ll \IHHHMMIM Ml WW. HUI ‘ I I“ H I! H} l L 3 ! 1 m4 .. Ill 0301 HTHS INFLUENCE Q5 fiNVtRQNMENTAL TEMPERATURE AN D E‘H‘r‘R-Q! D STAYUS 3N 313E iEEROfiUCfiVE GRGAN S. O?- \r‘ @9338 fiMALfi 33.3365 T’hmis 3m 3329 32353-39 cri- M. 3. 3mm ETA'E’E CQ' 3.333 “g ‘ if”; finlv~zg 3.43;” m . Griigdu 544.}; fang «unifies a1; 1%.;2". {32-39 This is to certify that the thesis entitled "Influence of Environmental Temperature and Thyroid Status on the Reproductive Organs of Young Female Mice" presented by Fouad Atalla Soliman has been accepted towards fulfillment of the requirements for M.S. degree in Physiology 52M Major professor Date May lg, 1950 0-169 INFLUENCE G.“ ENVIROMIEMAL TEMPERATURE AND THYROID STATES ON THE REPRODUCTIVE ORGAI‘B w YOUNG FEM-E MICE by FOUAD ATALLA 399nm ATHE'SIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and.App11ed.Science in.partial fulfillment of the requirements for the degree of MASTER CF SCIENCE Department of Physiolog and Pharmacology 1950 ACKI‘IGIIEDSEMEM‘S I would like to express my sincere gratitude for the help and guidance given me by Dr. E. P. Reineke, Professor of Physiology, Michigan State College. Dr. B. V. Alfredson, Head of the Department of Physiolog and Pharmac- ology, was very generous in providing the laboratory facil- ities of the department. Dr. Joseph Meites was also helpful in his valuable suggestions. My thanks are extended to Dr. Lois Calhoun, Head of the Departnent of Anatany, who gave me many helpful suggestions, and also permitted me to use labor- atory facilities for the histological work. I appreciate the fine work done on the photo- graphs by Mr. M. L. Gray of the Department of Animal Patholog. m appreciation, also, goes to Mr. J. Munroe for taking care of the animals. Finally, m deep appreciation is extended to the Egyptian Government for the Opportunity afforded me through the scholarship that made this work possible. 7 T) g 11 ,3...) ,‘.1 .v «2 TABLE OF COMN‘I‘S INTRODUCTION ......OOOOOOOOOOOO......OOOOOOOOO00.00.00.000 WWLITEMW ......O.........OOOOOOOOOO0.0.0.0...O The Effect of Environmental Temperature on Thyroid Aetiflty ......OOOOOOOOOOOOOOOOO......OOOOOOOOOOIOOOOO Thyroid Active Preparations GOitrogens eeeeeeeeeooeeeeoeeeoeooeeeeeeoeooeeeeeeeeee The Effect of Mild Hyperthyroidism on Growth . . . . . . . . . The Relation Between the Thyroid Gland and the Female ReprOductive SYStem eeeeeeeeeeeoeoeoeeoeoooeeeeeoeoeee 6 Eat 000000.00eeeeeeeeeeoeoeeeeee000.000.0000 Mouse 0.00.00...0.0......OOOOOOOOOOOOOOOOOCO 9 Rabbit 0.0.0..........OOOOOOOOOOO...0...... lo Gall-Bea Pig eeeoeeeeeeeeeeooeooeeeeeeeeeeoee cattle 00.000.00.00.........OOOOOOOOOOOO... MATERIALS AND METHOIB oeeeeeeeeeeeeeeeeooeeoooeoooo00000. Feeding ...00.000.00.00.........OOOOOOOO......OOOCOOC Temmmtm am Light eeeeeeeeeeeeeeeeeeoeeoeeeeeeeee Procedure eeeeeeeeeeoeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeee RBULE 00.0.00...0..0.0.00.0...O.....OOOOOOOCOOOOOOOOOOO Effects of Thyroprotein Feeding on Growth .. ... ... ... Effects of Thiouracil Treatment on Growth ........... Food and Water Consumption ......OOOOOOOOOOOO....0... 10 Kkfifiblfltfi 15 15 .eoooeoee00.000000000000000.00.90 ....O-II'IFIOI c-o..c00uo.e. 000.00.000.00...-coo-0010.000... ......‘I...'.OI Effects of Temperature .................... 15 Effects of Hyperthyroidism ................ 15 Effects of Hypothyroidism ..................16 Effects of Hyperthyroidism on Reproductive Organs .... 17 Effects of Ibrpothyroidism on Reproductive Organs ..... 19 DISCUSSION .............................................. 29 SUI-MARIAND CONCLUSIOIG eeeeeeeoeeeeeeeeeeeeeeeoeeeeeeeee 34 BIBLImRAPHY ............................................ 36 eeeeoOeeoteeeeeo ......IIIOOQOOO00.0.00... INTRQDUCTIGN Seasonal variations accompanied by: changes in such factors as temperature and light have been proved to influence the reproductive rhythm of female as well as male animals. The females permit copulation only during recurrent periods. On the other hand the evidence of such a relation between thyroid activity and the external environmental temperature and light has been proved. The thyroid gland is much more active in low temperatures and short days as those of the fall and {inter than in high temperatures and long days as those of Sumner. Such a sensitivity of both thyroid activity and re- production to changes in temperature and light irradiation suggest- ed an interrelationship between the tmrroid and reproductive organs. The purpose of this work is (l) to determine the effe ct of environmental temperatures on tlwroid activity and re- productive processes of female immature albino mice, (2) to find out the effect of mild hyperthyroidism induced experimentally by giving the mice minute doses of thyroprotein and (3) to determine the effect of hypothyroidism induced by giving thiouracil, on the rate of growth and reproductive activity of these mice. gym 0F QTEQTURE :23 Effect 3f Enviromental TemEmture 9_n Thyroid Activity. It is well known that the rate of thyroxine secret- ion is partially under the influence of the environmental tempera- ture. When the temperature is low (as in Winter) the rate of thy- roxine secretion is high in order to increase the rate of heat production to compensate the lost heat. When the temperature is high (as in Summer) the rate of thyroarine secretion is low and the rate of heat production is low since less heat is lost from the body.