period re- quired to reach sexual maturity. He offers two alternative expla- nations: (a) Normally, the thyroid exercises a stimulating influ- ence on the ovary, and the removal of this stimulus causes a length- ening of the cycle; (b) Ovarian activity varies with general body activity, and thyroidsctomy affects the estrus cycle through its effect on metabolic processes. Conflicting evidence was presented by Boise and Psreny (1928) when they reported that the thyroid hor- mone was an antagonist of the ovarian homone (thselin) as evidenced by the fact that subcutaneous injections of thyroid autocoid not only prevented heat in normal female rats and mice, but also inhi- bitsd the action of the ovarian hormone in castrate animals. A. subsequent «1..th by Van Horn (1933) confirmed these findings. He fed desiccated thyroid powder to ovarisctomised rats and ob- served their response to estrous by noting the cellular composition of vaginal smears. He found that approximately three times as much estrogen was required to produce positive estrus smears as was neces- sary before thyroid feeding. Richter (1933), reported that vaginal smears taken on thyroi- dectomissd animals with intact ovaries showed a persistence of corm- fied cells in the vaginal nears at all times, as if there was a 4 Marion Hanley Hill 5 tendency toward a prolonged estrual phase. At autopsy, the animls showed hypertrophied uteri. Contrary to what one might expect, that is, that hypothyroi- dism would have the opposite effect to hyperthyroidism, miller (1958) reported that thyroidectomy was without effect on the response of ovariectomised rats to estradicl bensoate. Evans and Long (1921‘) reported that thyroidectosw in rats affected neither the onset-of puberty, nor the length of estrual cycles. they also reported (19211:) that feeding of fresh thyroid gland substance had no disturbing effect upon the estrual cycles. Da Costa and Carlson (1935) reported that female albino rats fed 0.5 to l mgm. of desiccated thyroid daily showed slight accelera- tion in sexual maturation, while those fed 5 to 10 man. of thyroid daily showed a definite retardation in pubescence. Langham and Gustavson (1947) reported on the effect of the level of thyroid activity on response of ovariectomised rats to estrone. They found that daily administration of d,l-thyroxine by subcutaneous injection at dosage levels of l, 2 and 5 neg. per gram of body weight for 3, 6 and lo days decreased the estrus response of ovariectomised rats to 1.5 mag. of estrous, as determined by the method of vaginal smears. Both the weight loss and the decrease in sensitivity to estrone were found to be logarithmic functions of the thyroxine dosage. The authors also reported that the daily administration of thiourea at levels of 0.05, 0.1 and 0.3 per cent in the drinking Marion Hanley Hill 6 water of ovariectomized rate over a period of seventy days inhibited growth and produced marked hyperemia and enlargement of the thyroid gland. he estrus response of spayed rats to 1.5 mg. of estrone was first reduced and then increased as dosage with thiourea con- tinued. 5. Relation of Thyroid to Prom In the ovaries of cretin rabbits htun (1915) found his- tologically a decrease in the nmber of primary follicles, an in- creased sise of the follicular space and degenerative changes in the ova. lhese degenerative changes of the ovary were confirmed by Kunde, Carlson and Proud (1929). lhen so to 140 mg. of desic- cated thyroid were fed daily to cretin rabbits for a period of four to eight weeks, or until symptoms of hyperthyroidism became defi- nitely established, Kunde observed a decided increase in the number of developing Graafian follicles and primordial ova. In rabbits with severe hyperthyroidism, experimentally induced, the process of estrus, ovulation, fertilisation, migration and implantation take place, but in most instances the young are never born, resorption occurring during the latter two-thirds of the gestation period. Similarly, Weichert (1930) fomd that feeding 0.25 gm. to 0.5 go. of desiccated thyroid daily to pregnant rats caused resorption of young in some animls, abortion in others, and in some cases a prolonged period of gestation with the ultimate delivery of dead young. Marion Henley Hill Hoskins (1910) subjected pregnant guinea pigs to thyroid feeding with the intent of influencing the gland weight of their offspring. me variations in susceptibility to the ingested thyroid were demonstrated by the following results: One animal died of hyperthyroidism after eight days of feeding 0.01 gram per day, whereas another animal survived for the same period of time on a dose twenty-five times as great. The author incidentally noted that abortions were frequent in the surviving animals. Gudernatsch (1915) reported that thyroid substance ad- ministered to male or female rats, had a tendency to prevent preg- nancy, and when pregnancy occurred to produce abortion. 