E‘REMML $253 FGS‘FMML DEVELQPEE’M 0F TEYROEB FUNUEGK IR THE BQVENE shade for fine begs-ca at? M. '5. fiiCfifiGfiR Si‘m UREVERSEW Mama» V. hemmdez E9473 LIBRARY ; University thé‘g [M ABSTRACT PRENATAL AND POSTNATAL DEVELOPMENT OF THYROID FUNCTION IN THE BOVINE By Marco V. Hernandez Thyroid hormones play a very important role in regula- ting the metabolic processes of farm animals, eSpecially growth, reproduction and lactation. Normal thyroid function during fetal life must have a great influence on fetal devel- Opment and possibly on the future perfomance of the animal. This study contributes to a better understanding of bovine thyroid physiology during intrauterine and early postnatal life. Caesarean sections were performed in a total of U0 pregnant cows at either 90, 180 or 266 days of gestation. Maternal blood samples were taken from the jugular vein, uterine artery and uterine vein. Mixed arterial and venous fetal blood samples were taken at 90 days. At 180 and 266 days separate Samples were taken from the umbilical artery and umbilical vein. Uterine and umbilical samples were drawn through a polyethylene catheter attached to a 19 or 16 gauge needle. Fetal body weights and trimmed thyroid weights were recorded. In addition, jugular vein samples Marco V. Hernandez were taken from 19 newborn calves at days 1 - 7 after birth. Blood serum was analyzed for thyroxine (Tu) by a 125 - Abbott Labora- competitive binding method (Tetrasorb I tories). Protein bound iodine (PBI) determinations were also run on the same samples. In the 90 day fetal samples only T4 was determined. When the fetal thyroid weights (Y) and the fetal body weights (X) were plotted on log-log graph paper, these parameters were shown to be related by the equation log Y = - 0.h582 + 0.9h39 log X, with a correlation coeffi— cient of 0.9962. The regression coefficient of the equation (0.9H39) is not significantly different from 1.0. This demonstrates that from 90 to 266 days of gestation the bovine fetal thyroid gland grows in direct proportion to body growth. Fetal serum Tu values in Pg/lOO ml averaged 2.18 t 0.201, 11.66 t 0.499 and 17.16 t 0.702 at the first, second and third trimesters. The second trimester T4 value is more than 5 times (P410.001) that of the first trimester, and the third trimester value is about one-half higher (P<10.001) than the second. Maternal Tu values at the same stages averaged 6.67 t 0.205, 6.02 t 0.162 and 7.97 t 0.305 )Jg/lOO ml. The maternal serum Tu showed a significant increase (P‘10.01) during the third trimester. No significant arteriovenous differences were found in either the fetal or maternal samples at any trimester of gestation. Marco V. Hernandez At the second trimester of gestation 6 female fetuses had a significantly higher absolute thyroid weight of 2.268 t 0.103 gm than the value for 8 males of 1.752 t 0.111 gm (P<10.001). At the same stage the fetal female serum Tu value of 13.28 t 0.371 Pg/lOO ml was also significantly higher (P<:0.01) than the male serum Tu value of 10.05 t 0.693 Pg/100 m1. Mean serum Tu of neonatal calves was 16.92 pg/lOO ml at day 1 and declined eXponentially from day 2 - 5 according to the equation, log Y = 1.3959-0.0901h X (r = - 0.9997). It appears that Tu secretion was shut off during this period and the equation represents the degradation of extrathy- roidal T4 released earlier. The fractional degradation rate of 0.1872 daily is about 50% the value reported by Post and Mixner (1961) for l8-day-old calves. At the 7th postnatal day the serum T4 had a value of 7.29 Pg/lOO m1. T4 values closely paralleled the FBI values throughout this eXperiment. PRENATAL AND POSTNATAL DEVELOPMENT OF THYROID FUNCTION IN THE BOVINE By Marco V. Hernandez A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1971 ‘I (5“ 9.: ACKNOWLEDGMENTS I wish to eXpress my deepest gratitude to Dr. E. P. Reineke for his wise guidance, understanding and moral support throughout my program at Michigan State University. My sincere thanks to Dr. W. D. Oxender and to Mr. W. G. Ingalls for supplying the animal and serum samples used in this study. Special thanks are due to the Food and Agriculture Organization of the United Nations for the financial support that made this whole program possible. I am also indebted to Dr. Alvaro Munoz. F A O eXpert, for his encouragement and personal interest on my graduate studies. TABLE OF CONTENTS INTRODUCTIONOOIOOO0..000......OOOOOOOOOOOOOIOOOOOOOO LITERATURE REVIEW.0.0.0.0....IIOOOOOOOIOOOOOOOOIOOOO Anatomical Development of the Thyroid Gland.... Hormonogenesis by the Fetal Thyroid G1and...... Pituitary Regulation of Fetal Thyroid Function. Placental Transfer of Thyroid Hormones......... Distribution of Thyroid Hormone Between Mother and Fetus, Fetal Thyroid Hormone Concentration. Thyroid Function in the Perinatal Period....... MATERIAIS AND METHODSOOOOOOO0.0000000000000000000... Blood Sample Collection........................ Serum Thyroxine (T ) Analysis.................. a) PrinCiple 0f the TeStoooooooooooooooooo b) Procedure.............................. 0) Standard Curve and Ca1culations........ Protein- Bound Iodine (PBI) Analysis........... ComputationS................................... RESULTS 0 O O O O O O O 0 0 O O 0 O 0 O O 0 O O O 0 O O O O 0 O 0 0 O 0 O O O 0 O 0 O O O O 0 0 O DISCIJSSIONO I I O O O O O O O O O O O 0 O O 0 O O 0 0 0 O O O O 0 O O 0 O O O O O O O I O O 0 SUMMARY AND CONCLUSIONS 0 O 0 O O O O O O O 0 0 O O 0 O 0 O O O O O O I O C O I O REFERENCE LIST.0.0....0.00.00.0000000000000000000000 ii Page 1 LIST OF TABLES Fetal calf body and thyroid weight.......... Mean fetal and maternal serum T4 Pg/loo11110000000.0000000000000000000000.00.. Mean fetal and maternal PBI Pg/loo ml.O0.00.00.00.00...0.0.0.0....00.... Mean thyroxine degradation in newborn calves from days 2 to 5..................... iii Page 36 #2 “3 #8 Figure 1. LIST OF FIGURES EXponential relationship between fetal therid and bOdy weightSoooooooooooooo Maternal and fetal serum T4 during pregnancy............................. Maternal and fetal serum PBI during pregnancy.ooncoco.coco-00000000000000. Newborn calf serum T4 and PBI from days 1 to 7 after birthooooooooooooaoo Semilogarithmic plot of serum T4 and FBI of neonatal calves................ iv Page 37 39 40 45 46 INTRODUCTION The thyroid gland is a primitive organ that begins to function early in fetal life. Thyroid hormones are essen- tial for embryonogenesis in all vertebrates and continue to play an important role in regulating body functions through- out life. Considerable work has been reported on the development of thyroid function in mammals during the fetal and perinatal periods. Thyroid hormone formation in the fetus begins at about the end of the first one-third of pregnancy in most Species. However, there are many differ— ences between Species in the time at which thyroid hormone formation begins and also its level in the fetal as compared to the maternal circulation. There is a great deal of evidence that the fetus can supply its own thyroid hormone for some time before birth, but little or no direct proof as to when this hormone begins to have an essential biological function. Referring Specifically to the bovine, no reports have been found on the levels of circulating thyroxine in either the developing fetus or the newborn calf. It was reported by Lewis and Ralston (1953), that calves less than #8 hours old had higher plasma protein-bound iodine (PBI) levels than older animals. In the research to be reported in this thesis both serum thyroxine (Serum Tu) and PBI were determined on blood serum samples.obtained during Caesarian section, from the uterine and umbilical cord vessels in cows at the first, second and third trimester of pregnancy. Similar determina- tions were done in serum obtained daily from calves on the first to seventh day after birth. The data will be discussed in terms of fetal thyroid growth and development. probable maternal and fetal produc- tion of thyroid hormones, maternal and fetal utilization, possible placental transfer, and adaptation of thyroid function during the first seven postnatal days. REVIEW OF LITERATURE Anatomical Develogment of the Thyro1d Gland. The thyroid gland is derived from entoderm in the anterior portion of the embryonic alimentary tract. The thy- roid anlage appears as a medial unpaired evagination from the floor of the pharynx. The wall of the distal portion of this diverticulum increases in size and by active mitotic division of its cells becomes multi-layered and gradually eXpands until a bilobal form is reached; in the meantime. the stalked attachment narrows to form the thyroglossal duct. As the diverticulum becomes lobulated it migrates caudally and assumes a position on the anterior ventral surface of the trachea. The thyroglossal duct at the base of the tongue is later obliterated (Boyd, 1964; Turner p. 197, 1967). In the human, the thyroid anlage can be identified as early as three weeks after conception. By the seventh week of gestation the thyroid has reached the final paratracheal position. and between the seventh and twelfth week the thy- roid follicles begin to appear. Colloid appears only in fully organized follicles. (Boyd, 196“; French and Van Wyk, l96b). fiormonogenesis by the Fetal Thyroid Gland. Many workers have shown that when radioiodine is injected into pregnant females, it rapidly becomes available to the fetal thyroid. Using this technique, it has been possible to demonstrate that the stage of pregnancy at which thyroid tissue starts to accumulate iodide and synthesize its iodine compounds differs between species (Myant, 1964). In the mouse, (20 day gestation period) the fetal thyroid starts to accumulate iodide from the circulation around the 15th day. The ability to bind this iodide organically and the formation of colloid appears between the 15th and 16th days, whereas the formation of follicles and the ability to produce thyroxine appears between the 16th and 17th days of age (Van Heynigen, 1961). In the rat, (21-22 day gestation period) the ability of the fetal thyroid to store iodine begins around day 18 to 19 of gestation, which may be correlated with the first differentiation of follicles complete with lumen (Gorbman and Herbert, 1943). At day 21 of gestation the fetal rat is able to synthesize monoiodotyrosine, diiodotyrosine, 3,5,3' triiodothyronine and thyroxine (Geloso, 1956). In the rabbit, (30 day gestation period) the thyroid anlage appears around the 9th to 10th day of gestation: primitive follicles appear around day 17; at day 19 the fetal thyroid starts to accumulate iodine and between days 20 and 22 the colloid appears in the follicles. At this time the thyroid starts to synthetize thyroxine (Jost gt,g1., 1949; Waterman and Gorbman, 1956; Myant, 1958a). In the beagle dog, (63 day gestation period) the fetal thyroid does not secrete thyroxine before the 42nd day of gestation (Beirwaltes, 1967). The human fetal thyroid has the ability to accumulate demonstrable amounts of iodine by the 80th day and thyroxine has been identified by direct analysis of the gland between the twelfth and fourteenth weeks of gestation (Palmer gt‘gl., 1938; Chapman gt_al., 1948; Hodges gt a1., 1955: Costa gt 31., 1965). In the sheep (average gestation period 150 days) the fetal thyroid gland starts to accumulate iodine by the 50th day of gestation and the formation of thyroid follicles is observed histologically on the 52nd day (Barnes §t_§1., 1957). In the bovine (282 day gestation period), iodinated organic compounds have been detected in the fetal thyroid between day 53 and 70 of gestation, even before the forma- tion of follicles and the histological appearance of intracellular colloid. Between days 75 to 118 the devel- 0ping thyroid acquires a follicular structure; this gradual development is accompanied by a continuous increase in its organic iodine content (Koneff gt 31., 1949). It is clear that the fetal thyroid gland starts to accumulate iodine and to synthesize thyroxine early in pregnancy, but there are only a few studies where the amount of thyroxine actually present in the fetal circulation at different stages of pregnancy has been measured. Pickering and Kontaxis (1961), working with Macaque monkeys found fetal serum butanol-extractable iodine values of 1.2 Pg per 100 m1 of serum at 75 days of gestation (total gestational period in the monkey 150 days), after that, iodine values increased gradually approaching those of the maternal serum near term. Costa 23 