‘ Some of the work is cited here. Dempsey and Astwood (1943) were able to determine the rate of thyroid hormone secretion in rats and found it to be high in cold environments (1° C.) and low in hot environments (25° C. and 35° (2.). Reineke and Turner (1945) determined the amount of tbyroucine required to prevent an increase in the weight of the thyroid gland during the administration of thiouracil in White Plymouth Rock chicks at 2 weeks of age. Maxim secretion rate was observed during October and November. The thyroid secretion rate declined starting fran the month of February and remained at a low level during the Sumner. Mills (1918) and Kenyon (1933) exposed rats to a cold temperature and found that it increased the vascular activity of the thyroid and caused cellular hypertrophy that was believed to be induced by the metabolic strain. Iodine in a dose of 10 mcg. daily diminished or prevented this change. ' Further evidence was reported by Ring (1936) who measured the basal metabolic rates of rats kept at the temperature of 35° 0. and their basal metabolic rates when kept at the tempera- - 3 - ture of 6-80 C. It was greatly elevated when the animals were kept at the low temperature. It seemed possible that the inv crease in the metabolism.might be caused by the thyroid gland. Brolin (1946) confirms that the thyroid activa- . tion in response to cold is associated with an increase in baso- philic cells of the anterior pituitary and increased output of the thyrotropic hormone. Thygoid Agtive Preparations. Baumann (1895) discovered the presence of iodine in the normal thyroid gland. Kendall (1915) was able to isolate the active principle,thyroxine,from the thyroid gland and found that it contained a high percentage of iodine. Harington in 1927 produced the hormone synthetically. Crystalline thyronine was recovered from iodinated proteins by'Ludwig and.Mntzenbacher (1939) and Harington and Pitt Rivers (1939) but the potency of the iodine- ted protein was low. Reineke and Turner (1941, 1942) and Reineke (1949) prepared artificial thyroproteins hy the iodination of proteins such as casein, soyabean, protein and egg albumin. under optimal con- ditions of iodine input, temperature, and pH such preparations showed very high thyroidal activity. The actual hormone, thyroxine, ‘was recovered from these preparations and its identity was confirmed by microscopic structure, iodine content and spectographic analysis. They were highly active in stimulating metamorphosis of tadpoles. These preparations alleviated the symptoms of cretins ism.in thyroidectomized goats, elevated the metabolic rate of lab- - 4 - oratory animals, and increased milk production when fed to lactat- ing goats and dairy cattle. Qoitrggens. Before the use of goitrogens in inducing hypothyroid conditions, the usual technique was thyroidectomy, either complete or partial. Astwood et a1. (1943) found that the administration of sulfonamide compounds and thiourea caused an enlargement in the size of the thyroid. In 1944, Astwood and Bissell administered thiouracil to young rats and found a nearly complete disappearance of iodine from the thyroid gland in five days and a threefold increase in the size of the glands in 2 weeks. These effects were inhibited by hypophysectahy or by the injection of thyroxine, which proves that the thyroid enlargement occurs in response to excessive sec- retion of thyrotropic hormone resulting from decreased secretion of thyroxine (see a1so,‘Williams et al., 1944.and Paschkis et al., 1945) . Mixner et a1. (1944) tried both thiouracil and thiourea in chicks. ‘When 0.1 percent thiouracil was mixed with the feed they observed beginning enlargement of the thyroids by the fourth day and maximal enlargement by the twelfth day of thiouracil administration. By the injection of thyroxine to thiouracil-fed chicks the size of the thyroid was decreased. The amount of thyroxine that is necessary to maintain the normal size of the gland corresponds with the normal output of thyrox- ine fran the gland. The Etffect 2f Lug; mpgrthygoidism 33 m. The importance of the dose of thyroid employed in determining the results obtained was first emphasized by Parhon (1912) who suggested that small amounts of thyroid material are anabolic while a large dose is catabolic in effect. Moussu (1899) and Dott (1923) found that small amounts of thyroid tissue given to immature dogs always resulted in accelerating the growth rate. By feeding 1.9 mg. of fresh thyroid to immature male and female mice Robertson (1928) noticed an increase in their rate of growth. Koger, Reineke, and Turner (1943) reported that by giving 0.01 to 0.03 mg. of crystalline tim'roodne-sodimn daily or by feeding .04 to 0.32 percent thyroprotein in the ration of young growing mice their rate of growth was accelerated. Large amounts of thyrmcine or thyroprotein were toxic and inhibited growth. The gain was a true one with a high percentage of water and protein and decreased percentage of fat. In rats Koger and Turner (1943) found that with doses of 0.04 or 0.08 percent thyroprotein in their ration the growth rate was not affected or else was depressed. Chicks given mild doses of therprotein, fran 0.025 to 0.2 percent in their ration were reported by Irwin, Reineke, and Turner (1943) and Parker (1943) to gain more than the control animals during early life. Reineke et a1. (1948) reported that in pigs fed -6- thyroprotein at the level of 2.8 gm. per 100 pounds of feed there was a significant increase in the rate of gain. Maqsood and Reineke (1950) showed that thyroprotein when fed to immature male mice kept at 249 C. for a period of 4 weeks at 0.025 and 0.05 percent in the ration caused a signific- ant increase in the body weight gain when compared with the none treated group. Similar growth stimulation of male mice was also reported by Novak (1950). 