. .31."!- EFFECTS OF HYPER- AND HYPOTHYROiDISM 0N SERUM FSH AND LH EN 'GONADECTOMIZED AND INTACT MALE AND FEW RATS Thesis for the Degree of M. S. MlCHEGAN STATE UNIVERSITY. JOHN FREDERICK BMW 197 4 {8}" LIBRARY akth NDERS V.“ spa m an" mcmn . ABSTRACT EFFECTS OF HYPER- AND HYPOTHYROIDISM 0N SERUM FSH AND LH IN GONADECTOMIZED AND INTACT MALE AND FEMALE RATS By John Frederick Bruni Thyroidectomy superimposed upon castration in male and female rats increased both serum LH and FSH above castrate control levels. Administration of 2.5 ug thyroxine per lOO gm body wt. to castrate, thyroidectomized male and female rats returned serum LH to the castrate control levels, and lO ug/lOO gm body wt. reduced serum LH below the castrate control level. Both doses of thyroxine were equally effective in decreasing serum FSH to the castrate control level. Therefore, LH appears to be more sensitive to the inhibitory effect of'a high dose of thyroxine than FSH. Thyroidectomy in male and female rats with intact gonads pro- duced a reduction in serum LH and FSH. Administration of 2.5 pg thyroxine/loo gm body wt. returned serum LH levels to the intact control level, but failed to return serum FSH to the intact control level. Administration of lo pg T4/l00 gm body wt. did not increase LH above the intact control level in either sex. However, in the female rat, administration of lo pg T4/l00 gm body wt. increased serum FSH above the control level, indicating that FSH in the female is more sensitive to a high dose of thyroxine than the male. John Frederick Bruni Serum testosterone decreased both in the hyper- and hypothyroid state. An increase in weight of the accessory reproductive organs in the hypothyroid male rats suggests that the metabolism of testosterone is decreased and its half-life is increased. This smaller amount of testosterone present for a longer period of time may be more effective in depressing LH and FSH secretion and increasing male accessory organ weights. Paralleling the observations in the hypothyroid male rats, female hypothyroid rats also showed an increase in uterine weight, indicating a prolonged half-life for estrogen and a greater effectiveness in depressing LH and FSH secretion. EFFECTS OF HYPER- AND HYPOTHYROIDISM 0N SERUM FSH AND LH IN GONADECTOMIZED AND INTACT MALE AND FEMALE RATS By John Frederick Bruni A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1974 l as?! C" “0 ACKNOWLEDGMENTS Throughout the course of my master's program, I've received academic, financial and moral support from a great many people in and outside the laboratory. I feel that these peOple deserve recognition in that they have contributed significantly to my education. First, I would like to bestow my appreciation to my parents, Dr. and Mrs. John R. Bruni, for their financial assistance and concern throughout my entire education. A Second, I wish to extend my gratitude to Dr. Joseph Meites (my major professor and academic advisor) for providing laboratory facilities, equipment and financial support while working on my research problem, not to mention the thought-provoking questions which he instilled in me while reviewing my work. I would also like to extend my sincere appreciation to James A. Dibbet, a fellow graduate student. Jim was almost like a "second major professor." Upon entering the laboratory he taught me assay techniques, surgical techniques and experimental design, which made my work enjoyable and thorough. I would like to acknowledge Mrs. Claire Twohy, a wonderful person, who not only did my histology but also spent time teaching me histological techniques. ii My thanks goes to Steve “Naugahyde” Marshall, a fellow researcher and good friend. He developed and validated the Micro-RIA which enabled me to measure hormone levels in the male rat. I wish to thank Dr. G. D. Reigle, Dr. R. D. Pittman, Dr. R. A. Bernard, the members of my master's committee, and Dr. R. Echt for reviewing and constructively criticizing my thesis manuscript. Last, I wish to extend my appreciation to the remaining members of the laboratory for taking time to explain several procedures and concepts. They are a fantastic group of people and very amiable both inside and outside the laboratory. These pe0ple are: Marie Gelato, Gary Kledzik, Carol Bradley, Greg Mueller, H. J. (Buana) Chen, Mike Boudreau, Lindsey Grandison, Henry Huang and Karen Kowalski. TABLE OF CONTENTS LIST OF TABLES ........................ LIST OF FIGURES ....................... INTRODUCTION ........................ LITERATURE REVIEW ...................... Effects of Castration on Gonadotropins in Males and Females ........................ Thyroid-Gonad-Gonadotropin Interactions ......... Females . .1 ..................... Males ........................ Effects of Hyper- and Hypothyroidism on Female and Male Accessory Organs and Other Endocrine Glands . . . . Effects of Hyper- and Hypothyroidism on Reproductive Functions ....................... Females ....................... Males ........................ Effects of Castration and Thyroid Hormones on Pituitary and Thyroid Cytology .................. Anterior Pituitary Cytology ............. Thyroid Cytology ................... Role of Thyroid Hormones in the Metabolism of Steroid Hormones ........................ MATERIALS AND METHODS .................... Animals ......................... Surgical Procedures ................... Thyro-parathyroidectomy ............... Orchidectomy ..................... Ovariectomy ..................... Histological Methods ................... Preparation of Hormones for Injection .......... Blood Collection ..................... Radioimmunoassays (RIA) ................. Extraction and RIA for Testosterone ......... RIA for LH ...................... iv Page vi vii —'LO\O 00 01030)“) —l—l—l —-l #NN —l as RIA for FSH ..................... Micro-RIAs for FSH and LH .............. Statistical Analysis ................... EXPERIMENTAL ................ . ......... Effects of Hyper- and Hypothyroidism on Serum LH and FSH Levels in Gonadectomized Male and Female Rats Objective ...................... Materials and Methods ................ Results ....................... Conclusion ...................... Effects of Hyper- and Hypothyroidism on Serum LH, FSH and Testosterone, and on the Reproductive and Accessory Organs in the Male Rat ............ Objectives ...................... Materials and Methods ................ Results ....................... Conclusions ..................... Effects of Hyper- and Hypothyroidism on Serum LH and FSH, and on Reproductive Organs and Associated Endocrine Glands in the Female Rat ........... Objectives ...................... Materials and Methods ................ Results ....................... DISCUSSION . ' ......................... LIST OF REFERENCES ...................... Table la. lb. 2a. 2b. LIST OF TABLES Effects of hyper- and hypothyroidism on serum LH and FSH levels in female rats castrated for 30 days, thyroidectomized on day 3l and treated for l5 days with T Effects of hyper- and hypothyroidism on serum LH and FSH levels in male rats castrated for 30 days, thyroidectomized on day 3l and treated for 15 days with T4 ....................... Effects of hyper- and hypothyroidism on serum FSH and LH levels in female rats castrated and thyroidectomized for 30 days, followed by T4 or T4 and E.B. treatment for l5 days ........ Effect of hyper- and hypothyroidism on serum LH and FSH levels in male rats castrated and thyroidectomized for 30 days, followed by T4 treatment for 15 days .............. Effects of hyper- and hypothyroidism on the reproductive and accessory organs of male rats Serum LH, FSH and testosterone in hyper- and hypothyroid male rats ................ Effects of hyper- and hypothyroidism on female reproductive organs and endocrine glands ...... Effects of hyper- and hypothyroidism on serum LH and FSH in female rats .............. vi 4 00000000000000000 Page 29 30 35 36 43 45 52 53 Figure la. lb. 2a. 2b. LIST OF FIGURES Serum LH and FSH in female rats gonadectomized for 30 days, thyroidectomized on day 3l and treated for l5 days with T4 ................. Serum LH and FSH in male rats gonadectomized for 30 days, thryoidectomized on day 3l and treated for l5 days with T4 ................. Serum LH and FSH in female rats gonadectomized and thyroidectomized for 30 days followed by 15 days treatment with T4 or T4 and E.B. .......... Serum LH and FSH in male rats gonadectomized and thyroidectomized for 30 days, followed by l5 days treatment with T4 .................. Serum LH and FSH in intact, hyper- and hypothyroid male rats treated for 30 days with T4 ........ Serum testosterone in hyper- and hypothyroid male rats ........................ Serum LH and FSH in hyper- and hypothyroid female rats ........................ vii Page 32 34 38 40 47 49 55 INTRODUCTION Early investigations on the effects of hyper- and hypothyroidism on serum gonadotropins indicated that serum LH and FSH were decreased with thyroidectomy and increased with thyroid treatment (Reineke gt al., l953; Contopolous gt al., 1958; Smelser, l939). However, since the t al., 1968) development of radioimmunoassays (RIAs) for LH (Niswender and FSH, no reports have yet appeared on the quantitative changes in serum gonadotropins in hypo- and hyperthyroid states. With the availability of the more refined RIAs, it was of interest to determine quantitatively the effects of hyper- and hypothyroidism on serum gonadotropins. The experiments were performed in gonadectomized as well as in intact rats in order to determine the role of the gonadal hormones in mediating the response Of the thyroid on gonadotropin secretion. LITERATURE REVIEW Effects of Castration on Gonadotropins Th Males andEFemaTes Castration, steroid implants, placement of lesions, etc., have been employed to study the effects of gonadal steroids and castration on LH and FSH release. Various assay systems have been used to measure LH and FSH. Parlow gt_al, (1961) developed the ovarian ascorbic acid depletion assay to study LH. Ramirez and McCann (1965) used this assay to show an inhibitory feedback of testosterone on gonadotropin secre- tion. Contopoulos §t_al, (1958) noted that following gonadectomy and thyroidectomy, there was an increase in TSH, FSH and LH (ICSH) in the pituitary of male rats. Yamoto §t_al, (1970) and Gay gt_al, (1972) quantitatively demonstrated by use of radioimmunoassays (RIA) that both FSH and LH levels rose in the serum following gonadectomy in male and female rats. Gay gt_gl, (1972) showed that after castration a periodic release of LH and FSH persisted indefinitely. Yamoto gt_al, (1970) demonstrated a more rapid increase in serum LH after orchidectomy than after ovariectomy. Davidson (1969) noted that the gonadal steroids, estrogen, progesterone and testosterone, had an inhibitory effect on the release of gonadotropins from the pituitary ifl_viyg_and 1g vitrg. Kalra gt_al, (1973) showed by RIA that estradiol benzoate and testosterone propionate both inhibited LH release, and that male rats were more sensitive to testosterone propionate than female rats. They also showed that these steroids were equally effective in lowering serum FSH in the male rat. However, FSH was stimulated by low doses of estradiol benzoate in the male rat (0.5 and 10 pg per day). Lostroh (1969) demonstrated that both FSH and ICSH (Interstitial Cell Stimulating Hormone or LH) enhanced androgen secretion, as indicated by an increase in male accessory organ weights in the rat. One may conclude that both serum FSH and LH are elevated by castration but are differentially sensitive to varying concentrations of circulating gonadal steroids. Thyroid-Gonad-Gonadotropin Interactions Females Hyper- and hypothyroidism are known to affect the sensitivity of the ovaries to gonadotropins (Chadrashaker and Meites, 1949; Johnson and Meites, 1950; Laird gt_al,, 1970). These researchers noted that thyroidectomy increased the ovarian response to pregnant mares' serum (PMS), whereas administration of thyroxine (T4) inhibited this reSponse. Bischoff gt_gl, (1941) stated that thyroidectomy increased the metabo- lism of gonadotropins, and T4 was antagonistic to gonadotropic metabolism. Several mechanisms have been presented to explain the effects of thyroidectomy on serum and pituitary gonadotropins. Leonard (1936) reported that thyroidectomized rats had large ovaries and increased amounts of female sex hormones, as indicated by the increase in the uterine weight. He concluded that the thyroid gland has a differential effect on inhibiting LH and FSH secretion, as indicated by assays of the relative concentrations of the gonadotropins in the urine. Chu (1944) reported that thyroidectomy increased the growth of Graafian follicles but reduced LH concentration in the serum in rabbits, and this resulted in failure to ovulate following coitus. From these observations he concluded that the hypophysis of the thyroidectomized rabbit was devoid of ovulating hormone but contained increased amounts of follicle stimulating hormone (FSH). Chu (1945) further investigated the effects of thyroidectomy on gonadotropin content in the rabbit. He postulated that ovulating hor- mone was absent in the thyroidectomized rabbit, but by feeding these animals desiccated thyroid, the animals ovulated. This was attributed to a decrease in FSH and an increase in LH. From these observations, he concluded that FSH and LH were regulated by two different mechanisms. He believed the gonadal hormones stimulated the pituitary action on the thyroid, which in turn released thyroid hormones, and this inhibited the secretion of FSH by the pituitary. LH was believed to be controlled in the same manner except that the thyroid hormones stimulated the pitu- itary to elicit an increase in LH release. Through these observations he concluded that FSH and LH secretion were under direct regulation by the thyroid. Goldsmith gt_al, (1951), working with human patients, noted that thyrotoxicosis produced formation of a corpus luteum, indi- cating an increase in LH release; myxedematous patients were believed to be devoid of LH as indicated by irregular menstrual cycles. Employing another technique, Florsheim gt_al, (1967) demonstrated in unilateral ovariectomized rats that hypothyroidism induced by methimazole, methylthiouracil, pr0py1thiouracil and surgical thyroid- ectomy prevented unilateral ovarian hypertrOphy. He noted that methimazole had toxic effects. From these observations, Florsheim _t_al, (1967) concluded that the hypothyroid state limits the functional