a1. (1965), have done an extensive study of thyroid function in the human fetus, and have found PBI values of 2-3 pg/lOO ml of serum at the second trimester of gestation. At term the concentration of PBI was almost the same as in the maternal serum. Pituitary Regulation of Fetal Thyroid Function. Many workers proved that secretion of thyroxine by the fetal thyroid gland is regulated by the fetal hypOphysis and that the pituitary and thyroid of the fetus interact with each other in the same way as they do during postnatal life (Jost, 1959-60). It also appears that the differentiation of the thyroid gland from the early undifferentiated anlage does not depend upon the pituitary, but thyroid growth and secretion is definitely stimulated by the pituitary in the later stages of gestation (Jost, 1959—60; Sobel gt él°' 1960). The thyroid gland of rabbits decapitated ig_gtg£g fixes less iodine than those with intact pituitaries (Jost, 25 al., 1949). Pituitary control of the thyroid gland .begins in the fetal rabbit at about 22 to 23 days of gestation (Jost, 1959-60). Removal of the fetal pituitary gland by decapitation in near term rats, produces a decrease in size and number of thyroid follicles; injections of thyrotrOphin in these fetuses not only prevented thyroid atrOphy, but caused accelerated thyroid growth including an increase in height of the follicular epithelium. (Sethre, 1950; Sethre and Wells, 1951). Under normal conditions the placenta, is impermeable to maternal thyrotrOphin. This has been demonstrated in the guinea pig by Peterson and Young (1952), and in rats by Sobel gt 5;. (1960). In the human, a clinical case of a child born with congenital hyperthyroidism from a hypothyroid mother, has been interpreted as a fetal thyroid reSponse to excess maternal thyrotrOphic hormone, which might have crossed the placenta (Koerner, 1954). More evidence that fetal thyroid function is control- led by the fetal pituitary has been obtained by the admin- istration of goitrogenic drugs to pregnant females. Goitrogenic agents easily cross the placenta and block hormone production in the maternal and fetal thyroid (French and Van Wyk, 1964). Intact and hypophysectomized .w- ,- 'huh‘ ..‘ may .. .1- a... “a Pp.\\ l"~~ ."o H“ . A ., a. A. ‘ ' 2: u.‘ .. I pregnant rats were given a mixture of 5% KClOn and 0.05% pr0pylthiouracil in the drinking water. This treatment caused enlargement and follicular disorganization in the thyroid of all the fetuses, indicating deve10pment of a fetal goiter as a consequence of an increased secretion of fetal TSH in reSponse to decreased production of thyroid hormone by the fetal gland. Administration of thyroxine to the same rats prevented goiter development (Sobel gt_al., 1960). The same kind of thyroid enlargement was induced in fetuses of pregnant guinea pigs administered pr0pylthio- uracil (Peterson and Young, 1952). Myant (1964) reported that essentially the same effect has been observed in hamsters, rabbits, goats, humans and mice. Placental Transfer of Thyroid Hormones. It has been demonstrated that inorganic iodide easily crosses the mammalian placenta (Chapman gt 1., 1948; Koneff 1., 1949; Jost gt a1., 1949; Hodges t al a . a1., 1957; Van Heynigen, 1961; Costa et a1., 1965). $1 , 1955; Barnes 21 Also, there is an active iodide tranSport mechanism across the placenta, which is inhibited by sodium thiocyanate (NaCNS) (LongothetOpoulus and Scott, 1956). The problem of placental permeability to thyroxine has attracted the attention of many workers and with the avail- ability of radioactive thyroxine a great deal of progress has been.made in this area. A clear demonstration that thyroxine crosses the placenta and that the pituitary-thyroid axis is already working in the fetus, was made by the administration of pr0py1thiouracil to guinea pigs during the last three weeks of gestation. This treatment produced a hyperplasia of the fetal thyroid and pituitary that could be counteracted by thyroxine administration. It was also shown that injections of thyroxine alone into pregnant animals suppressed the deve10pment of both the fetal thyroid and pituitary (Peterson and Young, 1952). Another study in the guinea pig showed that thyroxine crosses from maternal to fetal circulation within five minutes after intravenous injections of thy- roxine-I131 (ContOpoulos gt al., 1964) The rabbit placenta is almost completely impermeable to thyroxine before the 19th day of gestation (Hall and Myant, 1956). The fetal/maternal ratio of butanol-soluble 1131 24 hours after the injection of radioactive thyroxine, in 20-days-pregnant rabbits, was about 0.05, this value increased to about 0.3 at the end of pregnancy (Myant, 1958a). The rat placenta is also permeable to thyroxine (Hoskins gt al., 1958). They recovered butanol-extractable I‘adioiodine from homogenates of fetuses from mothers injec- ted with thyroxine-1131. A decrease in weight of the fetal thyroids was demonstrated after hyp0physectomized pregnant 10 rats were injected with 100 Pg of thyroxine daily beginning at the 17th day of gestation (Sobel gt_a1., 1960). Radioac- tive thyroxine was detected in the fetal circulation five minutes after the intravenous injection of 1131 labeled thy- roxine into the mother (Cont0poulos gt_§1., 1964). The placentae of sows, ewes and ferrets are also per- meable to thyroxine. In the sheep and pig radioactive thyroxine could not be detected in fetuses of injected mothers before the 80th day of gestation. It was possible to detect thyroxine-I131 in the fetal ferret as early as 60 minutes after intravenous injections to the mother (ContOpoulos, 1964). In the human the tranSport of thyroxine across the 131 placenta was studied by administering I -labe11ed thyroxine to pregnant women at various times before parturition and the 131 was measured in the fetal and mater- concentration of PBI nal circulation at birth. It was found that a period of l to 3 days is required for an equilibrium to be reached between the mother and the fetus and that the concentration of radio- active thyroxine was 3 times greater in the mother than in the cord blood (Grumbach and Werner, 1956). In another. experiment, using the same procedures, it was found that the 131 concentration of organic I in the fetus was less than 10% 'that in the mother 14 to 53 hours following injection (Hirvanen and Lybeck, 1956). In some of the same eXperiments described above the Permmability of the placenta to triiodothyronine was studied. 