1132 Relation Between thg mg 5g y; 3.9% Repgoductive Szgtem. A relationship between the thyroid and the reprod- uctive organs has been postulated for a long time, especially'as regards the female generative organs. The large size of the thyroid gland in women was noticed by the early anatomists, and the periodical enlargement of the gland during menstruation and pregnancy has long been.known, as indicated in the report of Freund in 1883. One indication of such a relationship is that during the sexual cycle the mitotic activity of the thyroid epithelium corresponds to the different phases of the sexual cycle. Chouke et a1. (1935) noted that in guinea pigs the minimum activity of the thyroid corresponds to the follicular phase, and the maximal activity to the luteal phase. A.similar relationship was noted in the studies of Bing (1928) on the sexe ual cycle in women. Hunt (1944) also noticed that in young female rats the mitotic activity of the thyroid gland increased somewhat in early estrus, but was maximal in late estrus; it declined in met- estrus and attained its minimum activity during diestrus and pro— estrus.‘ Salter (1949) stated that the ttvroucine iodine is fixed in two general locations. The first is its accumulation in the pituitary and ovary. The second is its accumulation in the peripheral tissues primarily in the skeletal muscle. A survey of the literature shows little agreement on the relation of the thyroid status to the function of the gen- ital system of the female . Much of the disagreement can probably be explained by difference in thyroid dosage and age, species or strain of animals employed. As pointed out by Meites and Chand- rashaker (1949) rate appear to secrete more and mice appear to secrete less than an Optimal amount of thyroid hormone not only as Judged by their response to injected gonadotropin but also in their growth response. ' is}; Da Costa and Carlson (1933) gave immature rats daily doses of 0.5 to 1 mg. of desiccated thyroid and fmmd that it slightly accelerated sexual maturity as determined by the opening of the vagina; large doses of 5 to 10 mg. definitely retarded sexual maturity. Fluhmann (1934) reported that the feeding of 34 to 41 mg. of desiccated thyroid substance per 100 gm. body weight or 1.9 mg. thyrozdne per 100 gm. body weight to immature rats inject- ed with gonad stimulating extracts from either sheep pdtuitaries or human pregnancy blood resulted in markedly lessened increase of uterine and ovarian weight. He concluded that an overactive thy- roid inhibits ovarian function. In thyroidectomized rats Lengham and Gustavson (1947) showed that subcutaneous injection of dosages of 1, 2, and 3 mcg. of dl-thyroocine per gm. body weight for 3,6, and 10 days decreased the estrus response of ovariectomized rats to 1.5 mg. of estrone as determined by vaginal smears. Hayashi (1929) and Weichert and Boyd (1933) raported that desiccated thyroid when given to nature rats caused prolonga- tion of the diestrus periods to 13-24 days resulting in pseudopreg— nancy. Corpora lutea of large sizes were seen in sections of the ovaries. They suggested that hyperthyroidism stimulates the prod- uction of the luteinizing hormone of the anterior pituitary which is responsible for ovulation and the growth of the corpora lutea. Thee secrete progesteronewhich in turn inhibits the onset of estrus. Ershoff (1945) found that female rats raised to mat- urity on diets containing 0.5 and 1.0 percent desiccated tlwroid showed inhibition of ovarian developnent. Ovaries remined infant- ile both in weight and histologic appearance. In regard to hypothyroidism, Lane (1935), Ieonard (1936), she1ssr and Levin (194.1). and Stein and Lisle (1942) re- ported that thyroidectmw enhances the effect of the follicle o I . ‘ . ‘ ‘ O O a P . a . O- o ' . . o "I . r 0 .4— O . a ‘ I s v o a 3 . . . . ‘ . > C , , ' 3 * e A 4 - . o ' - .' (v.9 v” ‘ .1: ( I I . v r s O ’ O . o O O o . O . . - 9 - stimulating hormone in immature rate. Mann (1945) found that by the administration of 0.1 percent thiouracil to white rats their estrous cycle was prolonged. Rises and Pereny (1928) and Van.Horn (1933) found that in castrate rats the amount of estrogenic substance necessary to induce estrus was increased 3-5 times when the animals were pre- treated for six days with thyroxine. Langham.and Gustavson (1947) found that thyropara- thyroidectomy or the daily administration of thiourea resulted in an increase in the estrus response to 1.5 mg. of estrone. 3.2.1192; Cameron and Amies (1926) found that hyperthyroidism in- duced in mice by the administration of dried extracts of thyroid prolonged the estrous cycle as well as estrus itself. Stein et a1. (1947) found that the injection of 1 mg. of thyroxine into the ovarian capsule retarded mitosis in the germe inal epithelium. In male immature mice Butt (1949) showed that mild hyperthyroidism produced by giving 0.025 and 0.05 percent thyropro- tein in their diet stimulated the spermatogenic activity in the testes and epithelial proliferation of the mucous lining of the seminal vesicles. Meites and Chandrashaker (1949) concluded that mild hyperthyroidism induced by giving thyroprotein to young male mice increased the gonadotropic response, while in.hypothyroidism caused by giving 0.1 percent thiouracil in the diet the response was reduced. -10- Krohn (1947) noticed that during the administration of 0.3 mg. of propyl thiouracil to mature mice, the estrous cycle was either lengthened or disappeared. Mice fed thiourea (Dalton et al., 1945) or