capacity of the pituitary, decreasing FSH, and thereby accounting for the prevention of ovarian hypertrophy. Similarly, Hagino (1971) reported that the hypothyroid state reduces the pituitary content of gonadotropins, as indicated by a delay in ovulation. Laiduddin (1972) observed no greater ovarian hypertrophy in thyroidectomized or propylthiouracil treated rats than in euthyroid control animals. Van Dyke §t_al, (1933) and Stein gt_gl, (1942) made pituitary extracts from thyroidectomized rats and administered the extracts to normal rats. They found that the pituitary from the thyroidectomized animals were lower in gonadotropic hormones as compared with extracts from intact control animals. Reineke £3 al. (1953a) Soliman and Reineke (1954) and Janes (1954) examined the ovaries of therprotein-treated and hypothyroid rats. Reineke gt al, (1953a) noted that thyroprotein had no effects on the ovaries except for increasing the ovarian output of estrogen which was indicated by increased uterine weight. Hypothyroid animals also showed an increase in uterine weight but the increase was due to uterine edema. Reineke gt_gl, (1953a) and Janes (1954) reported that ovaries from rats which were either surgically thyroidectomized or treated with thiouracil contained very few or no corpora lutea, indicating a decrease in LH secretion. Subsequently they concluded that hypothyroidism elicited follicular growth rather than luteinization. However, when animals were fed desiccated thyroid, ovulation occurred and a large number of corpora lutea were present. In all the observations concerning the hypothyroid state, researchers noted that both estrous and menstrual cycles were lengthened, with prolonged periods of diestrus. Clemens and Meites (1969) investi- gated whether the constant estrous state in old female rats was due to a lack of thyroid hormones. They reported that administration of T4 yielded no significant change in these old rats. Males Hyper- and hypothyroidism are known to have effects on the male gonads and gonadotropins similar to the female. Cohen (1938), Smelser (1939) and Maqsood gt al, (1950b) reported that mild hyperthyroidism elicited an increase in testicular weight. Smelser (1939) noted that mild hyperthyroidism was stimulatory to the testis as observed by an increase in the weights of the seminal vesicles and prostate gland. Administration of larger doses of T4 produced a reduction in these weights. Cohen (1935) noted an increase in the pituitary potency of gonadotropins as a result of administration of a pituitary extract from hyperthyroid male rats to female rats. This yielded an increase in the ovarian response to pituitary extracts as compared to pituitary extracts from intact control animals. Hypothyroidism has exactly the opposite effect on the pituitary and plasma gonadotropins, and the testicular response to gonadotropins. Reineke g__gl, (1941), Stein (1942), Maqsood (1950a), Maqsood (1951) and Martinez (1973) showed that the testis was less stimulated by gonadotropins from hypothyroid animals than by gonadotropins from intact animals. Maqsood (1950a, 1951) reported degeneration of the seminiferous tubules in hypothyroid rats. There was atrophy of the interstitial and the spermatogenic cells as contrasted with the hyperthyroid animal where there was increased testicular secretion and spermatogenesis. The changes noted in the testis parallel those in the ovary. Smelser (1939) postulated that the action of testosterone, a male hormone, is inhibited by thyroxine as indicated by atrophic reproductive organs. Chopra gt _1. (1972) gave a possible explanation for the in- crease in LH and FSH in hyperthyroidism as measured by the RIA. They noted an increase in the testosterone-estrogen binding globulin (Te-BG) as a result of hyperthyroidism, accompanied by an increase in testoster- one blood levels. LH and FSH were not inhibited through the increase in testosterone because there was less unbound testosterone to feedback to the pituitary to inhibit LH and FSH. The male rat, like the female rat, shows an increase in gonadal weight with low levels of T4. At high levels of T4, the gonads of both sexes were decreased. In thyroidectomized rats, the ovaries are increased in size but the testis undergoes more radical changes and is markedly decreased in size. Effects of Hyper- and Hypothyroidism on Female and Male Accessory Organs and Other Endocrine Glands Van Dyke gt_al, (1935) noted an increase in pituitary weight and a decrease in body weight following thyroidectomy. Likewise, Martinez (1973) reported a significant increase in anterior pituitary weight following thyroidectomy. Most of the changes in the female reproductive system were given in the previous section. The increase in uterine weight with thyroid- ectomy is brought about through an increase in the amount of circulating estrogen or possibly a longer half life of estrogen or increased uterine responsiveness to estrogen. Reineke gt_al, (1953a) observed that the uterus of a thiouracil (T.U.) treated rats and mice had tall columnar epithelium but the uterine lumen was filled with water. The prolifer- ative uterus of the thyroprotein fed rats and mice with functional corpora lutea had a thickened endometrium with larger nuclei due to the increased estrogen and progesterone secretion. The male reproductive organs change with the functional state of the testis. As the testes are stimulated and secrete more testos- terone, the accessory organs increase in weight. Cohen (1935) reported that hyperthyroidism increased the weight of the testes, adrenals and pituitary but decreased the weight of the prostate and seminal vesicles, indicating decreased active testosterone or unbound testosterone (Chopra gt_gl,, 1972). Smelser (1939) also noted a decrease in weight of the prostate and seminal vesicles in T4 treated rats. Cohen (1935) and Evans gt_al, (1964) showed an increase in adrenal weight following thyroxine treatment. Evans gt_a1, (1964) also reported that the adrenal decreases in size following thyroidectomy. Thyroidectomy decreased the width of the zona glomerulosa with the greatest reduction in the zona fasciculata and medulla. The zona reticularis was less affected but became less vascular. Alternatively the changes in the hyperthyroid animal were exactly opposite; the zona glomerulosa was unchanged, the zona fasciculata and medulla were markedly increased, and there was very little change in the zona reticularis. One explanation for this could be due to changes in corticosterone-binding globulins or transcortin (Chopra gt_al,, 1972). Effects of Hyper- and Hypothyroidism on Reproductive FUhctions m The effects of hyper- and hypothyroidism are evident in changes in the estrous cycles, menstrual cycles, litter size, puberty and fertility. Thyroidectomy in the rat is known to directly affect the estrous cycle by lengthening diestrus (Freedman _t_al,, 1935; Bruce gt_gl,, 1957; Hagino, 1971; Bakke gt_al,, 1972). By feeding hypothyroid rats desiccated thyroid, Freedman _t__1, (1935) was able to restore the cycle. Aranaru gt_§l, (1946) studied hypothyroid monkeys and noted that they had amenorrhea and was able to reestablish the menstrual cycle by feeding desiccated thyroid. Likewise, in the human, Goldsmith gt_gl, (1952) studied