,.- -.=. _ ;_ ,f ~-scoMFAmsorF 0F gowns FEFAL AND FFATEFFNALzuj-ff _:1_ GROWTH HOWGNE LUFFFNFFFNG momma.-'_{-_:.;;._g.fgifgfzjg 0‘ Theszsforthebégvee 61PM) AND PRBLACTQN AI 90 380 AND 260 DAYS GESTATEON MICHIGAN STATE UNIVERSITY VVVVV I... 1971 - LIBRARY Michigan State University This is to certify that the thesis entitled Comparison of Bovine Fetal and Maternal Growth Hormone, Luteinizing Hormone And Prolactin at 90, I80 and 260 Days Gestation presented by Wayne Dwight Oxender has been accepted towards fulfillment of the requirements for Ph.D. dggreein Dairy Science Major r Date May L4: 1971 0-7639 ABSTRACT COMPARISON OF BOVINE FETAL AND MATERNAL GROWTH HORMONE, LUTEINIZING HORMONE AND PROLACTIN AT 90, 180 AND 260 DAYS GESTATION BY Wayne Dwight Oxender Growth hormone (GH), luteinizing hormone (LH) and prolactin were quantified by radioimmunoassay in 37 fetal pituitaries and in umbilical arteries and veins at 90 days, 180 days and 260 days of gestation. Also, blood samples from the jugular, the uterine artery and the uter- ine vein of pregnant cows were taken at the same intervals. Maternal GH increased from 5.7 ng/ml at 90 days to 10.0 ng/ml at 260 days. Serum GH averaged 2 to 5 times higher in the jugular vein sample than in the uterine ves- sels (P < 0.01). The jugular vein blood sample had more (P m 0.11) GH in cows with male fetuses (12.3 ng/ml) than in cows with female fetuses (7.3 ng/ml) at 260 days. The fetal anterior pituitary GH increased (P < 0.01) during gestation (4.2, 8.9 and 18.1 ug/mg at 90, 180 and 260 days, reSpectively). GH for male and female fetuses Wayne Dwight Oxender was similar at 90 and 180 days, while males averaged 12.1 and females 25.3 ug/mg at 260 days, revealed by a signifi- cant sex-age interaction (P < 0.01). Fetal serum GH levels increased (P < 0.01) with fetal age from 42, to 65 and to 103 ng/mg for 90, 180 and 260 days, respectively. Jugular prolactin in cows averaged 220, 145 and 365 ng/ml at 90, 180 and 260 days, respectively. At 90, 180 and 260 days, cows carrying male fetuses had signifi- cantly more jugular serum prolactin than cows carrying females (P < 0.05). Fetal pituitary prolactin concentration increased (72, 1150 and 2508 ng/mg for 90, 180 and 260 days, respec- tively, P < 0.01) with fetal age. Fetal pituitaries syn— thesized large amounts of prolactin (averaged 23.6 ug/mg/ 72 hr.) during incubation in vitro. Pituitaries from fe- male fetuses synthesized three times more prolactin than males at 180 and 260 days (P < 0.07). Prolactin levels in the fetal serum averaged 4, 43 and 61 ng/mg for 90, 180 and 260 days of gestation, reSpectively (P < 0.05). Maternal serum LH levels from all three blood sources were indistinguishable and averaged 0.75 to 0.89 ng/ml during gestation. The pituitary LH concentration increased with fetal age from 323, to 474 and to 535 ng/mg for 90, 180 and 260 days respectively (P m 0.06). Pituitaries from males at 260 days contained 33% more LH than females but this differ- ence was not significant. Fetal serum LH levels decreased Wayne Dwight Oxender from 3.00 to 1.28 and to 0.85 ng/mg for 90, 180 and 260 days of fetal age respectively (P < 0.01). Unlike GH and prolactin, female serum LH averaged 3.90 ng/mg at 90 days and was significantly higher than fetal males (1.46 ng/mg) at the same age (P < 0.01). The female serum LH also averaged higher at 180 days but at 260 days the LH levels in the male were higher. These sex-age differences in serum LH resulted in a significant interaction of these two factors (P < 0.01). Fetal pituitary and serum LH con- centrations were not significantly correlated (r = -0.03). At birth, serum GH averaged 36 ng/ml and LH aver- aged 0.36 ng/ml; and neither changed significantly during the week after birth. Serum prolactin, however, averaged 101 ng/ml at birth, decreased to 42 ng/ml by the second day after birth (P < 0.01) and remained relatively constant to day 6 after birth. The serum hormone gradients, maintained by the pla- centa between the fetus and the cow, and the fact that um- bilical arterial blood serum hormone levels were indistin- guishable from umbilical venous levels for GB, LH and pro- lactin, indicate that placental transfer of maternal GH, LH and prolactin is not a major source of fetal serum hor- mones. Furthermore, age and sex differences in the fetal pituitary hormones, in vitro synthesis of hormones and in fetal serum hormones all indicated a degree of fetal inde- pendence from maternal control. In fact, fetal influences on the maternal endocrine system were observed in this study. COMPARISON OF BOVINE FETAL AND MATERNAL GROWTH HORMONE, LUTEINIZING HORMONE AND PROLACTIN AT 90, 180 AND 260 DAYS GESTATION BY Wayne Dwight Oxender A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1971 BIOGRAPHICAL SKETCH of Wayne D. Oxender I was born in Constantine, Michigan on April 11, 1931 and received my elementary education at a small rural school. My secondary education was completed at Centre- ville, Michigan. I entered Michigan State College and com- pleted one year of the agricultural shortcourse program. I then went into partnership with my parents on a dairy farm working as partner and later as owner of a pure— bred Holstein herd and dairying Operation. The Holstein herd was dispersed in 1961. During my farming period I gained a wife, Joan, three daughters, Barbara, Belinda and Bethany and one son, Thomas. Desiring a veterinary medicine profession, I entered Michigan State University and received a B.S. in animal husbandry in 1966 and a D.V.M. in 1967. My experiences at Michigan State University led me to pursue postdoctoral research training. I was accepted by the Department of Dairy Science and Dr. Harold Hafs to continue my research training, shifting my major emphasis to endocrinology which I find intriguing. For the past two years I have been the ii recipient of a N.I.H. postdoctoral research fellowship. During my research training I have worked part time with the Large Animal Surgery and Medicine Department. The experiences of teaching veterinary students and veterinary clinical medicine have been rewarding to me. I am completing my Ph.D. degree with this thesis in June 1971 and anticipate the opportunity to continue as a researcher and teacher. iii Sc cl 51 91 a1 Re £6 E: 66 gr be DU ma f0 Th We 01 me ACKNOWLEDGMENTS I should like to thank the Departments of Dairy Science and Large Animal Surgery and Medicine and their chairmen, Dr. Charles Lassiter and Dr. Fayne Oberst re- spectively, for providing funds and facilities for my graduate studies. Also to be thanked for providing funds are my wife Joan, the National Institutes of Health General Research Support Grant FR-5623-02 and Postdoctoral ResearCh fellowships 5-F02-HD42436-01 and 5-HD42436-02. But to my friend and advisor, Dr. Harold Hafs must be extended a debt of gratitude for providing me support, encouragement and enthusiasm to complete my graduate studies. Also I am grateful for the advice and support of my committee mem- bers, Drs. R. L. Anderson, C. E. Meadows and C. K. Whitehair. This thesis, as the reader will realize, is not the output of one person, but involved the participation of many individuals. They include radioimmunoassay antisera for LH, GH and Prolactin provided by Dr. Lloyd Swanson, IDr. Roger Purchas and Dr. H. Allen Tucker, respectively. The purified hormones for radioiodination and standards were supplied by Dr. Leo Reichert and the N.I.H. Endocrin- ology Study Section. Providing assistance for R.I.A. methods were James KOprowski and Dr. Lee Edgerton. Dr. iv Be la ti Edward Convey assisted with the anterior pituitary incuba- tions and assay techniques. Several peOple including Dr. Lee Edgerton, Winston Ingalls, James Koprowski, Dr. Lloyd Swanson and Joseph Zolman helped collect samples. Drs. Chris Miller and David Morrow assisted with the surgeries and pregnancy examinations. Dennis Armstrong aided in pro- curing and transporting the animals. 7 Finally, I would thank my wife Joan and children Barbara, Belinda, Bethany and Thomas for assistance in the laboratory but more importantly for accepting my transient efforts as a husband and father during the completion of this thesis project. TABLE OF CONTENTS BIOGRAPHICAL SKETCH . . . . . ACKNOWLEDGMENTS . . . . . . LIST OF TABLES. . . . . . . LIST OF FIGURES . . . . . . LIST OF APPENDICES . . . . . INTRODUCTION . . . . . . . REVIEW A. B. C. OF LITERATURE. . . . . Historical . . . . . Fetal Surgery . . . . Maternal Hormones During Gestation l. Pituitary Hormones . 2. Steroid Hormones . . Placental Function During l. Hormone Synthesis. . 2. Transfer of Hormones. Parturition . . . . . Fetal Sexual Development. 1. Gonads . . . . . . Genital Tract . . . Hypothalamus . . . Mammary Gland . . . The Freemartin. . . UlubLAJN Fetal Pituitary Hormones. Gestation l. Pituitary Tissue Culture 2. Luteinizing Hormone (LH) 3. Growth Hormone (GH) . 4. Prolactin . . . . vi Page ii iv ix xi xiii \lU'l-b oh I'" won 10 10 11 13 14 16 18 19 21 22 24 27 28 29 31 H. I. Fetal Adrenals, Pandreas, Parathyroid and Thyroid . . . . . Neonatal Serum LH, GH and Prolactin MATERIAL AND METHODS . . . . . . . . A. B. C. D. E. F. G. H. RESULTS A. Experimental Design. . . . . . Experimental Animals . . . . . 1. Fetal . . . . . . . . . 2. Neonatal . . . . . . . . Surgery. . . . . . Blood Serum Samples. . Fetal Tissue Samples . Pituitary Tissue Culture 1. Equipment. . . . . . . 2. Culture Medium . . . . . . 3. Culture Method . . . . . . Pituitary Homogenization . . . . Radioimmunoassay (RIA). . . . . l. Luteinizing Hormone (LH). . . 2. Growth Hormone (GH) . . . . 3. Prolactin (P) . . . . . . AND DISCUSSION . . . . . . . Fetal Physical Data. . . . . . 1. Age, Body Weight and Crown-rump (CR) Length. . . . . . . .‘ 2 Adrenal and Gonad Weights . . 