thioura- cil (Morris et al., 1947) showed degenerated ovaries. Rabbit; . Hypothyroidism in rabbits , produced by thyroidectomy, seemed to increase the response of the females to gonadotropic hor- mone as noted by Barman (1940). Hofmeister (1893—94) found that the ovaries of thy- roidectomized rabbits contained very large follicles packed closely together. Chu (1944) showed that extracts fran pituitaries of normal animals when given to thyroidectomized animals showed more pronounced effect than when given to normal ones. Pituitary ex- tracts from thyroidectomized rabbits induced follicular growth, but ovulation did not occur after coitus. Hyperthyroidism pro- duced by thyroxine decreased the response to gonadotropic hor- mones in this species (Barman, 1940). Kunde et a1. (1929) induced severe hypertlnrroidism by giving thyroid substance and he found that estrus, ovulation, fertilization, and implantation occurred but resorption of the embryos followed and no young were born. Guinea fig: , The interrelationship between the thyroid and ovar- ian function was studied by Chouke et a1. (1937) with the con- 'clusion that the minimal activity of the thyroid gland corresponds to the follicular phase, and the maximum to the luteal phase. Cameron and Amies (1926) found that feeding the an- imals dried thyroid materials at doses of 1/40,ooo and 1/10,ooo of bocb' weight lengthened the estrous period without striking in- creases in the duration of the cycle. Cattle; After thyroidectomy in cattle Brody and Frankenbach (1942), and Spielman et a1. (1945) found that the normal physical signs of estrus and libido are absent. Spielman et a1. (1945) reported that fertile ova were produced in animals with marked modems. as evidenced by the birth of three normal calves from these animals, and that incomplete thy- roidectany has no effect on either mature or immature animals. In late pregnancy in a thyroidectomized goat, as de- scribed by Reineke and Turner (1941), and in cows by Spielman (1945) growth was restored due to the fetal production of thyroxine. Reineke, Bergnan and Turner (1941) reported that there is a decrease in the amount of gonadotropin in the pituitaries of thyroidectomized goats. Inmature female mice weighing 12-14 gm. were ob- tained from Rockland Farms , New City, New York. At their arrival .. 12 .. they were put in cages and kept in an air conditioned laboratory at the t91111391199311? 01' 24° C. for a period of one week to get acclimatized to the temperature and food. Feeding. Finely ground Purina Laboratory Chow manufactured by the Balaton Purina Canpany, St. Louis, Missouri, U.S.A. was offered ad libitum, in special trays to prevent its wastage, to get an accurate measure of food intake. Water was available at all times. Temperature and 2&3.- In the first and fourth experiments the animals were kept in an air conditioned laboratory at the temperature of 24° C. 1 1. For the other two experiments two incubators with glass tops were used. The first maintained at the temperature of 30° C. 1 1, and the other at the temperature of 35° C. 1 1. It was not possible to control the light, but in each experiment the control group was subjected to the same fluctuations in lighting as the experimental groups . Egocedure . The animals were divided into groups of 10 mice having similar average body weights. They were numbered indiv- idually by coloring them in different areas of the body with picramic acid. Therprotein containing 0.58 percent thyroxine (Reineke et al., 1950) and thiouracil were weighed to an accur- -13- acy of 0.1 mg. and mixed thoroughly into a weighed quantity of feed by means of a mechanical mixer. Food and water were changed and weighed accurately every day. The animals were weighed once each week. At the end of four weeks the animals were sacrific- ed by exposure to ether, then weighed. Their ovaries and uteri were dissected, cleaned carefully from surrounding tissue, and weighed accurately. In addition to the macroscopic examination of these organs, histological sections were made for microscopic study. The organs were fixed in Bouin's fixative, dehydra- ted in an alcohol series, and cleared with dioxane. Hematoxylin and eosin were used for staining. Vaginal smears were taken daily from certain groups in experiment four, towards the end of the experiment. They were stained with methylene blue. For statistical analysis of the data the following formulae were used: 1. For determining the standard error - I 2.2 n(n - l) SE= 2. For determining the significance of differences between the means of different groups the "t" value was calculated - M1 ‘ M2 t: -133- .820 03330 .. . 0.0.0 H 0.0 00.0 H 0.0.0. 00.0 a. 0:: 0 0H 0 E. mad + 00 «0030 0 N10 H «.0 .0.0 H 10H H00 H 0.3” 0 0H d E. ma 0 .N 3.0 H N.0 H0.0 H .13“ «0.0 H 13 0 0H ..0. n3. 030.0 m 00.0 H 0.0 0...“ H 0.00. 00.0 H 0.2” 0 0H .N .3. §N10 m 00.0 H 0.0 0.0” H HAN 3.0 H 0.3 0 0H .V H9380 HM . . 0 00m mo ogpwulmqama on... #0 0H 0.0052809 «0.0 H 0...... 00.0 H 0.0..” 0.10 H. 0.2 0H 0H .N E. 0.10 ..H 00.0 H ~.0 00.0 H 1mm 0.10 H 0.2” 0 0H .V .3. $00.0 0H 00.0 H 0.0 00.0 H 1.3.. $10 H 0.9” 0H 0H 0 H9350 0 .0 0mm no 83200809 93 p4 HHH poms—Hug 00.0 H .10 00.0 H 0.0m 0.10 H 0.3 0H 0H .V me $10 0 00.0 a 0.0 00.0 H 0.00 2.0 a 0.3 0 0H .0 00. 0000.0 0 00.0 H 0.0 0m.0 H 09$. #10 H 0.3 0H 0H .V Honmsoo 0 .0 00m mo gpwmlmmame 93 HM HH £853me 0.0.4 H 0.0 m...” H «.mm 0.10 H 0.9 0H 0H .\ me R10 0 ~10 H 0.0 0.0 H 0.3 0.10 H 0.9 0 0H .w ma $.10 .N 00.0 H 0.0 0.0 H m.m~ 010 H 0.3” 0 0H .N .3. $0.0 m 00.0 H 13” 0m.0 H .100. 410 H 0.3 0 0H q me umN0.0 N 00.0 H 0.0 N0.0 H 040. 0.10 H 0.0..” 