thyrotoxic as well as myxedemic patients and noted that either case there was amenorrhea, but the thyrotoxic patients had lO ovaries with active corpora lutea whereas the hypothyroid women did not. Similarly, Fisher gt_gl, (1936) treated dysmenorrhea, oligo- menorrhea and amenorrhea in women by feeding desiccated thyroid. Other investigators also have examined the effects of the thyroid state on the fertility of the female rat. Baker (1949) reported that hypothyroidism induced by thiouracil decreased fertility. Krohn _t_a1, (1950) noted that thiouracil caused resorption of fetuses, and that surgical thyroidectomy prolonged gestation and only a few young were born. These observations indicated that thiouracil may have toxic effects on the fetuses or on the uterine endometrium. Later Bakke gt_gl, (1972) implanted thyroxine into the hypothalamus of neonatal rats and noted a decrease in fertility. However, he failed to measure hormone secretion from the anterior pituitary. Janes gt_al, (1948) demonstrated that prolonged thyroxine treatment resulted in termination of gestation and resorption of the pups in the rat. Similarly, Kunde §t_al, (1929) reported that hyper- thyroidism in the rabbit elicited estrus, ovulation, fertilization and implantation, but 2/3 of the pups were resorbed in the last trimester of gestation. Reineke _t_al, (1953a) studied hyperthyroidism in the mouse and found no difference in litter size as compared to a euthyroid animal. Bruce gt_al, (1937) reported a 46% decrease in the litter size due to hypothyroidism. He also observed an increase in the length of gestation. One possible explanation for some of the effects of thyroid status on reproductive function was given by Reineke gt al, (1953b). 11 He noted that 113] incorporation was highest on the day of estrus and lowest on diestrus. He further noted that estradiol benzoate increased I131 131 incorporation into the thyroid, whereas progesterone decreased I incorporation, indicating that there might be'a reciprocal balance between the ovaries and the thyroid gland. 14.11.25. The thyroid status in the male is also known to influence the male's ability to sire litters. This may be mediated through the effects on sex drive or on the ability of the male to produce sperm. Cohen (1935) found that fertility and mating of the male rat was not affected by the hyperthyroid state. Likewise, Bruce gt_gl, (1957) noted that thyroidectomy had no effects on the fertility and ability of the male rat to sire litters. Young gt_gl, (1952) observed sex drive in the guinea pig and found no change due to hyperthyroidism. However, he observed that the percent of fertile matings was somewhat reduced. The relative percentage of fertile matings of the male is most likely related to the ability of the male gonad to produce sperm, and this is mediated largely through the gonadotrOpins. Bogart and Mayer (1916) reported that high temperatures decreased the semen volume and sperm number, accompanied by an increase in the number of abnormal sperm. When the same animals were given thiouracil they observed the same effect.‘ Likewise, Smelser (1939) and Cunningham gt_§l, (1941) noted that with high doses of thyroxine there was a decrease in spermatogenesis and an increase in rectal temperature. Therefore, 12 they concluded that the detrimental effect on spermatogenesis was due to an increase in temperature rather than to a toxic effect of thyroxine. They also noted a degeneration of the germinal epithelium. In contrast to these observations, Smelser (1939) and Maqsood §t_al, (1950b) noted that mild hyperthyroidism was stimulatory to spermatogenesis in the mouse. Not only did the number of mature sperm increase but the spermatid and Spermatocyte production were increased as well. Cohen (1935) reported not change in sperm production with either the hyper- and hypothyroid states. Effects of Castration and Thyroid Hormones on Pituitary and Thyroid Cytology, Anterior Pituitary Cytology The anterior pituitary (A.P.) is derived from an evagination of the oral ectoderm (Rathke's pouch). The pouch loses its contact with the oral cavity and later becomes a solid cell mass. There are three main cell types found in the A.P.: basophils, chromophobes and acidophils. The basophils comprise approximately 15% of the total A.P. cells, and secrete follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). Adenocorticotropic hormone (ACTH) is also believed to be secreted from these cells but the evidence for this is not conclusive as of now. The chromophobes com- prise 50% of the A.P. and are believed not to have any secretory activity. However, in the human the chromophobes have been shown to secrete ACTH (Rhodin, 1974). Some people believe that the chromo- phobe is a transitional type cell in that it may have the potential to 13 become an acidophil or basophil. Lastly, the acidophils comprise about 35% of the A.P. and are associated with prolactin (PRL) and growth hormone secretion. Purves and Griesbach (1956) mapped the rat A.P. according to the location of the thyrotrophs, FSH gonadotrophs and LH gonadotrophs. They noted that the FSH gonadotrophs were located on the peripheral area of the A.P. whereas the LH gonadotrophs and the thyrotrophs were scattered throughout the interior of the gland. They also noted that following thyroidectomy there was a degranulation of thyrotrophs and an increase in the overall number of basophils, which can be attributed to an increase in the number of gonadotrophs. Contopoulos gt_al, (1958) reported that gonadectomy in the rat elicits an increase in the number and size of the basophils with no effect on the acidophils. Likewise, thyroidectomy yielded a decrease in the number of acidophils accompanied by an increase in the number of basophils. From these observations and the observation that serum from thyroidectomized rats lacked gonad stimulating ability, they con— cluded that thyroidectomy produced a decrease in gonadotropic activity of the pituitary. Superimposing thyroidectomy upon gonadectomy yielded an increase in the gonadotropic potency of the A.P. as well as plasma, but thyroidectomy produced no further change than gonadectomy alone. Stein gt_al, (1942), Evans gt_al, (1964) and Foster _t_§l, (1969) also noted a decrease in acidophils and an increase in basophils after thyroidectomy. However, Foster gt_gl, claimed that there was a decrease in the gonadotrophs following thyroidectomy, but an apparent increase in secretory activity as indicated by an enlarged Golgi complex and increase in the number of mitochondria. 