3. Uterine and Seminal Vesicular Weights Maternal Serum Hormones During Gestation. l. Luteinizing Hormone (LH). . . 2. Growth Hormone (GH) . . . . 3. Prolactin. . . . . . . . Fetal Pituitary Hormones . . . . 1. Pituitary Content . . . . . 2. In Vitro Synthesis by Pituitary Tissue 3. Hormones in Fetal Blood Serum . vii Page 32 34 36 36 36 36 38 38 41 42 43 43 44 44 45 45 56 59 59 5‘9 61 64 65 66 7o 72 72 78 81 Page D. Neonatal Serum Hormones . . . . . . . 89 1. Luteinizing Hormone (LH) . . . . . 89 2. Growth Hormone (GH) . . . . . . . 90 3. PrelaCtj-n I O I O O I O O O O 90 GENERAL DISCUSSION . . . . . . . . . . . 91 A. Maternal-Fetal Hormone Interactions . . . 91 1. Growth Hormone (GH) . . . . . . . 91 2. Luteinizing Hormone (LH) . . . . . 93 3. Prolactin . . . . . . . . . . 94 4. Effect of Fetal Sex . . . . . . . 94 B. Fetal Pituitary and Serum Hormone Comparisons . . . . . . . . . . . 97 l. Pituitary Content. . . . . . . . 97 2. In Vitro Synthesis . . . . . . . 99 3. Serum Levels . . . . . . . . . 102 C. Fetal-Neonatal Serum Hormones . . . . . 105 SUMMARY AND CONCLUSIONS. . . . . . . . . . 108 BIBLIOGMPHY O O O O I O O O O O O O O 112 APPENDICES O I O O O O O O O O O O O O 128 viii Table 10. 11. 12. 13. 14. 15. 16. LIST OF TABLES Recovery of NIH-LH—BS from 100 pl of serum . Fetal body weight, crown-rump length and adrenal gland weight. . . . . . . . . Fetal testicular and seminal vesicular weights during gestation . . . . . . . Fetal ovarian and uterine weights during gestation . . . . . . . . . . . . Body weight of pregnant heifers . . . . . Jugular blood serum growth hormone (GH), LH and prolactin in cows carrying male or female fetuses. . . . . . . . . . . Blood serum LH in the cow during gestation . Blood serum growth hormone in the cow during gestation . . . . . . . . . . . . Blood serum prolactin in the cow during gestation . . . . . . . . . . . . Fetal anterior pituitary LH content. . . . Fetal anterior pituitary growth hormone content . . . . . . . . . . . . . Fetal anterior pituitary prolactin content . Net synthesis of LH synthesis by fetal anterior pituitary slices in vitro . . . . Net synthesis of growth hormone by fetal anterior pituitary slices in vitro . . . . Net synthesis of prolactin by fetal anterior pituitary slices in vitro . . . . . . . Average fetal serum LH . . . . . . . . ix Page 53 60 63 63 65 67 67 69 70 72 75 75 78 79 81 82 Table Page 17. Average fetal serum LH in the umbilical artery and vein . . . . . . . . . . 83 18. Average fetal serum growth hormone . . . . 85 19. Average fetal serum growth hormone in the umbilical artery and vein . . . . . . . 85 20. Average fetal serum prolactin levels . . . 86 21. Average fetal serum prolactin in the umbilical artery and vein . . . . . . . 87 22. Neonatal jugular serum growth hormone, LH and prolactin . . . . . . . . . . . 89 23. Some correlations between fetal pituitary hormone concentrations and net in vitro hormone synthesis. . . . . . . . . . 99 24. Some correlations between fetal pituitary and fetal serum concentrations of hormones . 103 25. Some correlations between fetal serum hormones and pituitary net in vitro synthesis . . . . . . . . . . . . 104 Figure 11. 12. LIST OF FIGURES Elution profile of iodinated luteinizing hormone (LH) after passage through Bio Gel P-60. The first peak represents iodinated LH and the second peak represents free iodine . . . . . . . . . . . . . Dose response curves for NIH-BS-LH standards and for bovine fetal sera, pituitary homo- genates and pituitary incubation media. . . Dose response curves for NIH-GH-BlZ standards and for bovine fetal sera, pituitary homo- genates and pituitary incubation media. . . Dose response curves for NIH-Bl-prolactin standards and for bovine fetal sera, pitui- tary homogenates and pituitary incubation media. . . . . . . . . . . . . . Comparison of crown-rump length and fetal age O O O O O O O O . O O O O 0 Levels of LH in fetal pituitary and blood germ“. O I O O O O O O O O O O 0 Levels of growth hormone in fetal pituitary and blood serum . . . . . . . . . . Levels of prolactin in fetal pituitary and serum. 0 I O O O O O O O O O O 0 Neonatal calf jugular serum prolactin . . . Levels of growth hormone in cow and fetal blood sera during gestation . . . . . . Levels of LH in cow and fetal blood sera during gestation . . . . . . . . ,. . Levels of prolactin in cow and fetal blood sera during gestation . . . . . . . . xi Page 48 52 52 58 58 84 84 88 88 92 92 95 Figure 13. 14. 15. 16. Fetal anterior pituitary growth hormone, LH and prolactin . . . . . Fetal pituitary content and in vitro synthesis of growth hormone, LH and prolactin . . . . . . Fetal blood serum growth hormone, LH and prolactin during gestation. Fetal and neonatal blood serum growth hormone, LH and prolactin . xii Page 95 100 100 106 LIST OF APPENDICES Appendix Page I. Composition of reagents used in radio- immunoassay. . . . . . . . . . . 129 II. Maternal physical and hormonal data on cows at 90 days gestation . . . . . . 132 III. Maternal physical and hormonal data on cows at 180 days gestation. . . . . . 133 IV. Maternal physical and hormonal data on cows at 260 days gestation. . . . . . 134 V. Physical and hormonal characteristics of fetuses at 90 days gestation . . . . . 135 VI. PhysiCal and hormonal characteristics of fetuses at 180 days gestation. . . . . 137 VII. Physical and hormonal characteristics of fetuses at 260 days gestation. . . . . 139 VIII. Hormone content of pituitary explants and pituitary incubation media . . . . 141 xiii INTRODUCTION What is the degree of fetal autonomy during develop- ment within the maternal environment? One problem en- countered by endocrinologists and reproductive physiolo- gists studying reproduction and developmental biology is whether the hypothalamo-hypophyseal system of the fetus participates in the maturation of the genital apparatus of the fetus. For instance, to what extent does the fetal hypothalamo-hypophyseal system and its secretions control somatic growth, the development of the gonads and the dif- ferentiation of the genital duct system? There is limited knowledge of the hormone levels of the fetal endocrine glands and the ability of the glands to synthesize and re- lease hormones in utero. As a consequence, whether the pre- natal endocrine secretions are identical to their adult counterparts or if the fetal reSponses to endocrine stimuli are similar to the response elicited postnatally are un- determined. Male or female develOpment appears to be gradual transition. Genetic sex of the individual is determined at conception, but gonads, genital ducts, accessory gland development and hypothalamic function can each be recognized at sexually indifferent stages. In other words, except for the genetic sex of an individual, endocrine secretions apparently determine at least in part male or female dif- ferentiation of the gonads, genital ducts, accessory glands and hypothalamus. Parts of sexual differentiation appear to be reversible while others, once differentiated, appear to be permanent. Major advances have been made recently in the area of endocrinology allowing determination of levels of hor- mone in fetal and maternal blood. Fetal pituitary hormone content and ability to synthesize hormones also can be quan- tified by recently developed radioimmunoassays (RIA). This has been a major breakthrough in endocrinology, including developmental biology. Whereas formerly, researchers quan- tified hormones crudely into "gonadotrophic activity," it is now possible to identify and quantify specific hormones in most endocrine organs and biological fluids. With the above questions about sexual develOpment and with the new techniques for quantifying hormones, this project was developed, initiated and completed. The bovine fetus was chosen as the subject of this project because of availability and convenient size for study. Endocrine develOpments of the bovine from the birth through puberty have been the subject of comprehensive studies in this laboratory in the past. This fetal developmental study was designed to extend our knowledge of the endocrine and reproductive changes of the bovine to the period before birth. The object was to study at least five male and five female fetuses, and their dams, near the end of each trimester of pregnancy. While this study may not provide final answers, knowledge gained from these studies may find practical application in a number of current endocrine malfunction problems in cattle. Furthermore, some human endocrine malfunctions lead to similar abnormal fetal and neonatal development. Increasing reproductive efficiency of animals would increase the supply of animal protein for food, with impact on malnutrition due to the lack of dietary protein. REVIEW OF LITERATURE A. Historical The fact is that animals, if they be subjected to a modification in minute organs, are liable to immense modifications in their general configura- tions. This phenomenon may be observed in the case of gelded animals: only a minute organ of the animal is mutilated, and the creature passes from the male to the female form. We may infer, then that if in the primary conformation of the embryo an infinitesimally minute but absolutely essential organ sustains a change of magnitude one way or the other, the animal will in one case turn to male and in the other to female: and also that if the said organ be obliterated altogether, the animal will be neither one sex nor the other. This quotation from Aristotle from over 2000 years ago (Smith and Ross, 1910) has to be one of the earliest published observations of endocrine function. Evolution of knowledge covering development of animal intersexes prior to 1779 was published in a treatise by Hunter and has been reviewed by Short (1969). McClung (1902) was the first to describe the genetic basis for sexuality. He found that all but one pair of chromosomes were identical in male and female cells. Fur- ther comparison of this non-identical pair of chromosomes revealed that chromosomes from male cells had one smaller chromosome in the pair of sex chromosomes, while the two chromosomes were identical from female cells. Later, Morgan and Bridges demonstrated that the autosomes also can in- fluence sexual develOpment in Drosophila (Bridges, 1939). 