0H 0H .N H9380 H .0 0.0.0 .00 onenesmmllfia 0%. 04 H 95538 1000 0cm 0.4 .00.“ng cum 0.4 H335 8303 .oz 580 .93 50: 1501.93 00.0993! 03.: mo 900.052 €390.30 00.0000 0:98 MUHZ ESE 0750» .8 9:30 .5835 Mnom zo M00950; Azm 02¢ Edam QHOEB be @0755ng H Sm: - 13 b - TABLE II INFLUENCE CF THYROID STATUS AND ENVIRONMENTAL TEMPERATURE 0N FOOD AND WATER INTAKE Group No. Dosage Food Intake Water Intake riment I 0 At the Temperature of 24 C. 1 Control 3.2 5.3 2 0.025% TP 4.6 8.9 3 0.05% TP 4.2 7.6 4 0.1% TP 4.8 9.4 5 0.2% T? 5.3 9.6 Egpgriment I; At the Temperature of 30° c. 6 Control 3.1 5.1 7 0.005% TP _ 3.4 5.3 8 0.1% TH 2.7 4.4 ggpgriment III At the Temperature of 353:9. 9 Control 3.4 4.9 10 0.005% TP 3.2 5.0 ll 0.1% TH 2.3 5.5 ..ch- Haggai: 03 ~10 H «.00 Hm 3. H «.0 0 3 E. .. 010 .. ma 008.0 m 0.3. 00.0 H 02:. 0..” 3. H 0.0 0 0H E. 010 .N 80 3.0 a «.03 0H 00. u 0.0 0 00 00. “00.0.0 0 «3 00.3” H «.00 mm 00. H 0.0 0 0H .2. 038.0 m .3 Que H 0.00 om 3. H 03 0 3 H9380 H 394.313.0433 3% 00 0.0 H n.0a 0m 00. H 0.0 0H 0a 000 3.3 H 0.00 om H0. « 0.0 0 0H 00.” 00:0. H 0.00 3 0..” H 0.0 0H 0H 00 02” H 0.3 an 0 H H 10 3 0H and 00.0 H 1.0m mm 04 H 10 0 0H 00 00. H 0.0a mm 0.0” H «.0 0.... 0H 33:00 a 838.08 05 0 3% SH 0.0 H 0.0m am 00. H 0.0 0.... 0..” .3. mud n «mm 00.0 H 040 mm 00. H 0.0 0 0H .3 R10 0 00H H023 H 0.0m 00 H0. H 0.0 0 0H my 000.0 0 0mm m0.0 H 0.0m 0m 00. H 0.0 0 0H my «000.0 m 00..” 00.0 H N98 0m :00. H 0.0 0H 0H 4 Hannah H .0 0.3.. «o 83.800800. 23 HH H 35% .0: 0000 .00 0H ...E 0000 .00 0H GHQ 8H\Owa 03.3 0% 8H O? 00.: 8g 002 ...5 230 05.30.: 00.823 .93 $25 5.30.00 000.554 Mo .3832 00.0qu 0:090 M00”: 309m 950% .3 @2000 Shoaommmm .6 EH8.» 20 Egg inBgmHBHm 92.4 99490 SHONE .5 mozmpflg HH mama“ RESULTS Effects 22 Temperature 933 m. The average gains in body weights of the three con- trol groups kept at the three different temperatures of 24° c. , 30° 0., and 35° C. were 7.8, 7.9 and 7.5 gm., respectively. No significant differences were noticed between them. Effects _o_f Twoprotein Feeding on m, In Experiment I in which the mice were kept at the temperature of 24° 0., group two which was given 0.025 percent thyroprotein in its food gave the maximal average gain in body weight of 12.2 gm. The other groups, given 0.05, 0.1, and 0.2 percent of thyroprotein in their feed, showed average gains in body weights of 9.0, 9.8, and 8.3 gm, respectively. The groups given 0.05 and 0.1 percent thyroprotein showed significantly in- creased rate of gain when compared to the control group. In Experiment IV where the animals were kept at the temperature of 24° C. a lower dose of 0.0125 percent of thyro- protein was used. This group showed an average gain in body weight of 8.5 gm., while the group given 0.025 percent thyropro- tein gave an increase in body weight of 9.2 gm. The gains in body weights of both these groups exceeded significantly the amount gained by the control group, which was 7.8 gm. In Experiment II and III where the animals were kept at the temperature of 30° 0. and 35° 0. the animals given 0.005 percent of thyroprotein showed an increase in body weight -15.. of 8.7 and 9.2 gm., respectively, while the average gains in body weights of the respective control group were 7.9 and 7.5 gm. Effects _o_f Thiouracil Treatment 933 93291115.}? In.Experiment IV at the temperature of 24° 0. feed- ing 0.1 percent of thiouracil gave an average gain in body weight of 3.4 gm. which is significantly less than the average amount gained by the control, at the same temperature and same conditions, which was 7.8 gm. In Experiment II and III at the temperature of 30° c. and 35° 0., respectively, groups fed 0.1 percent thiouracil gave the average gains in body weights of 6.1 and 5.6m. respectively which were significantly less than the average gains in body weights of the control group at the respective temperatures . Food and‘Water Consumption. Effects of Temperature: At the temperature of 24° C. the amount of food and water consumed per mouse per day was 3.2 gm. of food and 5.3 gm. of water, while the nice: kept at the temperature of 30° C. consumed 3.1 gm. of food and 5.1 gm. of water, and the one kept at the temperature of 35° c. consumed 3.0 gm. of food and 4.9 gm. of water. From this it appears that the amount of food and water consumption decreases with rise of temperature. Effects of Wrtflgoidismg In Experiment I the average daily food and water consumption of a mouse given 0.025 percent thyro- protein was 4.6 an. of food and 8.9 gm. of water. The animals given 0.05 percent therprotein consumed 4.2 gm. of food and 7.6 -16- an. of water, while those given 0.1 percent thyroprotein con- sumed 4.8 gm. of food and 9.4 gm. of water, and those given 0.2 percent therprotein consumed 5.3 gm. of food and 9.6 gm. of water. In Experiment II at the temperature of 30° 0. the aver- age daily food and water consumption of an animal given 0.005 percent thy-reprotein was 3.4 an. of food and 5.3 $11. of water. In mcperiment III at 35° 0. the amount of daily food and water consumption per mouse was 3.2 gm. of food and 5.0 an. of water, From this it appears that the amount of food and water increases by giving thyroprotein; the higher the dose the greater was the amount of food and water consumed by the animals. Effects _o_f motmoidism; While giving 0.1 percent thiouracil the amount of daily food and water consumption per mouse was 2.9 an. of food and 4.6 gm. of water at 24° 0., 2.7 gm. of food and 4.1. gm. of water at 30° 0., and 2.3 gn. of food and 5.5 gm. of water at 35° 0. This shows that a lvpottyroid animal consumed less amount of food and water than a normal