11 It was found that triiodothyronine crosses the placenta more readily than thyroxine in rabbits (Myant, 1958a) and in humans (Grumbach and Werner, 1956; Myant, 1958b). In all the Species studied it seems that the passage of thyroid hormones from mother to fetus follows the same . pattern. There is almost no thyroxine tranSport in the early stages of pregnancy and the placental permeability to this hormone increases as pregnancy advances. But even in late stages of pregnancy the rate of tranSport is Slow and limited (Hall.and Myant, 1956; Hirvonen and Lybech, 1956; Myant, 1958b; Osorio and Myant, 1960; French and Van Wyk, 1964; Glass gt 3.1., 1964). Up to now we have only considered the passage of thy- roid hormones from mother to fetus; we already know that the fetal thyroid starts to secrete thyroxine in the last quarter of gestation in common laboratory animals and at mid-gestation in other Species. If it is supposed that the placenta does not behave only as a one sided permeable membrane to thyroxine, then it is possible that thyroxine secreted by the fetus will cross the placenta towards the maternal circulation. The fact, that thyroxine crosses from fetus to mother has been demonstrated by some interesting studies. There is a clinical report about a myxedematous woman that became pregnant after she had been under thyroidin (desiccated thyroid) treatment for two years. At the beginning of pregnancy no myxedematous syptoms were present. 12 The thyroidin treatment was discontinued after the first three months of gestation, and surprisingly, no symptoms reappeared up to the time of delivery. Relief of myxedema symptoms was certainly due to the presence in the mother's circulation of thyroid hormone produced by the fetus (Zondek, 1940). Myxedema recurred in the mother after parturition. A similar result was reported in a goat that was thyroidectomized and subsequently became pregnant (Reineke and Turner, 1941). Body growth of heifers was retarded following thy- roidectomy but body growth was resumed during the last stages of pregnancy. Resunption of growth was attributed to the thyroid hormones that crossed the placenta from the fetus (Spielman gt_a1., 1945). In the rabbit (Hall and Myant, 1956), radioactive thy- roxine was detected in the maternal circulation at l to 4 hours after the injection of thyroxine 1131 into the fetus, but the amount did not exceed 7.1% of the dose. In rats, thyroxine I131 was injected into the perito- neal cavity of three fetuses in one uterine horn and within five minutes radioactive thyroxine was shown in the maternal circulation. Radioactivity was also detected in the blood of the uninjected fetuses of the contralateral uterine horn. Between 30 to 40 per cent of this radioactivity was in the form of thyroxine and the remainder as inorganic I131 (ContOpoulus gt,§l., 1964). I. d-uv‘n-uhuu :0. '1 U 13 Distribution of Thyroid Hormone Between Mothergagd—FEtus. Fetal Thyroid Hormone Concentration. It was established in the foregoing section that the passage of thyroid hormones across the placenta increases as pregnancy advances. This increase could accur in many ways; an increase in the permeability of the placenta, a decrease in the thickness of the membranes that separate the fetal from the maternal circulation, and an increase in the placental blood flow (Osorio and Myant, 1960). The thyroid hormones in the circulation are reversibly bound to thyroxine-binding proteins (TBP), and under physio- logical circumstances an equilibrium is maintained between bound and unbound thyroxine. The amount of free thyroxine is relatively low because of its high affinity for the serum proteins, eSpecially the thyroxine-binding globulins (TBG) (Robbins and Hall, 1960). In the human, for instance, the concentration of free thyroxine is less than 0.001 times the concentration of bound thyroxine (Robbins and Hall, 1957). French and Van Wyk (1964), suggested that protein- bound thyroxine probably is not available for placental tranSport and that only free thyroxine would cross the pla- centa. Based on this fact, they pr0posed that the transfer of thyroxine between mother and fetus would be determined primarily by the relative levels of free thyroxine on each side of the placenta; these levels in turn, would be controlled by the relative binding capacity of the two sera, 14 the thyroid hormone output and the thyroxine turnover rate of mother and fetus. The most important of these factors seems to be the difference in composition, affinity and binding capacity of the serum thyroxine binding proteins between mother and fetus (Osorio and Myant, 1960; Osorio and Myant, 1962). Myant and Osorio (1959), using electrOphoresiS and radioactive thyroxine, found that thyroxine binding proteins of the fetal rabbit (fetal TBP) are different from those of the adult rabbit (adult TBP). The fetal TBP'S make their initial appearance at about the 19th day of gestation, then gradually increase to levels characteristic of the adult at two weeks of post-natal life. The fetal TBP exhibits a diSplacement between theo<~2 and B-globulin, and the adult TBP between albumin and Oil-globulin. Myant and Osorio (1959) also demonstrated that the binding capacity of the serum TBP of the fetus at 25 days of age is about 10% that of the adult and the affinity of the fetal TBP for thyroxine is five times greater than that of the adult TBP. The same investigators mixed together equal parts of adult and fetal TBP with radioactive thyroxine, and using electrOphoresis, they found that at 25 days of age the fetal TBP binds about one third of the thyroxine, but it has a biinding affinity 5 times greater than the adult. At 28 days 01‘ age the fetal TBP binds as much thyroxine as the adult TBP (Osorio and Myant, 1960). 15 During pregnancy in women, the augmented blood levels of estrogen are an important factor in stimulating increased thyroxine binding capacity of the serum proteins, which reaches a level of about twice the nonpregnant values (Dowling gt a1., 1956; Robbins and Nelson, 1958; Russell 2£.