4 Fetal development studies received early assistance from Bouin and Ancel (1903) when they reported that the male fetal testis was a secretory organ. McCord (1915) was one of the first to demonstrate oxytocic activity in fetal pituitaries and epinephrine activity in fetal adrenal glands. The interesting natural phenomenon of the sterile bovine freemartin, a female born twin to a male, was hypoth— esized to be the result of hormone exposure during pre- natal development by the now classic studies of Lillie (1916) and Keller and Tandler (1916). Comparative endo- crinological studies of fetal development using insulin from the bovine fetal pancreas to depress blood glucose in a pancreatectomized dog have been reported by Banting and Best (1922) and by Hogben and Crew (1923). The latter authors investigated the cause of the pathological "bull- dog" fetus in Dexter cattle. The fetal thyroid gland from "bulldog" fetuses contains the same thyroid activity as normal calves when determined by metamorphic changes in axolotls (a small amphibian). B. Fetal Surgery As early as 1803 surgical procedures were developed to observe antenatal movements establishing the feasibility of surgery as a research technique (Swenson, 1925). To study the contribution of the fetal pituitaries to fetal development, Jost (1947) and Wells (1947) used fetal decapitation. Newer techniques for fetal hypophysectomy by using radioactive pellets (Hutchinson et_31,, 1962), electrocoagulation (Liggins et_al., 1967) and surgical ex- cision (Kraner and Parshall,l969) are more Specific, but remove all pituitary hormones simultaneously. A technique of marsupializing the fetus for com- plicated surgical techniques has been reported by Jackson and Egdahl (1960). Fetal surgical techniques for skin grafting, splenectomy, gonadectomy and thymectomy have been described by Kraner and Parshall (1969). Several surgical approaches for fetal immunological studies in primates have been successful (Parshall and Silverstein, 1969). Evidence that fetal endocrine activity may have far reaching consequences is abundant. For example, fetal adrenalectomy causes prolonged gestation in ewes (Drost and Holm, 1968). Another very successful surgical technique for 'studies of fetal endocrine development is catheterization of the fetal vascular system (Kramer and Parshall, 1969; Snow and Tyner, 1969). Chronic catheterization has been used to study fetal plasma corticosteroids (Bassett and Thorburn, 1969), insulin induced hypoglycemia in the ovine fetus and placental transfer of insulin (Colwill gt_aI., 1970), placental transfer of GH (Gitlin‘§E+§I., 1965), and placental transfer of LH and fetal reSponse to luteinizing hormone releasing factor (Foster, 1971). Placental trans- fer of drugs using the above techniques recently has been —————— repor nilam durin can be ment. tem Separe Conclt reported by Almond et_31. (1970); fetal levels of sulfa- nilamide were maintained at 60% of the maternal levels during constant infusion of the mother. Fetal metabolic studies and placental transfer in 3132 using radioactive compounds were reported by Diczfalusy and Mancuso (1969). An artificial fetal environment was developed by Zapol et_al. (1969) to study fetal metabolism of radioactive compounds. A synthetic fluid similar to amnionic fluids suspends the fetus in vitro, while oxygen- ated blood is perfused through the fetus via umbilical blood vessels. As indicated above, numerous surgical procedures can be used as aids to the study of fetal endocrine develOp- ment. C. Maternal Hormones during Gestation Zelenik (1965) reviewed the endocrinology of preg- nancy which must be viewed as a completely integrated sys- tem. Evaluating one hormone alone or one hormone system separately probably will continue to give isolated and in- conclusive information. The early influence of the developing bovine embryo on the maternal endocrine system was reported by Shemesh gt_§I. (1968). On day 19 after conception, the pregnant cow has proqesterone levels three-fold greater than the non-pregnant cow (Shemesh et_al., 1968). Thus, the devel- oping fetus affects the maternal endocrine system. Furthermore, the extent of this affect appears to be re- lated to the sex of the fetus; women with female fetuses have human chorionic gonadotropin (HCG) serum levels more than two-fold greater than women with male fetuses after 190 days of gestation (Brody and Carlstrém, 1965). Urine pregnanediol excretion is greater in women with male fetuses suggesting that sex differences of the fetal pla— centa may be responsible for differential metabolism of steroids (Rawlings and Krieger, 1964). Resko (1970) found higher plasma testosterone concentrations in primates with male fetuses than in those with female fetuses. Fetal sex influences on the maternal endocrine system are not the only fetal characteristics that have been reported. MacMillan (1970) found significantly re- duced human chorionic somatomammotropin in the plasma and placenta of mothers that delivered abnormally small babies. 1. Pituitary Hormones Maternal pituitary hormones are essential to main- tain pregnancy in most species during the first half of gestation. In early pregnancy, progesterone secretion is controlled by pituitary hormones, but placental hormones apparently replace this pituitary function later (Jaffe gt_aI., 1969). Pregnancy proceeds normally in females that are hypOphysectomized near mid-gestation in several species (Hutchinson §t_al,, 1962). Chez et_g£. (1970) hypOphysectomized primates in the second trimester of gesta- tion without causing abortions. in wc Chang peake dent the f lactiJ (Scha: 48 hox m adrena must a and es Vary C. interr‘ and pr: abort 1 after 2 ViEWQd Faiman et_aI. (1968) found no change in serum FSH in women during gestation. Pituitary FSH levels did not change in the sow during gestation, and while LH levels peaked on day 18, prOportionately little change was evi- dent during gestation (Melampy et_a£., 1966). Bovine serum LH averaged less than 1 ng/ml during the first 60 days of gestation (Schams, 1969). Serum pro- lactin increased sharply to 880 ng/ml before parturition (Schams and Karg, 1970), but this peak disappeared within 48 hours. 2. Steroid Hormones In the nonpregnant female, only the ovaries and adrenal glands produce steroid hormones while the placenta must also be considered during pregnancy. Progesterone and estrogen production by the ovaries during pregnancy vary considerably among species. Ovariectomy does not interrupt gestation in the guinea pig, cat, sheep, horse, and primates while the mouse, rabbit, goat and sow always abort following ovariectomy. The cow can be ovariectomized after 200 days of gestation without causing abortion (re- viewed by Amoroso and Finn, 1962). Stabenfeldt et_a£. (1969) found bovine plasma pro- gesterone levels increased nearly 50% from mid-gestation to term. Urinary estrogen elimination increases during the pregnancy period in humans and serum levels also increase as gestation proceeds (Diczfalusy and Mancuso, 1969; 10 Younglai and Solomon, 1969). Plasma corticosteroids_do not change in the ewe during gestation (Bassett and Thorburn, 1969). D. Placental Function during Gestation The placental permeability properties are unique. They can prevent transfer, selectively or actively trans- fer, or be completely permeable to a given biological com- pound. Considerable metabolic and synthetic activity is also associated with the placenta. In spite of the impor— tant placental functions, this specialized tissue is only a temporary organ. Its genetical derivation is from two sources. The endometrium of the mare had gonadotophic activity when the embryo was only 1.8 cm long (Catchpole and Lyons, 1934), and gonadotrophic activity was found in the incubation media after human placenta had been incu- bated in vitro (Gey et al., 1938). l. Hormone Synthesis Human chorionic gonadotropin (HCG) is the placental Ihormone that has been most widely studied. Aschheim and Zondek (1927) showed that it had gonadotrophic activity and since that time many studies have contributed to our present knowledge about HCG (reviewed by Brody, 1969). In addition to HCG, another protein hormone human chorionic somatomammotropin is synthesized by the human placenta. This hormone is sometimes called human placental lactogen (Gru: MacM: DOIIT‘FC bovir 1967; cate teror and h geste propel 5100113 k detErR mothez gcnado 11 (Grumbach et_aI., 1968; Gitlin and Biasucci, 1969; MacMillan, 1970). Human placental lactogen has both growth hormone and prolactin activity. Progesterone is synthesized in vitro by sheep, bovine and human placental tissues (Ainsworth and Ryan, 1967; Younglai and Solomon, 1969). In vitro studies indi— cate that human placental preparations metabolize proges- terone producing several derivatives. In contrast sheep and bovine placental preparations possess very little pro- gesterone metabolic activity (Ainsworth and Ryan, 1967). Estrogens are synthesized by bovine and goat pla- cental preparations in vitro indicating the presence of the necessary enzymes for aromatization of steroids (Pierrepoint eEJEqu969a; Ainsworth and Ryan, 1970). The placenta in most species, however, is a source of pro- gestins, estroqens and androgens. Precursors for these steroid hormones may come from either fetal or maternal sources . 