control animal. Temrature _a_._nd_ Reproductive gr_g_a_.g_s. Ovary weights of the animals kept at the three temperatures of 24° 0., 30° 0., and 35° 0. were 7.7, 7.2, and 8.7 mg., respectively (Table III). Uteri of the controls kept at 24° 0. averaged 21.2 mg. as compared to 19.0 and 29.9 mg. for the uteri of the controls maintained at 30° 0. and 35° 0., re- spectively. In view of the relatively large standard errors none of the differences between groups were statistically sig- -17.. nificant. Histological studies of the ovaries of nontreated animals kept at 24° 0. showed a normal picture of an ovary with follicles at various stages of develorment ranging from primor- dial to mature and atretic follicles (Figure l). (mly one animl showed corpus luteum formation. The ovaries of the animals kept at 300 C. and 35° C. showed an infantile picture with follicles in an early developing stage, and poor blood supply (Figure 2). The microscopic picture of the uteri of these three groups was that of an anestrus animal with poor blood supply and few small inactive uterine glands in their thin endometrium (Figure 5). Effects g Wig g_n Reproductive m. In Experiment I (24° 0.) animals given thyroprotein at the levels of 0.025 percent, 0.05 percent, and 0.1 percent had ovary weights of 7.8, 6.5, and 6.0 mg., respectively, compared to the control weight of 7.7 mg. None of the differences between groups are significant. The group fed 0.2 percent thyroprotein showed an average ovary weight of 4.8 mg. which was sigiificantly less than that of the control at the same temperature. In Experiment IV, the ovary weight of the controls and those given 0.0125 percent and 0.&5 percent terprotein were 4.7, 5.8, and 4.6 mg. , respectively; no significant differences were noticed between them. In Experiments II and III where the animals were kept at 300 0., and 35° 0. the animals given 0.005 percent thyro- -18- protein showed ovary weights of 7.3 and 6.9 mg., respectively. No significant differences in ovary weights were noticed between them and their respective controls. The ovary weights expressed as mg. per 100 an. body weight were less in the thyroprotein-treated animals than those of the controls. They showed a gradual decrease with increase in the dose of thyroprotein as shown in Table III. In contradistinction to the effects produced on ovary weights all of the animals given small doses of tmoprotein show- ed an increase above their controls in uterus weights. In Exper- iment I at 24° 0. the animals given thyroprotein at the levels of 0.o25, 0.05, and 0.1 and 0.2 percent showed average uterus weights of 59.0, 38.6, 31.9 and 36.2 mg., respectively while that of the control was 21.2 mg. In uperiment IV at 24° C. the average uterus weights of the mice given 0.0125 and 0.025 percent thyroprotein were 95.2 and 122.5 mg., respectively, while that of the control was 33.8 mg. In Experiments II and III at 30° 0. and 35° 0. the average uterus weight of the animals given thyroprotein at the level of 0.005 percent were 34.4 and 53.3 mg., respectively in comparison with those of their respective controls of 19.0 and 29.9 mg, The vagina of the therprotein-treated animals opened 10 days earlier than in the controls (Table IV). Vaginal smears taken from the groups given 0.0125 and 0.025 percent thy- -19.. roprotein also showed that the animals were going into estrous cycles. TQIE IV INFLUENCE (F THYROID STATUS 0N DATE OF OPENING OF VAGINA cm YOUNG FEMALE MICE Mriment I! - Dosage Control 0.012% 0.025% 0.1% 0.01251 '1‘? TP TP 4 TH -0.1% TB DayHr. DayHr. DayEr.DayHr. DavHr. Arithnetic Mean 13 . 9 - 8 2‘ 10 - 12 - Histological examination of the ovaries of these thyroprotein-treated animals showed corpora lutea, some with car-- pora lutea of two different ages as shown by their number and staining properties (Figure 3). This indicates that the mice had completed at least two ovulatory cycles. The ovaries led a very rich blood supply. The microscopic picture of the uteri showed thick endometrium with munerous well developed uterine glands, rich blood supply and stranal cells with rounded nuclei (Figures 6 and 7). ......Efrecte a: W as lea—“uctive mm Thiouracil administration caused a trend toward reduced ovary weights in all cases. The ovary weights of the -20- thiouracil-treated animals kept at 30° 0., and 35° 0. averaged 6.3 and 5.6 mg. , respectively, while those of their respective controls were 7.2 and 8.7 mg. In Experiment IV the ovary V weights of the group given thiouracil averaged 3.4 mg. while their controls averaged 4.7 mg. In group III and IV the thiou- racil-treated mice showed a significant decrease in their ovary weights when compared to those of their respective controls. In uperiment IV the average uterus weight of the thiouracil-treated animals was 74.9 mg., a significant-increase above the average control weight of 33.8 mg. At 30° C. and 35° 0., however, the uterus weights of the thiouracil-treated an- imalswere significantly reduced, as shown by the average values of 15.8 and 19.3 mg. for the thiouracil groups, compared to av- erages of 19.0 and 29.9 mg. for their respective controls. The ovaries of all the thiouracil-treated animls had a poor blood supply and were packed with large follicles but no corpora lutea were seen (Figure 4). The uteri of the animals kept at 24° 0. (Figure 8) showed a thick edematous en- danetrium with numerous leucocytes and tall epithelium. Vaga- al smears showed that the animals were in continuous estrus (cornified cells). The histological picture in the thiouracil-treated animals kept at both the temperatures of 30° C. and 35° 0. showed infantile thin-walled uteri, with thin endanetrium. Figure l. Ovary section of a normal mouse kept at 24° 0. Note (1) Cortex with Men follicles at different stages of development . (2) Medulle with rich blood supply. (3) Lack of corpora lutea. (1100) Figure 2. Ovary section of a mouse kept ‘1'. 