14 Castration and thyroidectomy both increased the number of basophils in the A.P. with an increase in the gonad stimulating ability following castration and thyroidectomy. Yet, thyroidectomy alone reduced the gonad stimulating ability of the pituitary. Several possible mechanisms have been postulated to exaplin the effect of thyroidectomy and the ability of the pituitary to incorporate amino acids or synthesize hormones. Matsuyaki (1970) showed that thyroidectomy caused an increased incorporation of C14 leucine in the pituitary whereas T4 administration decreased incorporation. Tonoue _t_gl, (1967) showed an increase in the ability of the pituitary to incorporate a-aminoisobuturic acid folloing thyroidectomy. Later Tonoue gt_al, (1967) demonstrated that there was also an increased incorpora- tion of C14--1eucine, alanine and glycine following castration, thyroidectomy and adrenalectomy. Thyroid Cytology The thyroid gland is composed mainly of follicles which contain a proteinatious substance, colloid. The colloid is composed of thyro- globulin and enzymes. Upon hydrolysis of the colloid, several amino acids are released: triiodothyronine (T3), the most active thyroid hormone; tetraiodothyronine (thyroxine, T4), the other metabolic active hormone; di- and mono-iodothyronine also are released from the colloid but usually are iodinated to form either T3 or T4. Hypothyroidism is known to increase the amount of colloid present in the follicle in addition to decreasing the epithelial cell height, indicating decreased secretory activity (Leeson and Leeson, 15 1970). On the other hand, hyperthyroidism reduces the amount of colloid but the follicles become larger due to increased height of the epithelium, indicative of increased secretory activity (Leeson and Leeson, 1970). The gonadal steroids are known to have an effect on the iodine turnover and secretion in the thyroid gland (Wolterink gt_al,, 1950; Engstrom gt_al,, 1952; Kumaresan gt_al,, 1966; Paschkis gt_gl,, 1948). Wolterink gt_al, (1950) showed that low doses of estrogen (l ug/animal) 131 I 131 increased output by the thyroid, where 10 and 100 pg per animal inhibited I output by the thyroid gland in the rat and mouse. Like- wise, Kumaresan and Turner (1966) showed that in the female rat, testos- terone elicited a 16% decrease in thyroxine production. Conversely, Engstom gt_gl, (1952) showed that serum precipitable iodine was in- creased in women treated with high doses of estrogen. Thus, they concluded that the tolerance for thyroid hormone was increased by estrogen or that thyroid binding globulin was increased. Kippen gt_al, (1936) ovariectomized rats and noticed an increased number of mitoses in the thyroid gland of mature rats, with no effect on prepubertal male or female rats. They also showed that the increase in the number of mitoses occurred only within the first week, followed by a return to normal with a slight increase in the amount of colloid present. 16 Role of Thyroid Hormones in the Metabolism of Steron Hormones The thyroid hormones affect the metabolism of the gonadal hormone, as they do the metabolism of the gonadotrOpins by the liver (Hagino, 1971). Progesterone, estrogen, and testoterone may either be metabolized more rapidly or stored in the form of their metabolites. Bradlow et 21: (1966) reported that there is no difference in proges- terone transformation in myxedemic or euthyroid patients. However, those patients receiving thyroxine showed an increase in the 5 a metabolites. Consequently, they concluded that the buildup of the 5 a metabolites unmasked a "latent enzyme" or expanded the compartment where the 5 a residues may act. Estrogen metabolism was found to be altered in the hyper- and hypothyroid state (Fishman §t_al,, 1965; Ruh gt_al,, 1970). Two metab- olites of estrogen, 2-hydroxyestrone and 2-methyloxyestrone, increase in hyperthyroidism and were found not to be present in hypothyroid humans (Fishman §t_al,, 1965). To determine whether estrogen has differential effects on its primary target organ, the uterus, in the hyper- or hypothyroid state, Ruh §t_al, (1970) studied 3H-17B-estradiol uptake by the uterus. They reported that there was decreased retention of 3H-l7B-estradiol in_vivg in thyroxine-treated rats and a decreased uptake in 21339, with no difference in the hypothyroid state. Testosterone and androstenedione are similarly affected by the thyroid (Rall §t_al,, 1964; Ruder et al., 1971; Chopra t 1., 1972) reported that testosterone-estrogen binding globulin is increased in 17 the hyperthyroid state. There was also an accompanied increase in testosterone, LH and FSH as measured by the radioimmunoassay. The increase in the gonadotrOpins is explained through the several fold increase in the testosterone-estrogen binding~globulins, thus leaving less free active testosterone to feedback on the pituitary and hypo- thalamus to reduce LH and FSH secretion. Rall gt_gl, (1964) also reported a reduction of steroid metabolism with hypothyroidism. However, serum LH, FSH and testosterone were not measured. Therefore, one cannot draw a quantitative conclusion as to the effects of hypothyroidism on testosterone transformation. MATERIALS AND METHODS Animals Mature male and female Sprague-Dawley rats weighing 200-225 grams were purchased from Spartan Research Animals, Inc., Haslett, Michigan. All animals were maintained in temperature controlled rooms (25°C:1°C) with artificial illumination (14 hours light from 5:00 AM to 7:00 PM and 10 hours of darkness). Wayne Lab Blox Pellets (Allied Mills, Chicago, Illinois) and tap water were provided ag_libitgm. All surgically treated animals were given a postoperative intramuscular injection of 0.2 m1 Longicil 5 (Fort Dodge Laboratories, Fort Dodge, Iowa) immediately following surgery. Thyroparathyroid- ectomized animals were maintained on 0.1% calcium lactate solution ad libitum for eleven days postoperatively, followed by tap water for the remainder of the experimental period. Estrous cycles were determined by removing daily vaginal smears. Only females showing at least 2 regular 4 or 5 day cycles were used in the experiment. Surgical Procedures All surgery was performed under ether anesthesia with clean but not sterile technique. 18 19 Thyro-parathyroidectomy A midline incision was made about one-quarter to one-half an inch in length on the ventral side of the rat approximately half way between the sternum and the first cervical vertebrae. The submaxillary salivary glands, subcutaneous fascia and the skin were retracted, exposing the sternoid muscle. The sternoid muscle was also divided in the midline parallel to the striations of the muscle, exposing the trachea, the thyroid and parathyroid glands. All tissue from the incision were held with small retractors throughout the operation. First the isthmus of the thyroid was severed using a small pair of scissors. Next the thyroid gland was dissected away from the trachea using fine curved forceps with great care to avoid injury to the carotid artery and the recurrent laryngeal nerves which lies deep near the thyroid on either side. Finally the subcutaneous fascia was sutured with silk and the skin closure was completed by means of autowound clips. Orchidectomy An incision was made perpendicular to the septum of the scrotum long enough to allow for the testis and epididymis to pass. A double ligature was placed around the vas deferens and its surrounding adipose tissue. The left and right testes and with their corresponding epi- didymis were removed, and the closure was made with purse-string suture and silk suture. 