él°v 1960; Russell gt a1., 1964). Serum of human fetuses, between 12 and 35 weeks of gestation, contains a Specific thyroxine-binding protein (TBP), that exhibits the same electrophoretic mobility as adult TBP. By the 24th week the maximal binding capacity of the fetal TBP is much less than the maternal TBP; however, the binding capacity rises towards the end of pregnancy (Osorio and Myant, 1962). At term, this capacity is one and one-half times greater than in the nonpregnant adult, but still about 50% lower than in the mother (Dowling 23 a1., 1956; Robbins and Nelson, 1958). It has been found in the human that at term the amount of fetal TBG is 29.1 pg per cent with a range from 20.2 to 38.7 Pg, as compared with the maternal TBG that ranges from 31.1 to 47.8 pg with a mean of 42.1 Pg; the last value is approximately twice the levels for nonpregnant females (Russell g§_al., 1964). The PBI of human mothers at term is greater than in nonpregnant adults, but it is approximately the same as in the fetus. The concentration of free thyroxine is signif- icantly higher in the fetus than in the mother (Robbins and 16 Nelson, 1958). It has also been shown that the fetal binding proteins are slightly but Significantly more saturated than their reSpective maternal proteins, resulting in a higher concentration of free thyroxine (Robin gt a1., 1969). The current thinking is that thyroxine crosses the placenta in its free rather than in its protein-bound form (Myant, 1964; Robin gt,a1., 1969), which supports the theory pr0posed by French and Van Wyk (1964) that the higher concen- tration of free thyroxine in fetal blood results in a positive net tranSport from fetus to mother. The existence of a free thyroxine tranSplacental gradient has been pr0posed by Robbins and Nelson (1958), but denied by De Mayer 23.31- (1966). All the functional changes that the thyroid undergoes during pregnancy contribute to increasing the concentration of endogenous hormone in the fetal blood towards the end of pregnancy (Myant, 1964). In the fetal rabbit the concentra- tion of serum protein-bound iodine at 18 days of age is so low that it is difficult to measure; afterwards it increases, equaling that of the mother's serum at 27 days of gestation (Myant, 1958b). In the Macaque monkey, the butanol-extractable iodine of the fetal serum, has a value of 1.2 Pg/lOO m1 at 75 days of age; it increases with pregnancy, reaching a value of 4.5 ‘pg at 150 days (Pickering and Kontaxis, 1961) and reaches the same value as in the mother at the time of parturition (Pickering, 1964). 17 In the serum of human fetuses removed by Caesarean section between 12 to 24 weeks of age, the concentration of protein-bound iodine is less than in serum of myxedematous patients (Osorio and Myant, 1962). The concentration of PBI at the second trimester was found to be 2-3 Pg/lOO m1 of serum (Costa 23 al., 1965); and at parturition, the mean PBI was 6.2 Pg with a range from 3.8 to 9.1 pg (Russell 33 31., 1964). Measurable amounts of iodine have been detected in fetal bovine thyroid at 60 days of gestation. Total and thyroxine-like iodine content increased steadily as the fetus grew. The rate of increase of these two iodine fractions was related eXponentially to fetal body weight, body length and calculated age. The amounts of iodine found in the bovine fetal thyroid with increasing age were greater than could be accounted for by mere increase in thyroid mass (Wolff gt al., 1949). Th roid Function in the Perinatal Period. In the human, the PBI concentration in blood samples taken from the mother during labor and from the umbilical cord soon after delivery have been found not to be signifi- cantly different (Danowski gt_a1., 1951; Man gt_gl., 1952; Pickering gt a1., 1958; Russell gt a1., 1960; Russell 23,a1., 1964). The thyroxine binding capacity was signif- icantly lower in the newborn (26.9 yg%) than in the mother 18 (39.6 Pg%), but both were greater than in the nonpregnant state (20.3 Pg%), (Russell gt_gl., 1960). Monkey fetal and maternal serum BEI also reaches the same level at the time of parturition (Pickering, 1964). Tracer quantities of radioiodine were injected into pregnant cows 1 week prior to parturition; at birth, the calf plasma showed radioactivity 10 times higher than that of the dam. The radioiodine in the calf plasma was in the protein-bound form, mostly as thyroxine (Monroe 23 a1., 1951). Two pregnant cows were sacrified 24 hours after radioiodine injection and it was found that the fetal thyroids contained twice as much 1131 as the maternal thy- roids and the concentration of I131 was six to seven times greater in the fetal thyroids. The fetal thyroglobulin was 27 times more radioactive than the mother's and analysis of the hydrolyzed thyroglobulins revealed that more than one- fifth of the radioactivity was due to newly formed thyroxine. The blood sera of the fetuses contained only slightly more radioactivity than those of the cows (Gorbman gt a1., 1952). In another eXperiment it was found that the fetal thyroids contained as much radioiodine as the maternal thyroids and were higher in concentration per gram. The concentration of 1131 in the calf plasma was over four times that in maternal plasma (Miller 23 El-v 1967). It has been reported that the newborn human baby eXperiences a sharp increase in serum thyroid hormone 19 concentration during the first two to three days of life. This is followed by a gradual decrease, approaching adult values by the 18th to 20th day of age (Danowski, 1951; Man, gt a1., 1952; Pickering 23 a1,, 1958). These high PBI values together with the finding of an increased thyroid iodine uptake, led to the suggestion that the newborn infant may eXperience a period of physiological hyperthyroidism (Van Middlesworth, 1954). Ponchon gt‘al. (1966) found that increased iodine uptake by the thyroid of the newborn is accompanied by a very active thyroxine clearance; the daily accumulation of iodine is up to ten times higher than in adulthood, and the total diSposal rate is up to three times faster in infants than in adults. Recent studies have demonstrated that the serum free thyroxine values are higher in neonatal life (Marks, 1965; Siersbaek-Nielsen and Molholmhansen, 1967), and