30° 0. Note the smell size of the developing Gratin follicles. (1100) Figure 3. Ovary section of s louse kept at 24° 0. end given 0.05 percent thyroprotein in the retion. Note (1) Extensive development of corpora lutea. (2) he presence of growing follicles. (1100) figure!» 0 Overyeecticsecfescuseheptetzl. 0.endgiven 0.1 percent thiouracil. Note the large number of Greefien follicles though they have not natured. (n20) -25.. Section of uterus of a control mouse kept at 24° 0. Note (I) Thinness of the uterus wall. (2) Low epith- elium. (1500) um 6e Sections free the endcnetrius of' a mouse kept at 24° 0. and fed 0.025 percent thyroprotein. not. the rounded strosal nuclei and distinct nucleoli in a thick endoutrius. This is a typical proges- terone response end is correlated with the luteis- isation observed in the ovary. (1500) -27- Figure 7. Section from the endonetriul of a sense kept at c 21. O. and given 0.025 percent thyroprotein. Note the well developed uterine glands. (1500) Figure 8. 0 Section of the endonetriun of a sense kept at 24 0. and given 0.1 peroem thiouracil in the ration. lots (1) The tall calmer epithelius with numerous (granules. (2) n1. pronounced ode-e. (3) The shrunken elliptical nuclei of the etronal cells. (1.) The pres- ence of morons leucocytes. (1500) ~28- -29.. DISCUSSION Growing female mice given mild doses of thy-repro- tein grew at a rate exceeding that of the nontreated animals. Fran this it appears that mice secrete thyroid hormone at a rate less than the optimal. Thiouracil further decreases the rate of growth and maturation. Food and water consumption were increased by giving thyr0protein, while they were decreased by giving thiou- racil. They were decreased also at higher temperatures. ' Hurst and Turner (1948) after estimation of the thy- roid output of male and female mice found that by giving 20 to 60 times their own thyroid output they could increase their rates of growth. A dosage of 80 times their own tlvroid secretion rate was found to be detrimental. to growth. It is generally agreed that the mouse is a relative- ly hypothyroid animl, as indicated in the work reported by Maqsood and Reineke (1950), Novak (1950) and Meites and Chandrashaker (1949). In this type a suitably regulated dosage of tm‘roidal substance will produce anabolic effects. In the work done by Butt (1949) on male mice, the optimal dosage of thyroprotein for growth was 0.05 percent of the ration while in our experiment it was 0.625 percent. with regard to the effect of temperature on growth we did not observe significant differences between the nontreated female mice kept at the temperatures of 24° 0., 30° 0., and 35° 0. -30... However, the animls kept at the high temperatures of 30° 0. and 35° 0. showed retarded mturation and their ovaries and their uteri were infantile. Hurst and Turner (1948) found that by lowering the environmental temperature below thermoneutrality in male and fe- male mice their thyroid secretion could be increased. They found that at 87° Fahrenheit the thyroid secretion rate in the female growing mouse was equivalent to 3.2 meg. of d,l-thyroxine per 100 gm. body weight per day while it was equivalent to 5.5 mg. of d,1-thyroxine per 100 an. body weight per day when kept at 80° Fahrenheit. Fran such results it appears that the thyroid secret- ion rate increases when the animals are kept in cold environments and decreases when kept in hot environments. Similar findings have been reported by many authors including Ring (1936, 1939) , Dempsey and Astaood (1943) and Reineke and Turner (1945). There is no doubt that there is a close hormonal re- lationship between the thyroid and the ovary. It has been noticed that hypothyroidism induced by removal of the thyroid leads to certain regressive changes in the ovary. Ovariectony also leads to increased activity of the thyroid gland (Franks and Ftassok, 1933) . The mechanism by which the thyroid influences gon- adal function is not yet known. However, there are three possible ways by which the thyroid can play a part in reproduction. The thyroid hormone is thought to be influencing the gonadal function -31.. by its effect on basal metabolic rate as suggested by Bennett (1926), Iee (1936) and Ross (1938). This suggestion agrees partially with the results that we obtained because there was a decrease in the ovarian weights following the decrease in rate of gain in the thiouracil-fed animals. 0n the other hand, though thyroprotein feeding increased the rate of growth, the ovarian weights did not vary fran those of the control animals. On the contary, the high dose of 0.2 percent thyroprotein decreased the ovarian weight while the body weight gains were more than those of the control. Engle (1931) states that there is a very low cor- relation between body weight and ovarian weight in albino mice at any aven life period. The thyroid gland probably has a direct effect on the ovary. Variable amounts of iodine were recovered from the ovaries of different species of animals and humans (Ruff, 1933). Carter (1932) suggests that differences in iodine contents report- ed by different authors are results of variations of the iodine content during the ovarian cycle. In newborn babies the average amount of iodine in the ovaries was less than those of