20 Ovariectomy Bilateral incisions 1/2 inch in length were made, approximately one inch rostal to the last rib and approximately 3/4 inches ventral to the spinal column, through the skin and lateral abdominal muscles. Each ovary accompanied by the uterus and adipose tissues were withdrawn. Pushing a pair of forceps through the mesentery near the oviducts, a ligature was placed around the fat to avoid damage to the uterus. The ovary was removed and care was taken to avoid excising the oviduct. The muscle was closed with silk, and the skin with stainless steel autowound clips. Histological Methods For histological examination of the testes, accessory organs, other endocrine glands, and the complementary female organs, organs were removed, cleaned, roll dried on a piece of tissue paper and weighed onia torsion balance. A11 tissues were fixed in Bouin's solution and stained with hematoxylin and eosin. Sectioning with a microtome at approximately 8 microns was done perpendicular to the longitudinal axis of most organs and parallel to the longitudinal axis of the ovary. The pituitaries were sectioned at 3-4 microns and stained with Masson's trichrome stain. Preparation of Hormones for Injection Sodium-L-Thyroxine Penthydrate (Nutritional Biochemicals Co., Cleveland, Ohio) solutions were prepared in 0.87% NaCl (saline) by 21 first adjusting the pH to 10 with 0.1 N NaOH so that the thyroxine would dissolve. The thyroxine (T4) solution was then titrated to pH 7.9 by using a pH meter and 0.1 N HCl. Finally the volume was brought to 10 ml by the addition of 0.87% NaCl, pH 7.9 to yield a concentration of 100 pg/ml or 10 pg/0.l ml. The T4 solution of 25 pg/ml was prepared by taking 2.5 ml of the 100 pg/ml solution and diluting to 10 ml with 0.87% NaCl, pH 7.9, yielding a solution of 2.5 pg/0.1 ml. Control animals and thyroidectomized animals (THX) received a daily injection of 0.2 ml of 0.87% NaCl (saline), pH 7.9. Estradiol benzoate (Nutritional Biochemicals Co., Cleveland, Ohio) was prepared from a stock solution of 50 pg estradiol benzoate (E.B.) per ml, by taking 2 m1 of the concentrated solution and diluting it with Mazola corn oil to yield a volume of 10 ml with a concentration of 10 pg/ml. Each treated rat was given E.B. in a 0.2 ml subcutaneous injection once per day. Blood Collection In all experiments, trunk blood was collected in 5 ml culture tubes. The blood was left at room temperature for approximately 5 minutes, then placed in a refrigerator for 4 hours. The blood samples were then centrifuged in a Sorvall-RC-ZB refrigerated centrifuge at 4°C at 3000 G for 20 minutes. The serum was harvested and stored at -20°C until assayed for LH and FSH. 22 Radioimmunoassays (RIA) Extraction and RIA for Testosterone The methods and materials for the testosterone were provided by the Department of Dairy Science at Michigan State University as estab- lished by K. Mongkonpunya and H. D. Hafs (1973). Duplicate aliquots of serum (0.1 ml) were placed in disposable culture tubes (16 x 100 mm). To account for procedural losses of testosterone, 2,000 dpm (disinte- grations per minute) of 3H-l,2 testosterone (New England Nuclear) was added to a third aliquot every fourth sample. The serum sample plus 3H-1,2 testosterone were vortexed for ten seconds and allowed to equilibrate. Serum was extracted by vortexing all samples in 2.0 ml nanograde benzenezhexane (1:2) for 30 seconds. All tubes were stored at -20°C for 4 hours to allow the aqueous phase to freeze. After the freezing, tubes were rapidly decanted into 12 x 75 mm culture tubes to avoid thawing of the aqueous phase. The tubes which were used to determine procedural loss were decanted into scintillation vials and allowed to evaporate. The radioactivity of these extracts were aver- aged to determine a single correction factor to account for loss of testosterone. The testosterone standard (Sigma Chemical Co.) was pipetted from a stock solution of 10 ng/ml in ethanol, in 3 sets (0.0, 0.025, 0.05, 0.1, 0.25, 0.50, 0.75, 1.0, 1.50 and 2.0 ng/ml). Then the testosterone standard and the serum extracts were dried under air. After all tubes were dried, the antibody provided by G. D. Niswender was diluted to 1:3000 with 0.1% PBS (phosphate buffered saline) gel. 23 Antibody (0.2 ml) was added to each tube, vortexed for ten seconds, and allowed to incubate at room temperature for ten minutes. Two hundred microliters of 3 H-l,2,6,7-testosterone (New England Nuclear) containing 30,000 dpm was added to each tube, vortexed and stored for 12-18 hours at 5°C. The bound and free testosterone were separated, using 1.0 m1 of 0.5% dextran 150 (Pharmacia, Uppsula, Sweden), and 0.25% carbon decolorizing neutral norit (Fisher Scientific Co.) in glass distilled water, and added to each tube. Contents are mixed, incubated on ice for 10 min. and centrifuged at 2,500 G for 10 min. at 5°C. A 0.8 m1 aliquot of the supernant was diluted with liquid scintillation fluid for a quantitative determination of radioactivity. The standards were plotted on 3 cycle graph paper and the serum testosterone for each sample was determined and analyzed statistically using the Dunnett's multiple range test. RIA for LH Serum LH concentrations were determined by radioimmunoassay by the method of Niswender _t_al, (1968). All serum samples were diluted with 0.1% gelatin in phosphate buffered saline (PBS) and assayed in two dilutions in duplicate. Each sample value was expressed in terms of a standard: National Institute of Arthritis and Metabolic Disease-Rat-LH-Reference Preparation-1 (NIAMD-Rat-LH-RP-l) obtained from the National Institutes of Health, Bethesda, Maryland. LH for iodination (LER-1056-C2) was generously provided by Dr. L. E. Reichert, 125 Atlanta, Georgia. The radioactive iodine, I , was purchased from 24 Amersham/Searle, Chicago, Illinois. The first antibody (Anti Ovine LH, A-OLH was prepared and characterized in the rabbit by Dr. Gordon Niswender, Colorado State University, Fort Collins, Colorado. The second antibody Anti Rabbit Gamma Globulin (ARGG) was produced locally through university owned sheep. This antibody was diluted and titrated in our laboratory. RIA for FSH The radioimmunoassay procedure for FSH was identical to LH. The NIAMD-Rat-FSH-Kit was used for the assay. The first antibody, Anti Human FSH (A-H-FSH), was provided by NIAMD and the National Pituitary Agency, Endocrine Study Section, University of Maryland School of Medicine. The standard preparation was NIAMD-Rat-FSH-RP-l and the hormone for iodination was NIAMD-Rat-FSH-I-l. The second antibody was prepared and characterized locally. Serum samples were assayed in duplicate and compared to the NIAMD-Rat-FSH-RP-l standard. Micro-RIAs for FSH and LH A micro-RIA was developed in our laboratory by Steve Marshall using the same reagents as in the other assays for LH and FSH. The total reaction volume was reduced from 1 ml to approximately 0.3 ml. The assay was validated by obtaining parallel curves with 2 reference preparations and cross reactivity with other anterior pituitary hormones was also examined. Samples were also done on the regular FSH assay and showed the same changes demonstrated by the micro-RIA. 25 Statistical Analysis Sample means (R) and standard errors (SE) were calculated. The level of significance of difference in serum levels and organ weights between the control groups and treated groups were deter- mined using analysis of variance (anova) for unequal group numbers. To compare all treatment groups with a single control group, the Dunnett's multiple range test was employed. Texts used for formulae were Sokal and Rohlf (1969) and Kirk (1968). EXPERIMENTAL Effects of Hyper- and Hypothyroidism on Serum LH and FSH Levels in Gonadectomized Male and Female Rats Objective The objective was to determine the effects of thyroidectomy and thyroxine (T4) on serum TSH and LH levels in the absence of gonadal steroids. Materials and Methods Experiment l.