4. Relation of glacid to Sperm Production Many factors enter into the picture of sperm production versus the thyroid gland. Observations show a rather distinct seasonal variation in sperm production. In sheep thyroidectomy of rams has been reported to depress sperm production, whereas in bulls normal semen and sperm were observed. Recent reports pre- sent definite qualitative data on the relationship of the thyroid to the production of spem. Berliner and Warbritton (1937) observed twenty-four rams during a two year period, and fomd a seasonal variation in sperm production, stunner being the low season. All Shropshires produced 7 Marion Henley Hill 8 semen of poor quality during the hot months but only the poorer Hampshires declined noticeably during the summer. It was sug- gested that poor sumner sperm production was due to the decline in thyroid hormone secretion due to the high temperatures pre- vailing during the summer. An intact Shropshire ram treated with thyroxine produced more normal sperm than usual, though in low concentration, during the summer. Two intact and one thyroidectomised Hampshire rams with high abnomality counts returned to normal after two doses (2 mg.) of thyroxine in August. Tlhey conclude that thyroxine therapy in a relatively normal animal with a less active thyroid can prevent the smumsr decline in normal spem production, or can restore sperm production early in the fall. A study was made by Petersen, Spielman, Pomeroy and. Boyd (1941) on a male Jersey, which was thyroidectomised at four months of age. lhey reported that thyroidectcmy of the male bovine causes a complete inhibition of libido, but had no effect upon speraogenesis or normality of the sperm. Oral administration of 25 grams of desic- cated thyroid over a period of three days restored normal activity and sexual behavior. Reineke (1946) reported on the effect of synthetic thyro- . protein on sterility in bulls. Fourteen bulls which had unsatis- factory breeding performance records, were fed thyrcprotein. Of this number improvement in vigor and libido was observed in ten. Marion Henley Hill The time required for an observable effect to occur ranged from seven to forty days. Definite evidence of improvement in the conception re- cord was obtained in only four cases. be limited conception records are suggestive, however, of an improvement in spermato- genesis. me thyroprotein was fed at the rate of about 0.5 to 1.0 gram per 100 pounds body weight. Da Costa and Carlson (1935) found in male albino rats fed 0.5 to 10 mm. of desiccated thyroid daily, a decidedly lower re- lative weight of the testes than in the controls. In most cases the sexual saturation of the gonads of the male rat was depressed by thyroid feeding (0.5 - 10 mm. per day). Martinez (1947) reported on the influence of varying levels of thyroid activity on semen production in the domestic fowl. no results showed that thyroprotein fed as 0.04 per cent of the ration causes a definite stimulation of spermatogenesis. Both the semen volume md the sperm concentration are increased markedly; conse- quently the total number of sperm per ejaculation is also increased. lilwsrth (1948) found that thyroprotein fed as 0.04 per cent of me ration causes a definite stimulation of spermatogenesis, in the domestic fowl. As compared to the control birds both semen volmne and sperm concentration increased to some extent with the re- sult that the total sperm per ejaculate increased rather markedly. In older roosters, similar but less pronounced trends were observed. 9 Marion Henley Hill 10 thyroprotein when given at higher levels of 0.08 and 0.16 per cent of the ration, depressed semen production and the total number of sperm decreased markedly. Hcskins (1916) reported only a very slight increase in weight of testes of rats fed desiccated thyroid. 