that the plasma. tyrosine and thyroxine are significantly higher than in euthyroid adults (Siersbaek-Nielsen and Molholmhansen, 1967). This confirms the impression of a physiological hyperactivity of the thyroid gland during the neonatal period. Fisher 33 a1. (1962), confirmed the presence of an elevated 1131 uptake rate very early in the newborn infant (12 to 24 hours after birth). They suggested that this phenomenon together with the higher PBI values at the same 20 age may be associated with an increased secretion of thy- rotrOphin (TSH) by the pituitary gland. They considered, among other factors, that the cooler temperatures to which the newborn is eXposed may be a powerful factor that, by way of the hypothalamus, stimulates TSH release. More recent studies conducted by the same authors (Fisher and Oddie, 1964; Fisher 23 g1., 1966; Fisher and Odell, 1969), confirm that the cold eXposure that the neonate experiences is the initial stimulus reSponsible for thyroid hyperactivity of the newborn. The mechanism of thyroid hyperactivity and hyper- thyroxinemia of the newborn infant have been further understood by the use of a sensitive and Specific TSH radioimmunoassay and the demonstration of a marked acute peak in serum TSH concentration soon after birth; the mean values increased from 9.4 pU/ml in cord blood to a peak at 30 minutes of 93 pU/ml, there was a rapid fall between 30 and 90 minutes, and values had dr0pped to 14 PU/ml at 24 hours (Fisher and Odell, 1969). In the goat, local cooling of the pre0ptic anterior hypothalamic region, the "heat loss center" caused a marked 131) from the thyroid gland with the development of an extreme increase in the release of protein-bound iodine (PBI hyperthermia (Andersson gt_a1., 1963). It has been reported that in the bovine the PBI concentration tends to decrease with age. Calves less than 21 48 hours old showed a PBI of 13.7 pg/loo m1 of plasma, while those between 2 days and 12 months averaged 7.2 Pg per cent (Lewis and Ralston, 1953). When the in vitro erythrocyte uptake (EU) of I131 labelled triiodothyronine was used as an indicator of thy- roid function in cattle, it was found that the EU values declined with age. Mean EU in calves under one month old was 13.5 and in heifers of 12-18 months old 6.2 (Thorell, 1965). Kossila (1967), published an excellent review, togeth- er with very important data of her own, on the deve10pment of bovine thyroid function from early fetal life to old age. Kossila's study is mostly based on the weight and basic structural components of the thyroid gland. The total weight of the fetal gland increased through- out gestation, but the rate of increase Slowed considerably near term. Colloid was first detected in the follicular lumen at the 84th day of intrauterine life. The amount of colloid increased from this stage until the end of gestation, being most marked from the 140th day to term. The percentage of glandular epithelial tissue was very high before the 100th day and showed a tendency to decrease as gestation pro- gressed. Between 160 and 195 days, and sometimes earlier, the absolute amount of epithelial tissue had already reached the level observed in newborn calves. The mean thyroid weight of 135 newborn calves was 22 7.52gm, with a percentage of epithelial.tissue of 18.84. Histologically, the activity of the thyroid appeared to be rather low at birth. Thyroid glands obtained from 55 calves over one month old, Showed an absolute thyroid weight increase, whereas the relative weight rapidly decreased during the first 12 months of age. Mean percentage of epithelial tissue was lower in calves under 6 months old than in those over 6 months old. MATERIALS AND METHODS For the study on fetal and maternal thyroid relation- ships, 40 pregnant Holstein Friesian heifers of known breeding dates were purchased from Michigan dairymen by Wayne D. Oxender DVM for use in his project on the develop- ment of the endocrine and reproductive systems in the bovine fetus. After being delivered to the Campus, the heifers were kept at the loose housing barn of the Michigan State University Dairy Department under the usual conditions of feeding and management until scheduled for an Operation. Dr. Oxender generously supplied serum samples for the T4 and PBI analyses and fetal thyroids were collected by Dr. E. P. Reineke. The serum samples for 19 newborn calves were kindly supplied by Winston G. Ingalls of the Michigan State University Dairy Department. They were taken daily at ages 1 to 7 days from calves born in the Michigan State University Dairy Herd. Blood Sample Collection. The blood samples were collected from pregnant cows divided into three groups according to the stage of gesta- tion. Sampling dates were timed to coincide with the end of the first, second and third trimester of gestation. 23 24 Caesarean sections were performed at the Veterinary Clinic of Michigan State University by Senior Veterinary students under the direction of Dr. Wayne D. Oxender. Depending on the side of pregnancy, the animals were .first clipped and surgically scrubbed with a germicide on the reSpective paralumbar fossae. Paravertebral or nerve blocking anesthesia was done by infiltration of 2.5% procaine hydrochloride solution with epinephine 1/50,0001. The first blood sample was withdrawn from the dam's jugular vein before initiation of surgery. Once the laparotomy Opening was made, the median uterine artery and uterine vein were cannulated with a 30 inch long polyethylene catheter attached to a 19 or 16 gauge needle (Minicath-l6, infusion set)2 and blood samples were withdrawn from each vessel. Afterwards, the uterus was brought up and hysterotomy was performed. In the 180-and 260-day-old fetuses, blood samples were taken from the umbilical artery and umbilical vein using the same kind of polyethylene catheters, but with a 16 gauge needle. In the case of the 90-day-old fetuses the blood was taken from the fetal aorta and the heart. After removal of the fetus, the uterus was closed with a Cushing suture pattern, using #1 chromic catgut. The peritoneum and muscle layers were sutured with #2 chromic lBio-Ceutic Laboratories, Inc. St. Joseph, Missouri 2 Desert Pharmaceutical Co., Inc. Sandy, Utah. 25 catgut. The skin closure was