2. Transfer of Hormones The type of placenta varies and the permeability properties and hormone metabolic activity appear to differ among many species. Since the placenta is selectively permeable to certain hormones it has been difficult to determine whether the hormone is from the fetus or the mother. Goodman and Wislocki (1933) could not detect any gonadotrophic activity in fetal fluids after pregnant cats an: ext int of He mat and wit pit pla cro tra. the hOrr l97( curE mOt} Plac SYnt te 12 and rabbits were injected with HCG or anterior pituitary 131I-Growth hormone extract. The technique for injecting into the umbilical cord and determining the distribution of the radioactivity was developed by Gitlin et a1. (1965). He showed that no radioactivity could be found in the maternal circulation. GonadotrOpins in umbilical cord and maternal blood serum were measured by radioimmunoassay (RIA) (Faiman et_al., 1968). Both of these studies agreed with the earlier conclusions of Mitskevich (1962) that pituitary hormones do not appear to be transferred by the placenta. Foster (1971) demonstrated that LH does not cross the placenta in either direction. However, Specific transfer of proteins must occur because insulin crosses the placental barrier readily (Mitskevich, 1962; Gitlin gt_§I., 1965; MacMillan, 1970) and maternal gamma globu- lins are transferred to the fetus but albumin cannot cross the placenta (Gitlin gt_al,, 1965). Thyroid hormone is also transferred by the placenta but thyroid stimulating hormone (Mitskevich, 1962) is not. Extensive studies using doubly labeled steroid hormones (Diczfalusy and Mancuso, 1969; Pasqualini gt_3l., 1970) showed that most steroid hormones and their pre- cursors appear to be transferred by the placenta from mother to fetus and from the fetus to the mother. The placenta appears to be the source of increasing estrogens synthesized from precursors supplied by the fetus during late gestation in humans. - I l .1. hp V‘_ “ivy—*1 . treat; that . (1969~ sulti. gen i: the cc 1969). steroi born t nancy Jost a, Steroig in the used dL reprodc fed the e Q” Stage 0 Conside VOlfiveS t 13 Fetal abnormalities produced by steroid hormone treatments of the mother during gestation also indicate that steroids are transferred to the fetus. Shane gt_§l, (1969) fed pregnant dogs testosterone and all of the re- sulting female offspring were pseudohermaphrodites. Estro- gen injections in pregnant beagles caused malformation of the coxofemoral joints in the pups (Gustafsson and Beling, 1969). Oral contraceptive pills contain a variety of steroids and have caused virilization of female children lxzrn to women who have taken contraceptives during preg- nancy (reviewed by Jones and Wilkins, 1960; Ferreira, 1969) . (host and Moreau-Stinnakre (1970) fed two contraceptive steroids to pregnant rats and produced genital anomalies 131 the female offSpring. However, not all steroid hormones used during pregnancy prove to be detrimental to the future reproductive performance of the offspring. Normal concep- tdxon rates were recorded for heifers from cows that were fend the synthetic progestin, melengestrol acetate (Schul et al., 1970). r The species, kind of steroid, level of steroid and stage of fetal develOpment are all factors that must be considered when steroids are to be administered to pregnant females. E. Parturition The final event for the developing fetus which in- VOlves the intimate interactions of the fetoplacental- maternal systems is parturition. Many workers have searched 14 for the factors controlling this event and several factors appear to be involved. Only a few of the more recent re- ports on the fetal contribution to the initiation of par- turition are listed here. Liggins gt_al.(l967) produced prolonged gestation in sheep by destruction of the fetal pituitary and reported that developmental failures of the fetal pituitary caused by ingestion of Veratum californicum F in pregnant ewes also delayed parturition. Fetal adenohypo- physealaplasia appeared to be caused by a genetic defect in Guernsey cattle and results in prolonged gestation. In addi- tion to adenohypOphysealaplasia, the fetal thyroid and adrenal cortex were hypOplastic in cattle (Kennedy et_al., 1957) and in humans (Benirschke, 1956). Fetal adrenalectomy also caused longer gestation periods in sheep (Drost and Holm,1968). Fetal lamb corticosteroid levels determined by umbilical catheterization revealed a six-fold increase in fetal corticosteroids from 130 to 150 days gestation (Bassett and Thorburn, 1969). Adams and Wagner (1970) reported plasma corticoid levels in the pregnant cow in- creased 100% the last four days of gestation. While the fetal adrenal apparently participates in initiation of partu— rition, how the fetal adrenal controls maternal uterine muscle remains to be determined. F. Fetal Sexual DevelOPment During the fetus's intra-uterine deveIOpment, the endocrine glands and the hormonal receptor organs diffe: for p: show u and St Early (1903) to the activi‘ Jost (L tary hc partici and Pic Uterine they pl. fetus? ln9 rece 15 differentiate, and at birth they are more or less ready for postnatal functions. Before birth, some fetal glands show unquestionable cytological signs of secretory activity\ and some organs seem to have been stimulated by hormones. Early observations and interpretations by Bouin and Ancel (1903), Lillie (1916), and Keller and Tandler (1916) led to the belief that the fetal testes produced androgenic activity. Subsequent studies by Wells (1946, 1947) and by p Jost (1947) using fetal decapitation and exogenous pitui- tary hormones concluded that the fetal pituitary and testes participate in sexual differentiation (reviewed by Jost and Picon, 1970). Do the fetal glands only prepare, during intra- uterine life, for their later postnatal functions or do they play an indispensible part in the development of the fetus? Do maternal hormones exert effects on the develOp- ing receptor organs? In experiments involving fetal castrations, organ tranSplants and exogenous androgens, Jost (1955) and Neumann et a1. (1969) concluded that the fetal testes have both an inhibitory and a stimulatory action on sexual differ- entiation. Additional research reviewed by Jost (1965) :supported the above conclusions. The castrated fetus .acquires feminine sexual features irrespective of genetic sex. 16 Three aSpects of fetal development that appear to be controlled by fetal pituitary trophic hormones are sexual differentiation, thyroid colloid synthesis and liver glycogenesis (Jost, 1966; Jost and Picon, 1970). l. Gonads Gonad differentiation begins with formation of primordial germ cells in the yolk sac lateral to the base of the viteline artery with subsequent movement of these I primordial germ cells into the embryo proper. The first distinct germinal epithelium is found in 28-day bovine embryos. Migration and elongation by mitotic division of the germ cells forms the genital ridge by day 30 in the bovine. Continued rapid expansion of the genital ridge results in a ballooning of the developing gonad. At about 40 days (Krehbiel, 1963; Erickson, 1966; Gier and Marion, 1969; Matschke and Erickson, 1969) the sexually indifferent stage of gonad deveIOpment ends and sex differentiation begins. By 42 days of bovine pregnancy, ovarian cortex is formed in the female or seminiferous cords in the male gonad and Leydig cells appear at 44 days (Gier and Marion, 1969). Macrosc0pic sex determination is possible at day 46 in the bovine embryo (Krehbiel, 1963). Macnaughton (1969) reported that the fetal rabbit gonad is differenti- ated at 15 days. The gonad differentiation appears to take place very early in fetal development and may be primarily controlled by genetic factors. l7 Bascom (1923) investigated fetal calf and pig testes and hypothesized that the interstitial cells were active in utero. Bovine fetal testicular extracts were a more potent source of androgens than adult testicular extracts (Koch, 1931). Ovarian and testicular extracts contained estrogenic activity but lacked androgenic acti- vity (Cole et_aI,, 1933). Wells (1946) used exogenous gonadotrOpin to stimulate an increase of Leydig cells in the fetal rat testis. Incubation of bovine ovaries with labeled steroid precursors (Roberts and Warren, 1964) indicated that fetal ovarian tissue is capable of some steroid transformations similar to the adult bovine ovary (Sweat et_aI., 1960). However, there is no evidence that fetal ovarian secre- tions are essential for sexual differentiation. In contrast fetal testicular secretion appears to be essential for normal male sexual differentiation. Benirschke and Bloch (1960) failed to demonstrate testo- sterone in testes in bovine fetuses that were around 110 days old. In contrast, Struck and Karg (1967) measured both testosterone and androstenedoine in fetal calves after 120 days of gestation. Testosterone levels of both fetal lamb (Attal, 1969) and calf testes (Struck and Karg, 1967) appear to peak in the second trimester of gestation and then decrease as gestation advances. Umbilical cord blood levels of testosterone are higher in males than in females in fetal rhesus monkeys (Resko, 1970). 18 The observation of periodic endocrine activity of fetal glands during develOpment was made by Jost (1947) originally and was supported by several other studies (Van Wagenen and Simpson, 1954). Fetal testicular secre- tion of androgens appears to be well documented, but their action and especially the integration of androgens with the fetal endocrine system remains obscure. 