5. GonadL 1331mm, Anterior HMs Relationship There is the possibility that the thyroid has an influence on the normal function of the hypophysis. Evans and Simpson (1950) observed that the gonad stimula- ting pr0perties of the anterior pituitaries from hyperthyroid rats were increased, while the glands from hypothyroid rats were less effective than normal. hey reported a qualitative relationship only. Reineke, Bergman and Turner (1941) reported on a group of eight male kids that were thyroidectcmised between the ages of five and twenty-four days, then killed at about four months of age, after growth stasis had appeared. lheir pituitaries were then assayed for gonadotropic hormone in comparison with groups of normals of the same weight (but younger) and of the same age (but normal weight). he gonadotrophic hormone was found to be present in low concentration and the testes showed lack of stimulation, averaging lighter than the youngest group of nomal controls. Marion Henley Hill 11 Bradbury (1947) reported that when estrogen is given to the innature female rat there is an increase in ovarian and pituitary weight in seventy-two to one hundred and twenty hours. he pituitary decreases in gonadotrophic hormone content in seventy-two to ninety-six hours. be less of pituitary potency precedes the increase in ovarian weight, indicating that effec- tive quantities of gonadotrcphin are released into the blood stream. Small doses of estrogen, l to 2 mcg. are sufficient to produce these effects. If rats are spayed the day before the initiation of estro- gen treatment, the pituitary does not decrease in potency in ninety- six hours even though the dose of estrogen is increased up to 200 The ovary accelerates or augments the estrogen stimulation of the pituitary of the infantile female rat. Baker and Everett (1947) reported that the injection of 2 meg. daily of diethylstilbestrcl into immature thyroidectomised rats for four days or into adult cvariectomised - thyroidectomised rats for ten days caused an increase in weight of the hypophysis, cellular enlargement, an accelerated mitotic activity among the acidcphiles and an increase in their number. Thus, it was demon- strated that this stimulating action of estrogen on the hypophysis is not mediated by the thyroid gland and that it may be elicited in the adult, as well as in immature rats. Marion Henley Hill 12 It may be well for the author to mention, in closing the review of literature, that as reported by Sadhu (1948), thyro- trcpic hormone has been assayed by the response in day-01d chicks and confined histologically by measuring the height of the epi- thelia in the thyroid acini. From the foregoing review it is evident that the thyroid secretion plays a part in the complex of hormones involved in reproduction in both the male and female. A report made by Schneider (1939), covering a review of the literature up to that time, on the effects of feeding thyroid substance, summarizes the subject somewhat in the following manner: thyroid feeding of the vertebrates shows that the thyroid which normally is an organ of internal secretion, may, if given orally, induce definite and con- siderable changes in the organism both immediately and over con- siderable periods of time. These changes, which are seen as so- celerations of metamorphosis in amphibians, or of either the -moulting cycle or feather production in birds, or of growth, or of milk or milk fat secretion in manuals, are all manifestations or expressions of the metabolic regulator function of the gland which is its basic job. myroid undoubtedly exerts its metabolic regulator activity on all protoplasm irrespective of the organism in which it occurs. Tne abundance of controversial evidence in the literature on thyroid feeding, or medication, prevents the drawing of any specific generalizations on the subject; hence there is need for Marion Henley Hill 16 carefully controlled experimentation in this field, with parti- cular emphasis placed on the importance of dosage in the physio- logical range of the animal. PROCEDURE lhe animals used were from a colorw of albino rats that have been kept in this laboratory for several years, desiyiated as the Michigan State College strain. Two groups of approximately thirty-six and sixty castrate femle rats, ranged in age from nine to fourteen months, and twelve to fourteen months respectively. Also one group of forty-two cas- trate female rats, three to four months old were used for a series of experiments, which were conducted to study the effects of age, and size of thyroxine dosage on the response of ovariectomized rats to estrone. The rats were fed a balanced stock diet, and drinking water was available at all times. lhey were kept in individual cages, under constant temperature of 75 degrees F. For the first four experiments the animals were divided into four groups of nine animals each. For experiments five, six and seven, a total of sixty rats were divided into groups of fifteen