made with Vetafil* 0.6 mm. Fetal thyroids were removed, trimmed and weighed. One part of each thyroid was sectioned and fixed in Dietrich solution for histological studies to be reported elsewhere. The remainder was frozen and stored for subsequent analyses also to be reported elsewhere. In each case blood samples of about 40 cc were col- lected into oxalated polyprOpylene tubes and placed in ice. After 10 to 30 minutes they were centrifuged in a refriger- ated centrifuge and the plasma was transfered into the same kind of polypropylene tubes with CaClZ added in amount sufficient to cause clotting. The samples were stored under refrigeration and within 24 to 48 hours the serum was separated from the fibrin clot. Serum samples for T4 and PBI determinations were kept frozen until the reSpective analyses were carried out. Blood samples from the newborn calves were withdrawn from the jugular vein into polyprOpylene tubes and were submitted to the same treatment as described above. Serum Thyroxine (Tu) Analysis .a) Principle of the Test. The serum thyroxine (serum T4) was measured by the Taitilfiasorb-125 method (Radio-Pharmacentical Division, Abbott \ * Ve Stafil Beugen, Haver-Lockhart, Kansas City, Mo. 26 Laboratories, North Chicago, Illinois). This method uses the principle of "competitive protein binding" that was first described by Ekins (1960), and further develOped and simplified by Murphy and Pattee (1964). A resin-Sponge to separate bound from unbound thyroxine was introduced by Nakajima gt_a1. (1966) and by Kennedy and Abelson (1967). Kaplan (1966) showed that 12SI-thyroxine could be bound in advance to the standard thyroxine-binding globulin solution, and that this treated solution is a satisfactory test reagent for thyroxine estimation. The principle of the test is described briefly as follows: There is only a small amount of thyroxine-binding globulin (TBG) in serum. The T4 binding sites can be readily saturated by the addition of small amounts of thy- roxine either labeled or unlabeled. If a small amount of 125I-thyroxine is added, the fraction which is protein-bound can be determined. As more unlabeled T4 is added, the amount of 125I-labeled bound thyroxine decreases, since both the labeled and unlabeled thyroxine compete for the same llinding sites. If instead of pure T4 a sample of depro- 'teiJuized plasma is added, the T4 which it contains competes fVDI‘ the thyroxine binding sites. When equilibrium is r“3€1<:hed, the resin-Sponge separates the TBG-bound thyroxine from the unbound thyroxine, and the amount of '11; contained in tII‘E’ Serum sample can be measured according to the fall in 27 bound isotOpe which it causes. b) Procedure. In the present study some minor modifications to the Tetrasorb-125 method were introduced. Serum samples were thawed and brought to room temperature. One ml was used for each determination. In some cases duplicates of the same sample were run. Two m1 of 95% ethanol were added and the solution was mixed for 30 seconds by means of a Vortex mixer. The tubes were tightly st0ppered. To facilitate maximal Tu extraction, the solution was allowed to stand for ten minutes and then centrifuged at 1000 g. for 20 minutes. One 0.3 ml aliquot of the supernatant liquid was transferred into polyprcpylene test tubes and evaporated to dryness by means of a mild stream of clean air, while the tubes were immersed in a warm-water bath (370 C). After complete drying, 1 m1 of 125 I-Tu-TBG was added to each sample by using a "syringe pipette" devised by Dr. E. P. Reineke. The tubes were gently shaken for a few moments and placed in a warm-water bath at 220 C for ten minutes to allow 23 better and constant equilibration between the labeled and. Lumlabeled thyroxine with the TBG molecule. The tubes were tTLell placed in an ice bath at 1.5-4O C. At the end of five m«'if'lutes in the ice bath, one Tetrasorb resin-Sponge was Placed in successive tubes at 20 second intervals and the air Was expressed by using the.Abbott plastic plunger. After 30 minutes of incubation one initial count (I) of 28 .the total radioactivity contained in each tube was obtained. At the end of exactly 1 hour of incubation, the reaction was st0pped in successive tubes at 20 second intervals by adding about 10 ml of cool (5-1000) glass-distilled water. Then, the washings were withrawn as soon as possible by depressing the Sponge 5 times with the Abbott aSpirator. Each tube was washed three additional times with cool glass-distilled water. Each tube remained in the ice bath until it was removed for the washing steps. The final radioactivity count (F) was taken using a well-type scintillation counter (Nuclear Chicago, Model Ds-5), and analyzer-scaler (Nuclear Chicagq.Model 8725). Finally, the percentage 125I-TI+ uptake by the resin-Sponge was calculated by using the following formula: R S. S U L k = F CPM - back ound CPM x 100 l e in p°nge p a e % 1 (opmi':"5581§§3558'15bmi ( ) c) Standard Curve and Calculations A standard T4 curve was run with the determinations of each day. The standard stock solution was prepared from crystalline free thyroxine purified by Dr. E.P. Reineke from monosodium thyroxine pentahydrate“. Ten mg of free T4 was dissolved in 95% ethanol with the aid of a few drOpS of a 0.5N NaOH solution and diluted to a concentration of *Baxter Laboratories, Morton Grove, Ill. 29 5 Pg/ml. The final concentration of the working standard was 0.05 pg/ml. The working standard was prepared aproxi- nmtely every 3 weeks and kept at 50 C in tightly closed siliconized glass containers. 