--Twenty-five female rats and forty-five male rats weighing 200-225 gms were gonadectomized (Gx) on the day of arrival. The castrated rats were allowed to remain in the animal rooms for 30 days. On day 31, 18 of the female rats and 34 of the male rats were thyro-parathyroidectomized (THX) and assigned to the following treatment regimes: (1) Gx controls, daily subcutaneous injection of 0.2 m1 of 0.87% NaCl, pH 7.9; (2) Gx and THX, subcutaneous injection of 0.2 ml 0.87% NaCl; (3) Gx and THX, 2.5 pg T4 per 100 gram body wt. at pH 7.9; and (4) Gx and THX, 10 pg T4/100 gram body wt. in 0.2 ml at pH 7.9. Fifteen days after thyroidectomy and treatment, the animals were decapitated, trunk blood was collected, and pituitaries were excised and weighed to the nearest tenth of a mg. on a Mettler balance. Serum was harvested and assayed by Niswender's LH (1968) radioimmunoassay and by the NIAMD-FSH-Kit. 26 27 Experiment 2.--Fifty-two female rats and thirty-six male rats (200-225 grams) were gonadectomized (Gx) and thyro-parathyroidectomized. On day 31 they were assigned to the same treatment regimes as in Experiment 1. However, there was on additional group in each sex: 7 intact male rats received 0.2 ml by subcutaneous injection of 0.87% NaCl and 10 females were Gx and THX and given 2 pg E.B. in 0.2 ml corn oil in addition to 0.2 ml of 2.5 pg T4 per 100 gram body weight, subcutaneously at pH 7.9. Results The quantitative values and levels of significance of serum LH and FSH are shown in Tables la and b and 2a and b for each corresponding experiment. The relative changes are shown in Figs. la and b and Figs. 2a and b. In both experiments, thyroidectomy superimposed on castration, yielded a significant increase in serum levels of FSH and LH above castrate control levels. Administration of 2.5 pg ofT4 produced a decrease in both serum LH and FSH to the euthyroid-gonadectomized levels, except for the LH values in the female rats which were thyroidectomized on day 31. There was a slight reduction in the LH concentration of the serum in these rats. Administration of 10 pg of T4 per 100 gram body wt. elicited a further reduction in serum LH with no further change in serum FSH as compared to the euthyroid-gonadectomized control animals. The group of intact male rats and female rats receiving 2 pg E.B. per day show the effects of testosterone and estrodiol benzoate on serum gonadotropins and pituitary weights. Note that estradiol 28 benzoate increases pituitary weight whereas testosterone is inhibitory to pituitary weight. Serum concentrations of FSH and LH are lower than the castrate control group further substantiating previous findings that testosterone and estrogen are inhibitory to gonadotropin secretion (Gay £3 31., 1972). In experiments 2a and 2b there is a significant decrease in the body weight of thyro-parathyroidectomized animals which received no thyroxine. This decrease represents the necessity of the presence of thyroxine to synergize with growth hormone to have normal body growth. Animals receiving 2.5 pg T4/100 gm body wt. showed no significant differ- ence in body weight compared to the castrate control rats. Animals receiving 10 pg T4/100 gm body wt. showed a decrease in body weight as a result of an increased overall metabolism. Conclusion These results demonstrate that thyro-parathyroidectomy super- imposed upon castration evokes a further increase in serum LH and FSH concentration above castrate control levels. Administration of 2.5 pg thyroxine per 100 gm body wt. reduced both serum LH and FSH to castrate control levels. Administration of 10 pg thyroxine further reduced LH but not FSH, indicating that LH can be influenced more by increases in thyroxine than FSH. Testosterone and estradiol benzoate are also shown to inhibit both LH and FSH secretion. Also testosterone is inhibitory to the increase in pituitary weight as a result of castration and estradiol benzoate increases pituitary weight over castrate control pituitary weights. These data are in contrast to most reports of a 29 60.17:. .S.vn? .m.mhmm s E em“ eom mama wpop m.ounp.o_ «km._Pauo.mmN .pz Anon Em oo_\ » m: o_t.x:h+.mumggmmu , 3 § mm“ mmo_ «OFNM comp m.oAH¢.__ kam.FFpum.mmN .pz anon Em oo_\ H a: m.~+.x:hz.mpmsummu E ammmpuwamF ammmanmmmm o._auo.m_ «m.oaum.mom Fumzt.xzpt.mpmcpmmu E o: mmm mmm R R2 ed A 5.3 and H “.mom £95.86 5m: + 3233 _E\mc _E\mc me Em mum; eo .0: 1mm Eacmm :4 Ezcmm .pz .a.< .uz xuom new ucmEummLH eh saw: mama mp Low cmpmmgu ccm Fm awn co uwNwEopumcwocxzp .mxmu om com vmumgummu mam; mpwsme cw mpm>mp :mm was :4 Ezgmm co Emvuwogxcpoaxc use icon»; eo mpummem .m_ m_nmh 3O .mo.v9$ .S.Vn_« .m.mHMM e 3: km“ amp, Emma NNFF ¢.opum.m «AF.NMHF.mNm .pz x6on Em oo_\ h m: o_+.x:h+.mpmcummu e 2: mm“ mmmp qufimqmF m.opum.w o.wouo.mom .pz xvon Em oo—\ H m: m.m+.xzh+.mpmgpmmo a: wow“ mFNN ANOFAHNmmN pkm.opuo.op hau.oppnw.mmm m:__mm+.xzhz.mpmgpmmu E 3ND: mmpmmom ¢.ou.m.m «90:9QO sz+3mbm3 _E\mc _E\m: me Em mums we .oc :mu Ezcmm :4 Ezgwm .uz .m.< .gz xuom ucm pcmspmmgh ah saw: mxmu mp Lee uwpmmcu use Fm xmu co umNVEopuwnwocxcp .mxmu om gee umumgpmmu mum; opus cw mpm>mF 1mm can :4 Ezcmm co EmwuwoLXLpoazz vcm -cwa»; we mpuwwwm .np mPnMF 31 Fig. la. Serum LH and FSH in female rats gonadectomized for 30 days, thyroidectomized on day 31 and treated for 15 days with T4. NIAMD-RP-l ng/ml 32 3000 s LH FSH 2000 - I I 6 / / / 1000 - ::: I; / a a / V / é / j 7? a? a; 61; / / / / / / / / / a / ; Gx Gx-THX Gx-THX Gx-THX NaCl NaCl 2.5 pg 10 pg T4/100 gm 14/100 gm body wt body wt 33 Fig. lb. Serum LH and FSH in male rats gonadectomized for 30 days, thryoidectomized on day 31 and treated for 15 days with T4. NIAMD-RP-l ng/ml 34 3000 4 LH ' FSH | | 2000 - 1 a”’ VI” / /,J [,1 C l ,,d /” //7 j a / /. . . f” p,a r” r” ,/I v” w" ::: ,zd 1" ’I/ ‘/,/ z/’ ”A / /i / / [’4 ,,d r/A r” / / / r,» z/’ 9"], 10 6 r’7 12”” fl / / Gx Gx-THX GX-THX GX-THX NaCl NaCl 2.5 g 10 . 9 T4/100 gm 14/100 gm body wt. body wt. 35 .5. v9. @5an Mo: 3.3.4 1 + .t. sew“ owe «Pmonmmm «m.o4”m.op a~.mpum.mmm ace: ea oop\¢h m: m.~4.x:h+.mumcpmmu a 8: mm“ mmo_ 4mm“ mam ¢.opne.m_ Em.o4”m.wem .43 Atom Em oo_\ H a: o_:.x:p+.mpmgpmmu e :: om“ mm~_ mop mmmp N.opuo.—P «N.¢4um.FmN .uz xuon oop\ h m: m.~:.x:»+.mpmgummu :: «me A E: «om: H 38. No .1. NE mé .1. WEN 5m: + x:_.+ 3333 8: 3.“me 271.3: N43149: mfmpmaom 8mz+32pm8 _E\mc Fs\m: me Em mpg: we .0: :m: Ezgmm :4 Eagmm .pz .a.< .43 Auom ucm acmEpmmgp pawsummcu .m.m use up go wk >4 umZoFFow mxmc mp Low .mxmu om com umesopumuwocx:u ucm nmumgummu mums mFmEmw cw m_w>w_ :4 can :m: Eagmm co EmwuwoLA:poaxc vcm -Lma»: Co muumeem .mm mpnmh 36 .SJe. 4%an .mpocpcoo mumgummu any on mums men mcomwgmasou umpoz .mo. v93. 4. E 8.. K: .3332 £532 .9: Roma _ .3: :38 Em 8: P a: 212:. 8.238 4 E 2. 22 8.. 82 .20.. _.2 .37. :2 .33 .484 E 8: H a: 933.3238 E $322 $2.32 .332 1;. RE 521:2. 3238 E 8.82 2.8: 3.0.3 9338 32:84 82.3238 84 .232 .22 .2... ma 1.23:3 52.335 FE\mc Fe\m: ms Em mum; $0 .0: :m: Esgmm :4 Ezcmm .p: .:.