each. In all cases, each of the four groups were designated as: Marion Hanley Hill 14 Group A, which received the lowest dosage of estrone; Group B, which received the middle dosage of estrone, and served as a con- trol group for Group D; Group C, which was treated continuously with the highest dosage of estrone; Group D, which received the same middle dosage of estrone as did Group B, and was injected daily with d,l-thyroxihe for ten days prior to the estrone injec- tion. The last dose of thyroxine was given at the same time as the rats received the various doses of estrone. Forty-eight hours later the response to estrone was detemined by the method of vaginal smears (Allen and Doisy, 1923). All D Groups in experiments one to six received daily for ten days prior to the estrone injection, 7 mg. of d,l-vthy- roxine per 100 gm. body weight. This thyroxine dosage is about twice the normal secretion rate (Reineke, Unpublished). It will be noted that these animals were being given this dose of thyroxine daily, almost continuously from November 8, 1947 to April 27, 1948; or for approximately six months. Group D in experiment number seven, had ten times this thyrozine dosage, or 70 mg. per 100 gm. rat. This amounts to twenty times the usual thyroid secretion rate. me estrone dosage was .1, .2, .4 end .8 mg. per 100 9:1. rat in the first four experiments, and continued at .4, .8 and 1.6 mcg. per 100 an. rat in experiments five, six and seven. Except in experiments one through four, all rats were primed on the fifth day of each experiment with 15 mg. per 100 gm. rat. Marion Henley Hill 15 lbs animals were weighed before each injection of estrone, and the rats in Group D were weighed at the beginning of each ex- periment, to lave a basis for the thyroxine dosage. In experiment eight, two groups of twenty-one rats were set up. One was treated for ten days with d,l-thyroxine at the rate of 7 meg. per 100 9:1. rat. lhe other group served as a con- trol. At the end of the fifth day all fortyhtm rats were primed with .4 mcg. estrone per 100 pi. rat. 'me tenth day all of the rats were again injected with .4 meg. estrone per 100 gm. rat. Vaginal smears were made previous to experimcnt one and experiment five, to determine that the animals were in the diestrus stage. Smears were taken again forty-eight hours after the priming dose to detemine the percentage of the animals in the positive stage; that is, proestrus, or estrus. Five days after the priming dose smears were nmde to determine that the rats were again in the nega- tive condition. Snare were made to determine the final results of the experiments thirty-six and forty-eight hours after the terminal injections of thyroxine and estrone. For individual smear classification, see Tables 1 to 8 in the Appendix. RESULTS Normally, according to Lee (1925) any infection, inanition or other abnormal condition is quickly shown by a lengthened diestrum. Marion Hanley Hill 16 If the rats are accustomed to being handled, the procedure of with- drawing samples of vaginal fluid does not affect tne estrus rhythm. 1. Response of Old Rats to Estrone and Effect of Thyzoxine Rats in the first seven experiments, having been spayed for about a year, were so depleted of estrogen that fine first four ex- periments are offered merely as proof that priming was absolutely necessary. Priming with 1.6 mcg. per 100 gm. rat, and final doses of .4, .8 and 1.6 mcg. per 100 gm. rat, for various groups worked out most satisfactorily. It was found necessary to give all dosages of both thyroxine and estrone, in proportion to the body weight of the rat, in order to obtain consistent results. In experiments one and two the rats were not primed and there was no response whatsoever, to .2 mg. of estrone. The rats in experiments three and four were primed with .6 mcg. of estrone per rat, and the response to .2 mg. of estrone was in each case 11 per cent, or one rat out of nine was in estrus. These results may be checked for individual response to estrone in Tables 1 to 4 in the Appendix. Since all response in the first four experiments was very low, or negative, the dosage was increased for experiments five, six and seven and continued in a uniform manner for comparative purposes. When both estrone and thyroxine were given at physiologically effective levels, the per cent of rats showing a positive vaginal res- ponse was increased above that observed with the same dose of estrone given alone (Figure l). The thyroxine treated group showed an average Marion Henley Hill 17 response of nine per cent above the controls