adults; it begins to drOp with the appearance of menOpause. Carter tried to determine the form in which iodine occurs in ripe ovaries of Echi- nus oschulentus. He could not detect thyrofine in am considerable proportion; but he found a substance which produced a marked in- fluence on metamorphosis in tadpoles. Orr and Leitch (1929) found iodine of the cow's ovary to be concentrated principally in the -32- corpora lutea. Tyndale ( 1937) observed that the tlwroid hormone has an inhibiting action upon the mechanism of gonad stimulation of mature hypOphysectomized rats and he claims that the tlwroid effect is a direct one and not mediated through the pituitaries. However, Johnson (1949) found that tolerable doses of tmropro- tein augmented the effect of gonadotrOpic hormone in fennle mice. Fran all of these observations it seems that the thyroid hormone at a certain critical point of secretion rate specific to every species favors the function of the gonadotmpic hormone. If present in more or less than optimal amounts it will impair ovar- ian flmction. . Allen (1939) suggested that changes in the follicle are initiatedbytlvroxine aswellasbymatm'inghormone ofthe anterior pituitary but that the latter only produces ruptm'e. The thyroid gland may exert its influence on the re- productive system through the pituitary gland. Cytological stud- ice or the rat pi‘hlitary by Severinghans et al., (1934), Zeckwer et al., (1935) and Goldberg and Chaikoff (1950) showed that after tkvroidectany there was an increase in the number of basophil cells while there was canplete elimination of the acidOphil cells. Similar results were proved to occur in the pituitary after ovar- iectmy. Basophilic cells are believed to have a dual func- tion, they secrete both thyrotropic hormone and follicle stimula- -33... ting hormone, Severinghaus (1937). When either of their target glands (ovary or thyroid) is absent the basophil cells seem to becane hyperactive and there is an increased output of both thy- rotropic hormone and follicle stimulating hormone , Severinghaus (1934). Under the influence of FSH the follicles will grow, but to reach maturity and ovulation they need the other fraction LH which is believed to be secreted by the acidophil cells. Severinghaus (1939) and Marine et al., (1935) confirmed the loss. of acidophils after thyroidectonw. This may explain wtw ovula- tion did not occur in our thiouracil-treated animals since they were presumably only under the influence of FSH which would stim- ulate the growth of the follicles and continuous estrus, but would not influence ovulation. Though there were large follicles in the ovaries of the thiouracil-treated animals kept at 30° 0. and 35° 0., the uteri were infantile. A good explanation for this may be that the temperature added to the thiouracil effect resulted in severe re- duction of the tlvroid homone which seems to be essential for the gonadotrOpin to induce its effect. In the rat, highly purif- ied preparations of follicle stinmlating hormone produced follic- ular groath but the uteri and vagina remained infantile (Fevold, 1941). It was believed that these follicles did not liberate effective amounts of estrogen, while by the addition of minute amounts of LB estrus was induced. - 31,- The number of acidophil cells, which are believed to secrete the luteinizing hormone, increases in mpertlvroid animls as noted by Severinghaus (1939). Weichert and Boyd (1933) found that luperthyroid rats showed persistent functional corpora lutea. Chouke et al., (1937) noticed that the maximal activity of the thyroid gland of the guinea pig corresponds to the luteal phase of the cycle. In our experiments we found thy- roprotein given in mild doses to young female mice accelerated their rate of maturation as indicated by the early opening of their vagina and the onset of ovulation denoting greater ovarian activity than in either controls or thiouracil-treated animals. SUMMARY WCLIB 3 Groups of young female mice were kept at an environ- mental temperature of 24° 0., 300 0., or 350 C. for a period of four weeks. Certain groups received either thyroprotein or thiou- racil as a percentage of their ration during this period. 1. Hyperthyroidism induced by giving mild doses of tl'mroprotein increased the rate of growth of these growing female mice. The animals kept at 24° c. and given thyroprotein at the levels of 0.025 percent showed the maximal gain in body weight. 2. mpothyroidism induced by giving thiouracil at the level of 0.1 percent of their food retarded their rates of growth. 3. No significant differences were noticed between the rates of growth of the animals kept at the three different temperatures of -35.. 24° 0., 30° c. and 35° c. 4. The animals kept at the temperatures of 30° c. and 35° 0. did not reach maturity by the end of the experiments as was indicated histologically. The ovaries showed young immature graafian follicles. The uteri were infantile and thin walled with thin endanetriuln. 5. The ovary weights in mg. per 100 gm. body weight tended to decrease in animals receiving thyroprotein. ThBy showed a gradual decrease with increasing dosage of thyroprotein. 6. TIU'roprotein-treated animals reached maturity earlier than their controls as indicated by earlier vaginal opening and the onset of regular estrous cycles. 7. Histological examination of the ovaries revealed corpora lutea in the groups receiving thyroprotein with the most cor- pore. present at the 0.025 percent level. 8. Thiouracil-treated mice either showed continuous estrm or anestrus. 9. 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