1 Based on the results obtained from previous studies in this laboratory (Wan, 1969; Lorscheider, 1970), it was decided that 3 concentrations of the working standard solution would be run for each standard curve, at l, 4 and 8 Pg/lOO m1 of solution. The three tubes were carried through the same procedure as the alcoholic serum extracts already described. The standard curve was obtained by plotting on linear coordinate graph paper, the T4 concentra- tion being eXpressed as Pg/lOO ml on the X axis and the % resin-Sponge uptake on the Y axis. In the same work mentioned above, it was found that in all the Species studied the serum T4 concentration did not exceed 12 pg/lOO ml, and it was established that in the range of 0-12 pg Tu/lOO m1 a perfectly linear relationship exists between T4 concentration and % resin-Sponge uptake. Thus, instead of reading values from the standard curve, the data were fitted by the method of "least squares" (Li, 1964) and the T4 values were calculated by use of the "linear regression equation" (Li, 1964). The calculations were made by using the following equation: xu= Y'a (2) Xu = Serum T4 (pg/100 ml) uncorrected for recovery. Y = % resin-Sponge uptake a = intercept of the Y-axis b = lepe of the standard curve. The ethanol extracts only 77.3% the T4 from the serum proteins. The results were corrected for extraction losses as follows: x=x(10°) (3) X0 = serum T4 (pg/100 ml) corrected for extraction recovery. As a routine control of the determinations, two duplicates of the same pool of steer serum were run each time. The mean T4 value for 24 determinations on this serum was 6.41 pg/lOO m1. In some cases the day's value for the steer serum was used as a standard to correct the experimental T4 values in order to get more consistent results. All equations and calculations were programmed on an Olivetti-Underwood Programma 101 desk computer. We (p311 Analysis . The PBI technique used in this laboratory is an adaptation of the alkaline ashing method of Barker and H’ L11"1131113ey(1950). Before analysing, the serum samples were 31 thawed and brought to room temperature. One ml of serum was pipetted into incineration tubes. Serum proteins were precipitated by adding 1 m1 of 10% ZnSO and 1 ml of 0.5N NaOH. The precipitate was washed L; three times with glass-distilled water. One ml of 4N Na2C03 was added to the precipitate, mixed well and dried overnight at 94-100O C. The sample was then incinerated in a muffle furnace for 2.5 hours at 615-6250 C. After ashing and cooling, the iodide from the ash was dissolved with 2 ml 2N HCl and 2 m1 7N H280“. Then the ash digest was diluted to a volume of 11 ml by adding 7 m1 of glass-distilled water. Two duplicate 5 ml aliquots of the dissolved ash solution were pipetted into colorimetric cuvettes and 0.5 ml of arsenious acid was added. The timed reaction with ceric ammonium sulfate was run for exactly 15 minutes at 370 C and then stOpped with brucine sulfate (Faulkner £3 21" 1961). The final readings were taken by means of a Coleman Spect0photometer (Model 6/35) set at a wave lenghth of 480 mu and adjusted to 100% transmittance through a glass- distilled water blank. A reagent blank and two standards containing 1 m1 of IC>dotrol*, or 1 ml of standard lamb serum were run each time and submitted to the same treatment as the serum samples. Iodine content was calculated by means of a standard curve ‘ it gagie Reagents, Inc. Miami, Florida (Distributed by Q 5L entific Products). 32 (intermediate range) prepared under identical conditions and using Hycel iodine standard stock solution* (1 pg/ml) with glass-distilled water to a final concentration of 0.02 pg/ml. In this standard curve, the net % transmittance (standard minus reagent blank) was plotted on the ordinate of linear graph paper, against iodine concentration in micrograms on the abscissa. Computations. When the fetal thyroid weights were plotted against body weight on log-log paper they formed a straight line. Thus they would be eXpected to fit the equation for a parabolic function. Y=aXb (4) In logarithmic form this equation may be written log Y = log a + b log X, .. (5) where log Y = log of thyroid weight eXpressed in gm X = body weight eXpressed in kg log a = log of the Y-axis intercept b = slope of the line (regression coefficient). AS described by Brody (1945 p. 398) two normal equa- . tions are needed to fit the data by the method of least squares: *Hycel, Inc. Houston, Texas (Distributed by Scientific Products). 33 I£(logY)=N£(loga)+bZ(logX). (6) II ( Log X.log Y ) = log a ( log X ) + b ( log2 X ) (7) The data were fitted and the line of best fit was drawn as shown in Figure No. l. The mean serum T4 and PBI of the newborn calves were plotted on semilog paper. The values from neonatal days 2 to 5 formed a straight line, and thus could be fitted by the equation, log Y = a + b X, (8) where log Y = log of T4 or PBI values ( pg/lOO ml serum ) X a days of age a = Y-axis intercept b = SlOpe of the line. The line of best fit for each slope was determined by the method of least squares (Li, 1964). Thyroxine biological half-life (t%) was calculated according to the eXpression: t8 = 0° 01 (9) where, 0.301 = log$55 and, b = log10 slope of the line. The thyroxine fractional turnover (TFTR) per day was calculated using the equation, 1 - e'x' (10) where X = 1n slope of the line, or b x 2.302, or .22éi2. The thyroxine volume of distribution (TVD) was calcu- lated using the value 22.0 (TVD per cent of body weight) reported by Post and Mixner (1961) in 18-day-old bull calves. The extrathyroidal thyroxine (ETT), T4 degraded daily and T4 degraded daily per 45.4 kg of body weight (Table 4) were also calculated. Significance of the differences between means were obtained by the Student (t) test (Li, 1964). RESULTS Primiparous cows 1% to 2 years of age were used for the eXperiment on thyroid deve10pment in the bovine fetus. The animals were in good heath and nutritional condition at the time of the Operation. The fetuses all showed body markings of the Holstein breed, except for fetus NO. 561 and fetus No. 564 that had breed characteristics of Hereford and Angus, reSpectively. After the fetus was removed from the uterus, it was weighed and tranSported to the necrOpsy building. There, it was decapitated and the thyroid gland dissected. Thy- roids were weighed to the nearest 0.1g. (on a Gram-atic balance in our laboratory). The means and standard errors of the body weight eXpressed in kg and thyroid gland weight EXpressed in gm are shown in Table 1. No significant difference in body weight between male and female fetuses was found at any trimester of gestation. The same was true for the thyroid gland weight at the first and third trimester. But, at the second trimester of gestation the mean female thyroid gland weight (2.268 gm) was significantly higher (P<;0.001) than the mean male thyroid gland weight (1.752 gm). 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