when both received .8 mcg. of estrone per 100 gm. rat. There is also evidence that the sensitivity to estrone is decreased in rats between eleven and fourteen months of age. Ten days previous to experiment seven, rats were injected daily with excessive doses of thyroxine (70 mcg. per 100 ya. rat). All doses of estrone remained the same in experiments five, six and seven. That is, Group A received .4 meg. , Group B .8 mcg., Group 0 1.6 mcg. and Group D .8 meg. per 100 gm. rat. However, in experiment seven, the response in Groups B, C and D were lower than in the two previous experiments; in experiments five and six Group 0 had a forty per cent response, and in experiment seven this dropped to thirty-three per cent. Group D in the last experiment showed a twenty-one per cent response, and in the two previous experiments thirty-three per cent. Group B in experiments five and six showed a positive response to estrone of twenty-six and twenty-one per cent respectively, which was less in all cases than the response in thy- roxine-treated Group D. Group A showed a twenty per cent response in experiment five, a fourteen per cent response in experiment six and in experiment seven the response was fifteen per cent, or two per cent above the results in Group B of the same experiment. 2. Regponse of Young Rate to Estrone It is interesting to note that in this particular strain of rats, approximately four times as much estrone is required by Marion Henley Hill 18 year-old rats, as by rats three months of age, for the same estrus response. This is shown in the following comparison: OLD RATS YOUNG RATS Per Cent Per Cent Dose 3: Response Dose Ag: Response .8 meg. 14 mo. 16 .l meg. 3 mo. 7 .8 meg. 12% mo. 21 .2 meg. 3 mo. 21 1.6 meg. 14 mo. 36 .4 mg. 3 mo. 35 Primed 1.6 meg. per 100 gm rat. Primed .4 meg. per 100 gm. rat. 3. Effect of Thyroxine on Estrone Resppnse in Young Rats In experiment sight, the control group of twenty-one rats showed a response of fourteen per cent above the thyroxine treated group (7 meg. per 100 gm. rat, daily for ten days). This is in agreement with the work done by Meyer and Warts (1938), and cor- roborates their contention that the demand for estrogen rises when thyroid is administered for more than a five day period, in three- month-old rats. Detailed data concerning this experiment may be found in Table 8 of the Appendix. 4. Effect of Estrone, ‘Jhyroxine and Age, on Weight of Old Rats A constant weight was maintained in rats ranging in age from nine to eleven and one—half months, during the first three experiments. This is readily observed in the graph (Figure 2), Marion Henley Hill 19 which shows the effect of estrone and estrone plus d,l-thyrcxine administration, and age on the weight of cvarieetcmised rats. This is taken to indicate that a tolerable dose of thyroxine was being given. However, somewhere between eleven and one-half and fourteen months, the weight decreased noticeably with the same dosage of thyroxine. It is therefore concluded, that at appro- ximately the same time tlmt sensitivity to estrone decreases, sensitivity to thyroxine increases. When an excessive dose of thyroxine (70 meg. per 100 gm. rat) was injected daily for ten days previous to the experiment, there was also a marked decrease in the weight. The animals at this time were thirteen and one- half to fourteen months old. For detailed data on individual weight see hbles l - 7 in the Appendix. Marion Henley Hill 20 50 PER CENT ESTRUS RESPONSE ITHYROX/NE DA/L Y 0 1 I / .3 l3. 5 I4 AGE, MON THS GROUP A .4MCG. £5 TRONE GROUP 3 .8MCG. ESTRONE GROUP c /.6MCG £5 TRO/vE GROUP 0 .GMCG. ES TRONE (PER /00 GRAM RA 7') ' Figure 1 The Effect of Estrone and Estrone Plus d,l-thyroxine Administration, in Relationship to Age, on Per Cent Response of Ovarieetcmised Rats. 21 Marion Henley Hill .Bam eeadaovecug a.“ 3303 do ewe use scaveapodudfied. 0393.311". seam enoavnm ens 0:3qu no 90093 SH N Paw?” Q .Q exec c9 gone .3: ......8q M2033 Sammie as . as 9.6 we we we no as: to: flex 2 Q 3 t E a. 1 _ _ _ a _ _ _ . — Q\m. Q; a z, .8: ca ,, .UU‘vaN l QNM. /. 5:6 mzficaxta a J. / x 1 96 / , 6%: xx a M I. w W .I‘ m .9 ... m .I||.....\¥L. .