2. Genital Tract Normally, the tubular and external genitalia of both sexes develOp to an indifferent stage and then differ- entiate. However, all degrees of intersexual development are possible between the normal male and female. In males, the Wolffian or mesonephric ducts develop into the epidi- dymis, vas deferens and seminal vesicles. The prostate gland and bulbourethral gland originate from the urogenital sinus. The Mullerian duct regresses in the male. In con- trast, the female infundibulum, oviduct, uterus and vagina all develop from the Mullerian ducts and the Wolffian ducts regress. Male or female external genitalia differentiates from a common origin. Much research interest has centered on genital duct differentiation and its exogenous control. Possibly, the existence of the bovine freemartin (see Freemartin develop- ment) has had a stimulatory effect on research in this area. é.§l§ll§§ an .1.‘ . , 19 Price and Pannabecker (1959), in a unique experi- ment, used embryonal genital tracts cultured in vitro to discover that testosterone would prevent female organo- genesis. Androgens and anti-androgens have been used by Neumann EE_El° (1969) to induce intersexuality. Androgens or testicular secretions appear to be necessary for normal male genitalia develOpment. The need for more precise information on hormone caused ab- normalities is evident. 3. Hypothalamus If one assumes that the male and female pituitary are similar and that only the anterior pituitary of the female releases gonadotrOphic hormones in a cyclic pattern while the male gonadotrophic release pattern is tonic, then higher brain centers must control pituitary gonado- trophic release (Harris and Jacobsohn, 1951). The hypo- thalamus apparently is responsible for female cyclicity and indirectly for estrus behavior (reviewed by Young, 1961 and 1966; Levine, 1971). Sexual differentiation of the hypothalamus appears to involve irreversible alteration of sensitivity to steroid hormones (Barraclough, 1967). The age at differentiation varies with species. The age in the hamster is 1-3 days postnatally (Gorski, 1968; Alleva et_a£., 1969), but in the guinea pig it occurs prenatally (Young, 1961). Differ- entiation of the hypothalamus appears to be prenatal in 20 species such as pigs and calves that have more mature fetuses at birth (Zimbleman, 1964). Androgens seem to be necessary to prevent the development of the female cyclic pattern of gonadotrophic secretion. Females exposed to androgens during the criti- cal age~period are said to be androgen sterilized. Andro- gen sterilized females do not show evidence of cyclic activity as judged by vaginal smears. Females that have been so sterilized fail to mate, ovulate or conceive and may show male behavioral aggressiveness (Harris and Levine 1965). Dorfman (1967) could prevent the androgen sterili- zation of female rats by simultaneous administration of progesterone. Estrogen exposure to male fetuses or neonates causes testicular atrophy presumably through interruption of gonadotrophic stimulatory mechanisms possibly at the hypothalamic level. This action of estrogen is prevented by progesterone (Dorfman, 1967). Eguchi and Morikawa (1968) approached the fetal gonadotropin secretion problem by the use of parabiotic twin studies. If they used intact female fetuses and de— capitated males they observed testicular atrophy suggesting that the male fetal pituitary releases gonadotropins. Large doses of an oral contraceptive (Lynestrenol and Mestranol) to lactating hamsters caused permanent sterility in the nursing offspring (Czyba §E_§I., 1969). These studies suggested that the estrogenic activity of 21 the contraceptive agent may have acted directly on the neo- natal gonads to cause sterility in the case of the nursing hamsters. Hypothalamic control of pituitary function has been studied by controlled deafferentiation of hypothamic areas with the Halasz knife (Halasz and Gorski, 1967). Deafferentiation studies have shown that cyclic LH release in females is controlled by brain centers other than the median basal hypothalamus (Halasz and Gorski, 1967). The sexual differentiation of the hypothalamus by exposure to steroid hormones at critical periods seems well established in rats and in a few other species. In addition, marked behavioral patterns of humans appear to be influenced by hormonal stimuli (reviewed by Levine, 1971). 4. Mammary Gland Mammary gland development is also sexually depen- dent; however there are some stages in development which appear to be reversible. Cupceancu gt_aI. (1969) reported that administration of several synthetic progestogens to pregnant rats prevented normal mammary gland development in both female and male fetuses. Similar results were ob- tained by Jean (1969) and by Jean and Delost (1969a) using androgens and estrogens on pregnant mice. Prolactin did not cause fetal mammary gland abnormalities when given to pregnant mice (Jean and Delost, 1969b). 22 5. The Freemartin The freemartin appears to be a unique abnormality in sexual differentiation of the bovine and has been the subject of numerous investigations. Early scientists attempted to determine the cause and more recent efforts have failed experimentally to produce a freemartin. Hunter in 1779 reported on intersexes in several species but it was not until 1916 that a fetal hormone was theorized to be the cause of freemartinism. Lillie (1916) and Tandler and Keller (1916) independently arrived at the conclusion that the cause was hormonal transfer to the female fetus from the male fetus. A classic paper by Lillie (1917) compared his findings with all the previously advanced theories. A pOpular theory at that time was that the sterile female was a genetic male or monozygotic twin. Lillie's conclusions were: (1) the twins were of separate zygotic origin, (2) vascular anastomosis of placental ves- sels was present, (3) the sterile female was limited to the bovine because of the type of placentation, and (4) the fetal testis developed earlier than the ovary. Injec- tions of dyes showed a common placental circulation between :he male and female fetuses of the bovine (Lillie, 1917), n sharp contrast to the separate circulatory systems ob- :rved in sheep fetuses even when twin fetuses share a tyledon (Mellor, 1969). Chaplin (1971) did an extensive stological study of freemartins and substantiated the 23 wide variability observed in freemartin gonads and geni- talia development. According to Willier (1921) the degree of transformation of the freemartin ovary into a testis histologically was related to the degree of masculiniza- tion of the external genitalia. Studies of the metabolic activity of freemartin gonads in vitro were reported by Hoffman and Martin (1968) and by Pierrepoint et_aI. (1969b). The former authors reported synthesis of androstenedione from progesterone while the latter authors found that the rate of metabolism of dehydroepiandrosterone was intermediate to that of the normal ovary and testes. Administration of several androgens at varying levels and periods of gestation all produced similar re- sults (Mason et_§I., 1958; Hurst, 1962; Jost et_al., 1963; Jainudeen and Hafez, 1965). All of the above researchers observed some masculinization of external genitalia of the developing female fetus. However, in contrast to the free- martin, the ovaries were always normal. Chimerism of germ cells in freemartin gonads has been reported by Ohno (1969). Fechheimer et_al. (1963) and Stewart (1965) hypothesized that this germ cell mosaicism in the gonad caused the freemartin anatomical defects by local action on the germinal ridge. Ohno (1969) favored the hormonal theory as a cause of freemartin development. 24 It is possible that hormonal influences from the male twin testes and germ cell Chimerism with its effect on the germinal ridge each contribute to the development of a freemartin. The etiology is still unknown. G. Fetal Pituitary Hormones Male rat pituitaries when transplanted into females release gonadotrOpins in a cyclic manner according to Harris and Jacobsohn (1951). The evidence suggests that the pitu- itary does not participate in sexual differentiation but that sexual differences in secretion and content of pitui- tary hormones are controlled by factors outside the pitui- tary. Hypothalamic releasing factors, inhibitory factors, pituitary hormones and gonadal hormone feedback mechanisms are all controls on the pituitary function. Early research on bovine fetal pituitaries by McCord (1915) indicated the presence of oxytocic activity in 56-day fetuses. McCord suggested that the fetus was under the influence of its own glands in utero. Smith and Dortzbach (1929) used pituitary extracts from l7-cm fetal pigs to cause precocious sexual development in immature mice and extracts from 9-cm pig fetuses to stimulate growth in hypOphysectomized rats. He concluded that GH and gonadotroPins were separate hormones and both were present in the fetal pituitary. Gonadotrophic activity of fetal pituitary extracts have been recorded for the horse (Hellbaum, 1935) and for the cow (Bates et al., 1935). i; 25 Bates also found prolactin and thyroid stimulating activity in the bovine fetal pituitary, indicating an autogenous hormone source for the fetus. Another accident of nature is the human anencephalic fetus. These fetuses may develop without a brain, hypo- thalamus and/or pituitary. Hypoplasia of the fetal adrenals and testes are common pathological changes associated with anencephalic humans and indicate the lack of trophic hor— mone stimulation (Benirschke, 1956; Blizzard and Alberts, 1956; Brewer, 1957; Zondek and Zondek, 1965). Liggins and Kennedy (1968) noted hypOplasia of the adrenals, testes and thyroid when the sheep fetal pituitary was removed. A reciprocal functional relationship between the hypophysis and the adrenals during fetal development was demonstrated by Kitchell and Wells (1952). Electron microsc0pic studies by Hatakeyama (1969) confirmed activity of the hypothalamic- pituitary-adrenal axis in the human fetus by mid-gestation. Mitskevich (1962) also concluded that the fetal pituitary- thyroid function is