< .93 xvom new acmEuomep .nm 324.2 mxmu mp Low ucmsummgp :2 >4 umzoppow .mzmu om Lo. umN_Eopumcwog>:p ucm umumgpmmu mum; opus cw mFm>mF :m: ucm :4 534mm :o Emwwwogxcgoaxc ucm lama»: $0 pummmu 37 Fig. 2a. Serum LH and FSH in female rats gonadectomized and thyroid- ectomized for 30 days followed by 15 days treatment with T4 or T4 and E.B. NIAMD-RP-l ng/ml 38 3000- 2000- I 1 l / / / 1L / i / / 10004 / / § 1/ z/’ ,// ,/d r /// K / / / / / / / / / / / / / / / / / / / / / § § § / 10 6 11/ 11 / 10/ FT; 4 C C C / Gx Gx-THX Gx-THX Gx-THX Gx-THX NaCl NaCl 2.5 pg 10 pg 2.5 pg T4/100 gm T4/100 gm 14/100 gm body wt. body wt. body wt. 39 Fig. 2b. Serum LH and FSH in male rats gonadectomized and thyroidectomized for 30 days, followed by 15 days treatment with T4. NIAMD-RP-l ng/ml 3000 —» 4O LH _FSH 2000 a 1 1 I T/ / / / 1 / 1000- } é '; /‘ / / / "é / / / l/ / / / / / / / / / / / K 7/ 7/ 7/ 7/ / / 1/ / / / / / / GX Gx-THX Gx-THX Gx-THX NaCl NaCl 2.5 pg 10 pg T4/100 gm T4/100 gm body wt. body wt. 41 reduction of gonadotr0pins after thyroidectomy. However, other researchers did not examine the effects of hyper- and hypothyroidism on serum gonadotropins in the absence of gonadal steroids (Reineke gt_al,, 1941, 1953; Chadrashaker gt_al,, 1949; Cohen gt_al,, 1935). Effects of Hyper- and Hypothyroidism on Serum LH, FSH and’Testosterone, and on the ReproductiVe and Accessory Organs in the Male Rat Objectives Early work has shown that there is a decrease in pituitary and serum gonadotropins after thyroidectomy (Smelser gt_al,, 1939; Reineke, 1941; Martinez, 1973; Warren Chen, personal communication) and an in- crease with mild hyperthyroidism (Chopra gt_al,, 1972). Smelser (1939) showed that low doses of thyroxine stimulated testicular growth whereas high doses inhibited testicular growth. Takkar (1969) demonstrated that prostate weight increased after thyroidectomy but the prostate follicles were distended with secretion and showed reduced epithelial height. Evans gt_al, (1964) noted that the adrenals atrophied after thyroid- ectomy, with the greatest reduction in the zona fasciculata and medulla. The zona reticularis was less affected whereas the glomerulosa was enlarged as compared to the intact rat. This experiment was performed to determine the effects of hyper- and hypothyroidism 0n the serum levels of LH, FSH and testosterone as measured by RIA. 42 Materials and Methods Thirty-six male Sprague-Dawley rats, weighing 200-225 grams each, were purchased from Spartan Research Animals, Inc., Haslett, Michigan. Twenty-seven rats were thyro-parathyroidectomized and the remaining 9 served as intact controls and were given 0.2 ml 0.87% NaCl, pH 7.9. The 27 thyroidectomized (THX) rats were assigned to the following treatment schedule for 30 days: (1) THX plus 0.2 m1 daily by subcutaneous injection of 0.87% NaCl, pH 7.9, (2) THX plus a daily subcutaneous injection of 2.5 pg T4 per 100 gm body wt. in 0.2 m1, pH 7.9, (3) THX plus a daily subcutaneous injection of 10 pg T4 per 100 gm body wt. in 0.2 ml, pH 7.9. At the end of 30 days, 24 hours after the last injection, the rats were sacrificed, trunk blood was collected in 5 ml culture tubes. Pituitaries, accessory organs and glands were excised, weighed and fixed for histological examination in Bouin's solution. Serum was harvested, and stored at -20°C until assayed for LH, FSH and testosterone. Results Thyroidectomy in the male rat decreased serum LH from the intact control level of 24.80::6.31 ng/ml to 8.5:t3.0 (p <.05), whereas the replacement dose of 2.5 pg T4/100 gm body wt. returned serum LH to the control level (Table 3). Ten pg T4 per 100 gm body wt. did not elicit a change from the control level. FSH values were also decreased by thyroidectomy, from approximately 300 ng/ml to 200 ng/ml (p‘<.05). Administration of 2.5 pg T4 per 100 gm body wt. did not increase serum 43 .mo.v%£ .SJA? 4.3m... 2A4 E .3; Avon «o.m«m.mm «Am.wmno.mmm m.-um.nmm m.omfip.¢mm oo.owmm.m m.oum.m N.NPHN.mNN Em oo_\ H m: op + . um~wEopumuwogmzh Amv a .4; Econ m.mhm.om .m.m¢p~.mmm m.mmpp.mom m.FNHm.PnoF no.mwem.m m.op¢.op m.mFNo.nmm Em oo_\ h m: m.m + umNVEopumc_ogmzh :24. «m.FH~.mN ao.mmhn.mmm m.npm.¢~m o.m~am.¢opp mo.ou¢m.m R«N.opw.o_ «m.mfln.¢- umeEopumuuwcugfi $4... N.~Hm.om ~.__Ho.omm 0.2mHN.—mm o.m¢pm.mppp OP.OA_o.m m.op_.m mm.ompm.~mm Focpcou ”wane“ mE mE mE mE mE mE Em mam; mo .o: mpmcmgu< mmpowmm> mumpmocm mexuwcwaw mmpmmh .pz .m.< .43 Eve: vcm acmEpmmgh mcpEmm 111M11L11111111111111111E1111111111111111111111 11" mum; mme mo mammgo zgommouum use m>wuu=uogamg wsp co Emwuwogaspoaxz can 1gmax: mo mpummmu .m mpnmh 44 FSH to the intact control level, but 10 pg returned serum FSH to the intact control level. Serum testosterone was decreased in both hypothyroidism (THX) and hyperthyroidism (10 pg/lOO gm body wt.), from 4.6::0.5 ng/ml to approximately 2.3 ng/ml. All quantitative values (R tS.E.) are given in Table 4 and the relative changes are shown in Fig. 3. The effects of hyper- and hypothyroidism on the accessory reproductive organs and glands are tabulated in Table 3. The greatest changes other than the 19% increase in pituitary and body weights as a result of thyroidectomy were (1) a 72% increase in weight of seminal vesicles and a 50% decrease in weight of the adrenals due to thyroid- ectomy, and (2) a 31% decrease in weight of the seminal vesicles and 30% increase in adrenal weight due to T4 treatment. Histological examination showed pronounced changes in the zona fasciculata and medulla, as reported previously by Evans et al. (1964). Conclusions These results indicate that thyroidectomy reduces serum LH and FSH and that T4 in physiological doses returns serum LH to intact levels but 10 pg T4 was required to return FSH to control level. High doses of thyroxine did not increase serum gonadotropins further. Also LH seemed to be more sensitive to T4 than FSH because serum FSH did not return to the control level until 10 pg per 100 gm body wt. was given, whereas LH returned to the intact control level with administration of 2.5 and 10 pg 14 per 100 gm body wt. The decrease in testosterone by both the hyper- and hypothyroid state may be explained through the amount 45 .mo.vn_.f¢ .m.mNMm 4N4 ..m.o . E.N mm . omm A H m_ .4; atop Em oo_\e» m: o_ + x12 E AEV m.o a m.m ..A_ . om2 o . EN .4; Econ Em oo_\ H m: m.N + xzh ARV kkm.o H m.N krm N mom «Am H w Fonz + xzh REV m.o . 4.. 2m . 2cm an . EN WFOLDEOU SJMSEH _E\mc _E\mc _E\m: mpwg mo .0: mcogmpmoummp Eagmm mpm>o_ :m: Eagmm mpm>mF :4 Eacmm ucm acmEummgh mum; mme vwogxguoamg ucm 1gma»: cw acogmpmopmmp use :m: .:4 Ezgmm .m mpnmp 46 Fig. 3. Serum LH and FSH in intact, hyper- and hypothyroid male rats treated for 30 days with T4. 47 - 3OO FE\:mu ~1mm1ozo .43._o:mgu< .43 .E.< .43 moo: nco 4:0E4om44 mucopm wc4guoncm uco mcomgo m>44uzoogam4 mPoEmm co Em4c404>54oqzc new 13mm»: 40 m4ummmm .m anoH 53 Table 6. Effects of hyper- and hypothyroidism on serum LH and FSH in female rats Treatment and Serum LH Serum FSH no. of rats ng/ml ng/ml Intact controls 12 3 1a 25 i 2 (7) THX + saline 6 i l** 16 i 2** (7) THX + 2.? pg T4/lOO gm body wt. 10 i 2 16 i 2** 7 12 :1 73 1' 9* THX + 10)pg T4/100 gm body wt. 7 3?: S.E. *P<.Ol. **P<.05. 54 Fig. 5. Serum LH and FSH in hyper— and hypothyroid female rats. 1., ll... ESE/322:22:44:454:14 RM. . 133444544444: mm. o m m- TIE/52:44:44; no 5 1 7 .1 7O JV ‘f 30 .4 ILL 4. fiwé/fi/éé/fi/fi/fi/Z;4:Ki Intact Controls NaCl 0 2 10 -4 _E\on 4-nm-nz