\. m «Unix/JV. Marion Hanley Hill 22 p;scussxon (It is evident from “the results of these experiments that much of the apparently contradictory evidence in the literature, with regard to the influence of thyroid function on reproduction, can be explained if the critical nature of the thyroid dosage level in determining the physiologic effect, is kept in mind. It was not realized that the dose of thyroxine must be near the animals can secretion rate, until Dempsey and Astwood (1943) reported on the determination of the rate of thyroid hor- mone secretion at various environmental temperatures. his paper was followed by a report made by Reineke, Miner and Turner (1945) on the effect of graded doses of thyroxine on metabolism and thyroid weight of rats treated with thiouraeil. It is evident that the im- portance of a tolerable dosage of thyroxine was not taken into ac- count , by the excessive amounts of thyroxine which were administered by the early investigators. As a setter of fact, it is impossible to determine just what. relation these dosages of thyroid substance actually bear in relation to the animal's own thyroid secretion rate, or tolerance level, because their thyroidal potency was not reported. The results of the present investigation show that there is a difference in the response to estrone of old and young ovariectomized rats, put on a tolerable dosage of thyroxine for ten days. The mecha- nism of thyroid action in this relation is not thoroughly established. Evidence of a relationship between the anterior pituitary, the thyroid Marion Henley Hill 23 and the gonads was reported by Evans and Simpson (1930). ‘lhey observed 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. Reineke, Bergman and Turner (1941) reported that thyroidectomised animals show a decrease in the gonadotrophic hormone of the hypophysis. There is also evidence of a direct effect of thyroxine upon the gonads. Richter (1933) found a direct effect upon the length of the estrus cycle; while DaCcsta and Carlson (1933) reported an acceleration in sexual maturation. Reiheke (1946), Martinez (1947) and Wilwerth (1948) reported on a direct relationship between thy- roxine and an improvement in spermatogenesie. This paper presents results which indicate that the sensitivity to estrone of the rat, a year or more old, decreases with age. his is presumably cor- related with a decline in thyroid function. Rats nine months to a year, mew a slight increase in estrus response when given a tolerable dosage of thyroxine for ten days, but never reach the per cent of res- ponse obtained in young rats (three to six months of age) 3 so evidently, thyroid is only one factor in the changes of senescence. The estrus response of both young and old rats treated with thyroxine at a level equivalent to twice the normal secretion rate re- mained the same, while the weight of rats given an excessive dose de- creased. Although the mechanism by which this is accomplished is not clearly understood, it has been observed that during the senescent period, that is, somewhere between eleven and fem-teen months, the Marion Hanley Hill 24 sensitivity of the rat to estrogen decreases, and thebsensitivity to thyroxine increases. n1. results mentioned in this paper seem to indicate that if the anterior pituitary, which nature devised to keep in perfect check the entire endocrine system, is not over influenced by one or more of the glandular secretions, the glands continue to function in a favorable, or normal manner. SUMMARY AND CONCLUSIONS 1. Young rats given tolerable doses of thyroxine (7 meg. per 100 gm. rat), over a period of ten days, show a decrease in response to estrone . 2. Nomally, with increasing age, thyroid secretion declines. The estrus response is increased in old rats (age thirteen to fourteen months) when given a tolerable thyroxine dosage (7 mcg. per 100 gm. rat), for a period of ten days. 3.. The estrus response and body weight are both decreased in old rats, when excessive doses of thyroxine (70 meg. per 100 go. rat) are administered daily for a ten day period. 4. It is observed that somewhere between eleven and one-half and fourteen months, the sensitivity to estrogen decreases, and the sensitivity to thyronne increases. Marion Hanley Hill 25 APPENDIX Marion Hanl ey Hill 26 APPENDIX Table 1. The vaginal smear ratings of rats in experiment 1 taken 48 hours after injection with estrone. These rats were not "primed” prior to the trial. the body weights were taken in grams immediately before the estrone injection. GROUP A C B D * Estrone D08. ms. 0'2 0.8 0.4 0.4 Veg. Body Vag. Body Vag. Body Vag. Body Rat No. Smear It. Smear Ht. Smear Wt. Smear Wt. l D 410 D 400 D 410 E 400 2 D 390 D 380 D 390 P 380 3 D 370 D 360 D 360 P 360 4 D 350 E 360 D 350 D 350 5 D 340 D 340 D 340 D 340 6 D 320 D 330 D 340 D 340 7 D 320 D 310 D 320 D 420 8 D 300 D 300 D 300 Mi 300 9 D 280 D 320 D 280 D 290 AVe BOdy Wt e and Per Cent 0 344.4 11 343.3 0 343.3 33 353.3 Estrus It Injected subcutaneously with 7 meg. per 100 gm. rat, d,l- thy- roxine, for 10 days prior to the estrone injection. Rats born February 1947; spayed May 1947. Experiment dates 11-18-47. APPENDIX Marion Hanley Hill Table 2. The vaginal smear ratings of rats in experiment 2 taken 48 hours after injection with estrone. prior to the trial. before the estrone injection. These rats were not ”primed” The body weights were taken in grams inediately 27 GROUP 0 B D e Estron: D080 meg. 0’05 002 0e]. 