established during gestation in the rabbit. Bioassays demonstrated GH, LH, FSH, TSH, MSH, ADH, prolactin and oxytocin activity in the fetal pituitary of various species (reviewed by Levina, 1968 and Macnaughton, 1969). Fetal pituitary FSH was detected in the rat (Corbin and Daniels, 1967), in sheep (Mauleon and Reviers, 1969), and in man (Levina, 1968). Fetal serum FSH levels were 26 reported by Faiman §E_§I, (1968) for umbilical cord blood samples but whether the fetal serum FSH originated in the dam was not determined. Fetal pituitary LH concentrations were reported for man (Levina, 1968), for sheep (Mauleon and Reviers, 1969; Foster, 1971), and for cattle (Karg, 1967a, 1967b). Mauleon and Reviers (1969) found pituitary LH Content in the male similar to that in the female fetus using bioassay, how- ever Foster (1971) found that LH levels of males were higher than those of females using RIA. The LH-FSH ratio in fetal pituitaries differs in males and females for humans (Levina, 1968) and for sheep (Mauleon and Reviers, 1969). When human fetal pituitaries were transplanted into adult hypo- physectomized rats (Levina, 1968), gonadotrOpin secretion of the fetal human pituitaries stimulated the rat gonads. Serum levels of LH in fetal lambs increased after injection of porcine hypothalamic extract (Foster, 1971) indicating the fetal lamb pituitary is capable of respond- ing to releasing factors. RIA techniques will allow more precise research on individual pituitary hormones and their release patterns in the fetus. Each fetal hormone should be compared to the related endocrine environment to more accurately under- stand fetal pituitary function. 1. Pituitary Tissue Culture Pituitary synthesis and secretion can be studied in tissue cultures. Gey gt_al. (1938) incubated fetal pituitary tissues and tested the incubation medium for gonadotrOphic activity. He could not detect activity in a bioassay. However, he reported that microsc0pic examina- tion indicated slight gonadotrOphic activity. Better methods of pituitary explant culture have since been de- veloped (Meites et_al., 1961; Piascek and Meites, 1967). Meites gt_aI. (1961) reported rapid synthesis and release of prolactin by rat pituitary eXplants. Radioactively labeled amino acids were incorporated into GH, FSH, LH, and TSH during incubation of human fetal pituitary tissues (Gitlin and Biasucci, 1969). In their study, GH was the earlist pituitary hormone shown to be synthesized by human fetal pituitary cultures. Labeled GH was synthesized in cultures of 60-day fetal pituitaries. Gailani et_a£. (1970) detected GH, LH and TSH in human fetal pituitary culture media using RIA. Growth hormone measured in the media by RIA also was capable of producing growth of the rat tibia, thereby showing a correlation of the immunological and bio- logical assays. Pituitary tissue culture techniques have been im- portant methods to test for releasing factors and steroid hormone feed-back controls on the anterior pituitary. Caution must be used in interpreting in vitro results with the in vivo pituitary function. 28 2. Luteiniz‘i‘nLHO'moie (LH) GonadotrOphic activity in fetal pituitary extracts- from the pig (Smith and Dortzbach, 1929; Melampy fl” l966),tme horse (Hellbaum, 1935), the sheep (Mauleon and Reviers, 1969; Foster, 1971), the cow (Bates 3131., 1935; Karg, 1967a, 1967b) and in man (Levina, 1968; Rice et al., 1968) have been reported. Gitlin and Biasucci (1969) used immunoelectrOphoretic techniques to identify in vitro syn- thesis of labeled LH by human fetal pituitary explants which had been incubated with radioactive labeled amino acids. Immunofluorescent techniques were used by Dubois and Mauleon (1969) to identify LH in anterior pituitary cells of a 49- day lamb fetus. Foster (1971) used RIA to quantify LH in fetal sheep pituitary homogenates and found that pituitary LH increased faster in males than in females. This increase in fetal pituitary LH concentration with increased fetal age agrees with the bioassay data for sheep (Mauleon and Reviers, 1969) and also for the cow (Karg, 1967a, 1967b). In early studies researchers were unable to detect gonadotropic activity in fetal blood and serum using bio- assays. Foster (1971) used RIA to measure the serum LH levels in fetal lambs from 55 days through parturition and detected increased LH up to 100 days and then a decrease as parturition approached. I found no reports of fetal bovine serum LH in the literature. Sexual differences in fetal pituitary LH concentra- tion have been observed in the cow (Karg, 1967b) and in the 29 sheep (Foster, 1971) . Mauleon and Reviers (1969) found a sexual dimorphism in LH:FSH ratios in fetal lamb pituita- riessfinular to that observed by Levina (1968) in human fetalgfltuitaries. RIA and catherization of develoPing fetuses makes possible a more accurate determination of 1M during fetal develOpment. 3. Growth Hormone (GH) In a review, Jost (1966) concluded that GH is not required for rabbit fetal growth, but is required for syn- thesis of glycogen by the fetal liver. These conclusions were derived mainly from studies on the changes following decapitation of the rabbit fetus. Heggestad and Wells (1965) studied prenatal growth of decapitated rat fetuses and concluded that the rat fetus requires GH for normal growth. However, exogenous GH would not cause added growth in normal fetuses. The fetal monkey appears to grow at a nearly normal rate after hypophysectomy (Chez et al., 1970). Decapitation of sheep fetuses appeared to retard bone growth, although most of the other parts of decapitated fetuses developed normally (Liggins et al., 1967; Liggins and Kennedy, 1968). Nanagas (1925) studied the body growth patterns of human anencephalic and normal fetuses and discovered disprOportionate growth of the anencephalic fetuses. Smith and Dortzbach (1929) found that anterior pituitary extracts from 9- to ll-cm pig fetuses stimulated 30 growuuin hypothysectomized rats. Biologically active GH also was detected in human fetal pituitaries by the rat tibia test (Rice et al., 1968). Pavlova et a1. (1968) used a hemagglutination inhibition test to quantify GH in human fetal pituitary homogenates and correlated these results with histological studies of the anterior pituitary. They found trace amounts of GH at 60 days of gestation and in- creasing quantities up to 140 days, paralleling an increase in pituitary acidOphils. Immunofluorescent staining of fetal lamb anterior pituitary tissues identified GH in the tissues from 58-day or older fetuses (Stokes and Boda, 1968); differential staining of pituitary tissue revealed two types of acidOphils, one which produced prolactin and the other GH. Immunofluorescent techniques also were used to study bovine fetal pituitaries (Meneghelli and Scapinelli, 1962). They found that lOO-day fetuses had GH-containing acidophils which increased in number as the age of the fetus increased. Another method of studying fetal GH is in vitro incubation of pituitaries. Brauman et a1. (1964) cultured human fetal‘pituitaries for five weeks and found that GH production decreased as the incubation time increased. Radioactive amino acids were incorporated into human growth hormone by 60-day fetuses using in vitro pituitary tissue cultures (Gitlin and Biasucci, 1969) . Biological and 31 inmmnological human GH activity was present in the culture nediacfiffetal anterior pituitary cell cultures for periods Cd'incubation as long as 150 days (Gailani et al., 1970). The plasma GH levels of fetal lambs were 10-fold higher than in the mother and increased from 40 ng/ml at 110 days to 120 ng/ml at 140 days of gestation (Bassett et al., 1970). HypOphysectomy of fetal lambs resulted in disappearance of plasma GH, and exogenous GH given to fetal lambs was degraded at a rate similar to that of endogenous GH (Bassett et al., 1970). I conclude that fetal plasma GH is higher than maternal plasma GH, and although fetal GH has similar biological activity to that in adults, its function in fetal develOpment has not been established. 4. Prolactin In 1935, Bates et al. reported that the bovine fetal pituitary contained two to three times more prolactin activity than older cattle. In contrast, Reece and Turner (1937) reported bovine fetal pituitaries contained less prolactin activity than older calves. Lyons (1937) mea- sured prolactin activity in urine from new born human babies and put forth the premise that "witches milk" or :mammary gland secretions by many newborn babies is caused by high prolactin levels in the fetus (reviewed by Smith, 1959).. Smith (1959) also stated that premature babies seldom have mammary gland secretions at birth. 32 nmmnofluorescent location of the prolactin- containing acidOphils in fetal sheep pituitaries could not be observed until 80 or more days of gestation; they were always less prevalent than GH acidophils (Stokes and Boda, 1968). Human fetal pituitaries produce more prolactin than GH in vitro. GH and prolactin are thought to be separate hormones in human fetal pituitary (Brauman et al., 1964). Fetal plasma prolactin levels do not seem to have been reported by previous researchers. Fetal Adrenals, Pancreas, H. Parathyroid and Thyroid The fetal adrenal gland is capable of reSponding to exogenous ACTH, atrOphies when the fetal pituitary is removed and is capable of most steroid transformations typical of adult adrenal glands (reviewed by Jost, 1966; Kitchell and 1969; Pasqualini et al., 1970). Macnaughton, Wells (1952) used adrenalectomy and cortisone implantation to establish that a reciprocal relationship exists between the adrenals and hypophysis in the fetal rat. Bovine fetal adrenals cultured in vitro can syn- thesize carbon-l4 corticosteroids from carbon-14 acetate Bassett and Thorburn (Chouraqui and Weniger, 1969, 1970). (1969) detected a six-fold increase of corticosteroid in sheep fetal plasma from 130 days to 150 days of gestation. During this same period maternal plasma corticoids remained :onstant. 33 Adrenal medulla hormones have been identified in the bovine fetus (McCord, 1915; Comline and Silver, 1966) . Comline and Silver (1966) found that the fetal effluent blood from the adrenal vein contained increased catecho- lamines in response to anoxia. Thus the evidence indi- cates that the fetal adrenal gland functions long before birth. Bovine fetal pancreas was the source of insulin I that Banting and Best isolated in 1922. That discovery was possible because the endocrine pancreas become func- tional before the exocrine pancreas begins to produce proteolytic enzymes. Both insulin and glucagon have been isolated from the fetal pancreas and hyperglycemia elicits insulin release in the fetus (reviewed by Macnaughton, 1969). Several authors have speculated that fetal insulin production in response to hyperglycemia in pregnant dia- betic mothers is beneficial in controlling diabetes. How— ever, fetal consumption of glucose may help to control maternal hyperglycemia (Macnaughton, 1969). Macnaughton (1969) reviewed the fetal parathyroid research and concluded that fetal parathyroid is active in calcium metabolism before birth in many species. Fetal hypothyroidism appears to be associated with ental deficiencies and retarded ossification (reviewed by 35“: and Picon, 1970). Hogben and Crew (1923) tried to :tablish that a thyroid deficiency in the Dexter cattle 34 was the cause of the syndrome known as "bulldog dwarf." Their bioassay results did not show a difference between normal and "bulldog" fetuses. Thyroid stimulating hormone (TSH) was found in fetal bovine pituitaries by Bates et al. (1935), who indicated that the developing fetus is auto— genously supplied with pituitary hormones. Thyroid hor— i mone injected into fetal rats decreased fetal pituitary TSH, indicating a functional pituitary-thyroid axis in the fetus (reviewed by Jost, 1966; Jost and Picon, 1970) . The first record of bovine fetal serum thyroxine levels was reported by Hernandez (1971) on the same animals used in this thesis study. According to Hernandez, maternal thyroxine levels increase about 19% during gestation while fetal thyroxine level increases from 2.18, 11.66 and 17.16 g/100 ml for 90, 180, and 260 days of age, reSpectively. The thyroid increased in size in direct prOportion to fetal body weight. Females had larger thyroids and more serum thyroxine than males at 180 days of gestation (Hernandez, 1971) . Thus, bovine fetal thyroid appears to be meta- bolically active. I. Neonatal Serum LH, GH and Prolactin Macmillan and Hafs (1968) reported LH plasma levels f 0.48 Lug/liter in male calves less than 7 days old, and Female 1 average LH pituitary concentration of 0.76 ug/ml. lves less than 7 days old had average pituitary LH con- ntrations of 2.44 ug/ml (Desjardins and Hafs, 1968). 35 Foster (1971) used RIA to quantify LH in neonatal lambs; males had serum LH levels less than 0.3 ng/ml and females less than 0.5 ng/ml during the first 7 postnatal days. Serum LH levels in female lambs increased to 3 ng/ml by day 18 postnatally, while the male lamb showed no increase to day 18. Neonatal serum GH averaged 33.5 ng/ml in umbilical Purchas blood from humans at birth (Grumbach et al., 1968) . et a1. (1970) reported that serum GH averaged 32 ng/ml in The bulls less than 7 days old using RIA techniques. pituitaries from the same bulls as above had pituitary GH concentrations of 6 ug/mg by bioassay and 2 ug/mg by RIA (Purchas et al., 1970). Serum prolactin values for bovine neonates do not appear to be available, however, Reece and Turner (1937) reported prolactin values of 111.4 bird units per calf pituitary and Lyons (1937) found prolactin activity in urine from babies less than 7 days old. MATERIAL AND METHODS A. Experimental Design This research project was designed to study changes in hormone levels and physical changes in the reproductive organs of the bovine fetus during deve10pment. These develOpmental changes were then used to study interactions of maternal and fetal endocrine systems during gestation. A third phase of this project was a study of changes in the bovine endocrine system which occur during the week following birth. For the purposes of these studies the gestation period was divided into trimesters and a minimum of five male and five female fetuses were collected near the end of each trimester. This sampling procedure permitted statistical analysis of the fetal age and sex differences. In addition, blood samples were taken from ten calves at birth and daily for one week, to determine hor- mones during the early neonatal period. B. Experimental Animals 1. Fetal Pregnant primiparous Holsteins, 18 to 26 months ld, were purchased and transported to Michigan State Uni- :rsity. Date of conception was determined by dates of 36 37 natural breeding or artificial insemination and verified by rectal palpation. Rectal palpation of the pregnant uteri were performed when the heifers were 25 to 45 days pregnant. Independent estimations of day of pregnancy were made on each heifer by the investigator and by another veterinarian, and then the two“ estimates were averaged. If this value was not within three days from the reported breeding date for the heifer she was not used in this study. Animals without breeding dates were used if the individual estimates of the length of pregnancy were within five days of each other. Rectal palpation of the embryonic vesicle has proved to be an accurate method of determining length of pregnancy from 28 to 45 days after conception. At Michigan State University, the animals were maintained on pasture until two or three days before sched- uled surgery when they were moved into the Dairy Barn and housed in stanchions. At this time, each animal was pal- pated to determine if the pregnancy had developed normally and to determine which uterine horn contained the .fetus. Eighteen to twenty hours before hysterotomy, each animal was transported to the Veterinary Clinic and placed in a >ox stall where feed was restricted until after surgery. After surgery, the heifers were housed overnight the Veterinary Clinic. They were returned to the dairy rn the following day. Postoperative observation was con- nued for 7 to 10 days. 38 2. Neonatal The calves used for the neonatal study were from primiparous Holsteins that had been artificially inseminated with semen from purebred Holstein sires. The pregnant heifers were observed every 2 to 3 hours as parturi- tion approached. Blood samples were drawn from the jugular vein of the calves within 2 hours of birth and daily thereafter for 6 days. C. Surgery Forty surgeries were performed. During the surgery the pregnant heifer was restrained in stocks. A 40-m1 blood sample was drawn from the jugular vein immediately after the animal was in stocks just prior to surgery. Either the left or right paralumbar area nearest the fetus was prepared for surgery by clipping the hair and scrubbing the area three times with a solution of Betadine. The selected paralumbar fossa was anesthetized with Procaine (Bio-centic Laboratories, St. Joseph, Missouri). The local anesthetic block for the incision within the paralumbar fossa extended 10 cm parallel to the vertebral lumbar transverse processes on the dorsal margin and 12 cm rertically on the anterior margin of the paralumbar fossa. 16- to 30—cm incision was made in the abdominal wall in .e anesthetized region. Both the middle uterine artery and vein were cated in the abdominal cavity by palpation and theterized with a polyethylene 30-inch catheter 39 (Standard Minicath, Desert Pharmaceutical Co., Inc., Sandy, Utah) attached to a 19 gauge x 1.25 inch needle. Forty m1 arterial and venous blood samples were drawn using 50 m1 syringes attached to the catheters. In some cases difficulty was experienced with catheterization of the middle uterine vein and venous samples were obtained from large veins on the uterine surface. The average elapsed time between the collection of the sample from the jugular vein and the uterine vein was 68 minutes and for the uterine artery 71 minutes. After collection of the uterine blood the gravid uterine horn was manipulated into the abdominal wall inci- sion and held in place with two pair of vusullem forceps. A 10- to 25-cm incision was made in the greater curvature of the gravid horn with a scissors. The 260—day fetal blood samples were obtained by catheterization of the um- bilical vessels with the fetus remaining in utero. The connective tissue covering of the umbilical cord was dis- sected bluntly with scissors, exposing the four blood ves- sels. Umbilical arteries and veins could be identified readily by the definite pulse in the former. The smaller 180-day fetuses were removed from the uterus, leaving the placental attachment intact. While the fetus was held aside the cow, the umbilical vessels were catheterized in the same manner described for the 260—day fetuses. 40 Obtaining adequate blood samples from the relatively small 90-day fetuses proved difficult. The fetus can easily be removed from the uterus leaving the placental attachment functional. Catheterization of the umbilical cord vessels, however, yielded only 1 or 2 ml of blood. Cardiac puncture or decapitation also provided insufficient volumes of blood. Opening the fetal thoracic cavity and catheterizing the thoracic aorta permitted the collection of 10 to 20 ml of blood from the 90-day fetuses. The average time between the collection of the maternal jugular veinous blood until the umbilical vein and artery were sampled was 73 and 77 minutes reSpectively. The 90-day fetuses were more difficult and required an average of 90 minutes between collection of the maternal jugular and fetal blood samples. In all cases, after the fetal blood samples were 'taken the fetus was removed from the uterus (260 day) and 'the umbilical cord severed. The uterine incision was in- 'verted and closed with a double row of sutures (Chromic «satgut #1) using a Cushing pattern and the uterus was then replaced in normal position. The peritoneum, abdominal nnascle layers and subcutaneous tissues were closed separ- atuely in layers using a continuous suture pattern (Chromic curtgut #2). A synthetic suture (Vetafil Bengen, Haver Imxckhart,Kansas City, Missouri) in a continuous lock stitch pattern was used to close the skin incision. 