0.1 Vag. Body Vag. Body Vag. Body Vag. Body Rat No . Smear Wt . Smear Wt. Smear Wt. Smear Wt. 1 D 415 D 420 D 435 D 390 2 D 410 D 400 D 400 D 360 3 D 400 D 385 D 380 D 360 4 D 355 D 370 D 375 D 350 5 D 360 D 350 D 365 D 345 6 D 370 D 345 D 360 D 340 7 . D 365 D 330 D 330 D 420 8 D 330 D 330 D 315 D 310 9 D 290 D 340 D 325 D 300 Per Cent ~ Estrus and 0 366.1 0 363.3 0 365.0 0 352.8 .‘Ne BOdy Its It Injected subcutaneously with 7 meg. per 100 m. rat, d ,l-thyroxine for ten days prior to the estrone injection. Rats born February 1947; spayed May 1947. Experiment date: 1-18-48 Marion Hanley Hill APPENDIX Table 3. The vaginal smear ratings of rats in experiment 3 taken 48 hours after injection with estrone. These rats were ”primed” with .6 meg. of estrone per rat five days prior to the trial. The body weights were taken in grams immediately before the final es- trone injection. 28 GROUP A G B D e Estrone D080 m . 0.2 Gas ~ 004 0.4 _ Rat No. Smear Wt. Smear Wt. 'Smear _ Wt. Smear Wt. 1 D 420 P 430 D 445 M 380 2 E 420 P 415 D 410 P 340 3 M 420 M 400 D 400 M 365 4 M 360 M 380 D 390 P 355 5 D 380 M 360 D 380 M 350 6 D 380 M 360 D 380 M 340 7 D 380 M 345 D 335 P 420 8 M 340 E 350 D 325 M 310 9 D 310 M 355 D 350 M 310 Per Cent Extrus and 11 378.8 33 377.2 0 379.4 33 352.2 AVe BOdy Wte II- Injected subcutaneously with 7 meg. per 100 a. rat, d,l-thy- roxine, for ten days prior to the final estrone injection. Rats born February 1947; spayed May 1947. Experiment date: 1-30-48. Marion Hanley Hill APPENDIX Table 4. The vaginal smear ratings of rats in experiment 4 taken 48 hours after injection with estrone. These rats were ”primed" with .6 meg. of estrone per rat five days prior to the trial. The body weights were taken in grams immediately before the final as- trone injection. 29 GROUP A C B D It Estrone . Dose meg. 0‘2 008 0.4 0.4 Vag. Body Vag. Body Vag. Body Veg. Body Rat No. Smear Wt. Smear Wt. Smear Wt. Smear Wt: 1 D 410 M 420 D 430 D . 380 2 D 410 M 400 M 410 M 350 3 E 400 D 390 P 590 D 365 4 M 360 M 360 D 375 D 345 5 D 370 M 350 M 370 D 350 6 D 380 D 360 D 380 E 320 7 D 360 D 350 D 340 M 410 s D 320 E 330 D 525 M 320 9 D 300 M 360 D 350 D 300 Per Cent Estrus and 11 367.7 11 368.8 11 374.5 11 348.8 Av. Body Wt. 1* Injected subcutaneously with 7 mcg. per 100 gm. rat, d,1-thy— roxine, for ten days prior to the final estrone injection. Rats born February 1947; spayed May 1947. Experiment date: 2-16-48. Marion Hanley Hill APPENDIX Table 5. The vaginal smear ratings of rats in experiment 5 taken 48 hours after injection with estrone. These rats were "primed” with 1.6 meg. per 100 gm. rat five days prior to the trial. The body weights were taken in grams immediately before the final es- trone injection. 30 GROUP A L c B D t Estrone 0.4 1.6 0.8 0.8 Dose meg. Vag. Body Vag. Body Veg. Body Vag. Body _Rat No. Smear Wt. Smear Wt. Smear Wt. Smear Wt; l D 420 E 440 E 445 E 385 2 dead dead D 420 D 430 P 360 3 E 390 D 370 D 420 dead dead 4 M 370 D 380 D 360 D 345 5 M 365 P 360 P 365 D 360 6 D 380 D 360 D 380 E 320 7 E 375 D 340 D 340 M 420 8 E 325 E 320 P 320 D 340 9 D 300 D 360 D 360 E 300 10 D 390 M 370 D 365 D 360 11 D 340 D 320 P 360 D 350 12 D 340 P 320 M 340 P 340 13 D 320 E 340 D 320 M 320 14 D 320 P 325 D 300 M 285 15 D 325 D 340 D 290 D 1 280 Per Cent Estrus and 20 354.3 40 357.7 26 359.7 33 340.4 AVe Body Win“; 4' Injected subcutaneously with 7 meg. per 100 gm. rat, d,1-thy- roxine, for ten days prior to the final estrone injection. Rats Nos. 1 - 9 born February 1947; spayed May 1947. Rate Nos. 10 - 15 born February 1947; spayed February 1948. Experiment date: 3-12-48. Marion Hanley Hill APPENDIX Table 6. . The vaginal smear ratings of rats in experiment 6 taken E hours after injection 'with estrone. Bless rats were "primed” with 1.6 meg. per 100 gm. rat five days prior to the trial. The body weights were taken in grams immediately before the final es- trone injection. 31 GROUP A C B D * Estrone 0.4 1.6 0.8 Gas Dose meg. Vag. Body Veg. Body Vag. Body Vag. Body Rat No. Smear Wt. Smear Wt. Smear Wt. Smear Wt. 1 D 440 s 440 P 440 D 360 2 dead dead P 430 D 440 M 340 5 s 390 D 360 graze” 425 dead dead 4 D 380 D 380 D 340 M 540 5 M 380 P 370 D 370 P 550 6 M 390 D 370 D 400 D 300 7 M 380 D 350 M 340 P 410 8 E 330 P 320 D 330 E 320 9 D 310 M 360 P 360 D 280 10 M 400 M 370 D 350 D 350 11 M 350 D 340 D 540 D 550 12 M 350 P 340 M 340 M 310 15 D 330 M 360 M 550 P 330 14 D 520 D 330 P 310 s 280 15 M 520 P 340 D 290 D 270 Per Cent Estrus and 14 362.1 40 364.0 21 360.0 33 327.9 Av. Body Wt. It Injected subcutaneously with 7 mcg. per 100 gm. rat, d,1-thy- roxine, for ten days prior to the final estrone injection. Rats Nos. 1 - 9 born February 1947; spayed May 1947. Rate Nos. 10 - 15 born February 1947; spayed February 1948. Experiment date: 4-3-48. Marion Hanley Hill APPENDIX Table 7. The vaginal smear ratings of rats in experiment 7 taken 48 hours after injection with estrone. These rats were “primed" with 1.6 meg. per 100 gm. rat five days prior to the trial. The body weights were taken in grams immediately before the final es- trone injection. 32 GROUP M _ A C _* B D s Estrone Dose meg. 0'4 1-6 0.8 0.8 Vag. Bedy' Vag. Body' Veg. Body Veg. Body Rat No. Smear Wt. Smear Wt. Smear Wt. Smear Wt. l D 440 D 440 E 440 M 350 2 dead dead E 440 D 450 E 320 3 D 395 D 370 D 425 dead dead 4 P 380 D 380 D 340 D 340 5 D 380 E 370 D 360 E 320 6 D 385 D 360 D 390 D 260 7 D 385 E 340 D 350 P 400 8 E 330 P 310 D 340 IM 310 9 D 310 E 360 P 370 D 270 10 D 400 D 370 D 360 D 340 11 D 350 D 320 D 345 D 330 12 D 360 D 330 D 350 D 300 13 D 330 D 360 D 320 D 320 14 D 320 D 340 D 310 D 270 15 D 320 D 340 D 280 D 260 Per Cent Estrus and 15 363.2 33 362.0 13 360.3 21 313.6 .Av. Body Wt. * Injected subcutaneously'with 70 meg. per 100 gm. rat, d,1-thy- roxine,for ten days prior to the final estrone injection. Rats N68. 1 - 9 born February 1947; spayed May 1947. Rats N68. 10 - l5 born.February 1947; spayed February 1948. Experiment date: 4-27-48. Marion Hanley Hill 33 APPENDIX e 8. The vaginal smear ratings of rats in experiment 8 taken 48 hours after injection with estrone. These rats were ”primed” with estrone, .4 meg. per 100 gm. rat five days prior to the trial. Group Control Thyroid Treated * Estrone D086 meg. 0e4 0e4 Rat No. Vaginal Smear Vaginal Smear 1 P D 2 D P 3 P D 4 P P 5 P D 6 P D 7 D D 8 P P 9 P D 10 P D 11 D D 12 D P 13 P P 14 D D 15 D D 16 D D 17 D D 18 P P 19 D D 20 D P 21 D D Per Cent Estrus 4701 33e3 * Injected subcutaneously with 7 meg. per 100 gm. rat, d,1-thy- roxine, for ten days prior to the final estrone injection. Age of rats - 3 months; spayed at 2 months of age. Experiment date 2 6-14-48. Marion Hanley Hill APPENDIX Table 9. Procedure for making up solutions of estrone and d,1-tfiroxine used in injecting the rats. 1. Splution of Estrone* in Corn Oil Step No. Dilution 1 Stock Solution (original made up 11-14-47) 1000 meg. per 2 es. 500 meg. per 1 cc. 2 Stock Solution 500 meg. per 1 cc. original stock solution 1 cc. of No. l and 9 cc. of corn oil 50 mcg. per 1 co. 3 ’ 0.1 mcg. per.05 cc., take 1 cc. of No. 2 and 24 cc. of corn oil 4 0.2 meg. per .05 cc., take 1 cc. of No. 2 and 11.5 cc. of corn oil 5 0.4 mcg. per .05 cc., take 1 cc. of No. 2 and 5.25 cc. of corn oil 6 0.05 no . per.05 cc., take 143-- ee. of No. 5 and lug cc. of corn oil 7 1.6 meg. per .05 cc., take 12.8 cc. of No. 2 and 7.2 cc. of corn oil It Estrone obtained from Upjohn Company, Kalamazoo, Michigan 2. Solution of djl-thyroxine Step No. 1 Weigh out X meg. d,1-thyroxine 2 Wash off ooverglass with small amount of 1 Nermal NaOH (1N sol. 1 gm. per liter) 3 Shake until thyroxine dissolves and add several drops HUI. (to neutralize) until slightly cloudy Marion Hanley Hill 35 4 Add about one drop NaOH to redissolve 5 100 ml . flask Strength of solutions used in foregoing experiments: 7 mcg. per 100 go. rat 10 times as much d,1-thy- .01 cc. per 10 go. roxine to make 70 meg. per 1 cc. per 100 gm. rat 100 gm. rat Marion Hanley Hill APPENDIX Table 10. Standards used for classification of vaginal mmears in.the rat, as reported by Asdell (1947). Stage I Small, round nucleated cells only. Duration about 12 hours. PROESTRUM ° (P) Stage II Cornified cells only. Mating mostly in this stage. ESTRUS (E) Stage III Late cornified stage, abundant cheesy smear. Mating no longer allowed, as a rule. Stages II and III last about 27 hours. Stage IV Cornified cells and leucocytes. Duration about 6 hours. METESTRUM (M) Stage V Epithelial cells and leucocytes. Duration about 57 hours. DIESTRUM (D) 36 Marion Hanley Hill 37 BIEIOGBAPHY Marion Hanley Hill 38 BIRJOGRAPHY Allen, E., and Doisy, E.A. 1923. An ovarian hormone. 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