41 The postOperative care was minimal. More than half of the animals sloughed the placental membranes by seven days after surgery. Otherwise antibiotic infusion of the uterus was necessary to prevent septicemia. Four- teen days after surgery, all placental membranes had been sloughed. Skin sutures were removed 10-14 days after sur- gery and the animals were turned out to pasture. D. Blood Serum Samples All blood samples, except the 90-day fetal samples, were put into centrifuge tubes containing oxalate (31.7 mg) and kept on ice. The blood was centrifuged in a cold room (4°C) within 30 minutes of collection. After centri- fugation the plasma was transferred to another centrifuge tube containing CaCl2 (27.8 mg) and stored 24 to 48 hours at 4°C. The samples were then centrifuged to remove the fibrin precipitate, and the serum was transferred to a 20-dram plastic vial and stored at -20°C. Because of variable volumes, the 90-feta1 blood samples were collected with oxalated catheters and syringes and transferred to non-oxalated centrifuge tubes. The amount of CaClzwas adjusted to the sample volume and processed identically as described above. E. Fetal Tissue Samples The fetus was weighed and crown-rump length mea- sured within five minutes of removal from the uterus. The 42 anterior pituitaries were removed after decapitation. Each pituitary was bisected into right and left hemipituitaries. One half was put into Bouins fixative for histochemical studies. The whole pituitary was not weighed because it was necessary to bisect it in situ for histochemical studies by Dr. E. Baker, University of Michigan, and these studies will be reported in a subsequent publication. The remaining hemipituitaries from 90-day fetuses were stored at -20°C for hormone assay. Two or three central slices were removed from the 180- and 260-day hemipituitaries before storage at -20°C. The total weight of these slices, which were used for the in vitro incuba- tions, averaged 2.7 mg per pituitary. Gonads, adrenals, uteri and seminal vesicles were removed and weighed. An average of 20 minutes elapsed be- tween severing the umbilical cord and complete processing of all fetal samples. The gonads from each fetus were sectioned for electron microsc0pic study by Dr. R. Saacke, Virginia Polytechnical Institute, and also incubated in ‘vitro for hormone metabolic studies, both to be reported ‘in.subsequent publications. F. Pituitary Tissue Culture Tissue culture requires chemically clean Special- :ized equipment and strict asceptic technique. The method used is described in detail below. 1. Equipment Stainless steel incubation platforms and all glass- ware were initially washed in Alconox detergent (Alconox, Inc., New York, N. Y.) followed by ten rinses in tap water. Thereafter materials were cleaned with a tissue culture detergent (Micro-Solv, Microbiological Associates, Inc., Bethesda, Maryland) followed by three distilled water rinses. Water used for rinsing of glassware and other materials was distilled over glass and deionized. All glassware and other equipment were sterilized by autoclaving at 15 psi for 30 minutes. Sterile equipment was transferred to a sterile glove box (Germfree Labora- tories, Inc., Miami, Florida) where they were stored until used. This chamber was also used for pituitary dissection and transfer of slices to the culture vessels. An ultra- violet light on the ceiling of the chamber was turned on 30 minutes prior to use to aid in ascepsis. Surgical instruments for sterile dissection were stored in 70% ethanol. Similarly, 70% ethanol was used to disinfect the dissection areas and the transfer chamber floor. 2. Culture Medium The medium used for the culture was TC 199 (Difco, Detroit, Michigan). This was purchased as a ten-fold con- centrate and prepared in 25-ml quantities for culture; 44 2.5 ml of concentrated TC Medium 199, 1.0 ml Penicillin-G— Phosphate (1.7 mg/ml), 2.0 ml 2.8% NaHCO3, and 19.5 ml deionized water. 3. Culture Method Disposable, sterile organ culture dishes with ab- sorbent rings, measuring 60 x 15 mm (Falcon Plastics, Inc., Los Angeles, California) were used for anterior pituitary tissue culture. Stainless steel screen platforms (15 x 15 mm) were placed into these dishes. Lens paper wicks were placed on the screen platforms. One ml of TC 199 culture medium placed in the center well just covered the top of the platform. By this arrangement, pituitary ex- plants were exposed to the gaseous environment yet received adequate nutrition from the culture medium. The gas environment for all cultures consisted of and 5% CO . Gassing was continuous at a flow rate 2 2 of 300 ml/minute. Humidification of the gas was accom- 95% O jplished by bubbling it through sintered glass filters sub- Jnerged in distilled water before entry into the culture chamber. Explants were incubated in a culture oven for '72 hours at 37°C. At this time the explants and medium vnare separately recovered and stored at -20°C until assayed. G. Pituitary Homogenization In preparation for hormone assay, pituitaries were minced on a wax block, placed in a test tube containing 45 2 m1 of phosphate buffered saline (PBS) (Appendix I.B.l)t and homogenized by sonication (Sonifier Cell Disruptor, Model W185D, Heat Systems-Ultrasonics, Inc., Plainview, New York). The tube containing the pituitary slices was immersed in ice water during sonication to prevent over- heating. Sixty to eighty watts were applied intermittently for a total of three minutes. The anterior pituitary slices obtained from the incubation procedure described above were processed in the same manner. A11 pituitary homogenates were stored at —20°C until assayed. H. Radioimmunoassay (RIA) l. Luteinizing Hormone (LH) The bovine LH assay was similar to the assay re- ported by Niswender gE_al. (1969). LH antibody was developed by repeated injections of NIH-LH—BS into guinea pigs (Appen- dix I.C.l). Purified bovine LH used for iodination (LER- 1072-2) was supplied by Dr. Leo Reichert (Emroy University, .Atlanta, Georgia). This preparation had an LH potency of 1.66 NIH-LH-SI units/mg and showed no FSH activity when tested at 3600 pg in the Steelman-Pohley assay. It had a 'thyroid stimulating hormone (TSH) contamination estimated at 0.021 USP units/mg. a. Radioiodination.--Purified bovine LH (LER-1072-2) liad been previously dispensed into l-ml vials (2.5 ul of a l ug/ul solution in glass distilled water) and stored at -20°C. These vials were thawed immediately before iodina- trion and the iodination procedure was performed at room 46 temperature. Twenty—five ul of 0.5 M sodium phosphate buffer at pH 7.5 (Appendix I.A.1) was added to the hormone and mixed. One mCi of 1251 (50 mCi/ml, Iso-Serve Division of Cambridge Nuclear Corporation, Cambridge, Massachusetts) was added, and the contents gently mixed. Forty ug cloramine-T (Eastman Organic Chemicals, Rochester, New York) (Appendix I.A.3) was added to the vial, the vial was stoppered, and the contents were gently mixed by finger tapping. The reaction was stopped at exactly two minutes by adding 125 Hg sodium metabisulfite (Appen- dix I.A.4). After thorough mixing, 25 ul of 2.5% bovine serum albumin (BSA, Nutritional Biochemicals, Inc., Cleve- land, Ohio) in 0.01 M phosphate buffered saline (PBS) pH 7.0 (PBS~2.5% BSA) was added to diminish the loss of hormone adhering to the glass vial. A l x 12 cm glass column.packed with Bio Gel P-60 (Bio Rad Labs, Richmond, California) was equilibrated previously Iby passing 0.05 M sodium phosphate buffer pH 7.5 (Appen- --- Male 10.4:3.0a 12.113.5 Female 37.5124.0 36.5110.0 Average 24.0112.5 23.216.2 a Mean 1 standard error, n = 5 or 6. I conclude that bovine fetal pituitaries synthesize prolactin in vitro and that female pituitaries probably synthesize more prolactin than pituitaries from male fetuses. 3. Hormones in Fetal Blood Serum The data on peripheral hormones were analyzed statistically in two steps. The first step involved a two- factor (sex and age) analysis of variance for the 90, 180 and 260-day fetuses. Second the 180-day and 260-day fetal data were analyzed by using three-factor (fetal sex, age and source of blood) analysis of variance. The two step procedure was necessary because data were not available on arterial and venous hormone levels for 90-day fetuses. ‘a."Luteinizing Hormone (LH).--Fetal serum LH (Table 16) for 90-day fetuses averaged 3.0 ng/ml and de- creased to 1.28 ng/ml at 180 days and to 0.85 ng/ml at 260 days (P < 0.01). Females averaged higher serum LH than males at 90 and at 180 days (P < 0.01). The 90-day female 82 TABLE 16.--Average fetal serum LH.a Days Gestation Sex 90 180 260 *7 ------------ (ng/ml)---------------- Male 1.4610.15b 1.14:0.23 0.8810.12 Female 3.910.60 1.4710.19 0.8310.18 Average 3.010.5 1.2810.15 0.8510.10 a180 and 260 day values represent the average of umbilical artery and vein. bMean 1 standard error, n = 5 to 8. fetuses (3.9 ng/ml) averaging about 170% higher than 90-day males. The 260-day males, however, had higher LH than the females, causing a sex-age interaction (P < 0.01). Foster (1971) reported serum LH levels in fetal sheep of comparable ages. Serum LH for 55-, 100- and 139- day sheep fetuses were 0.25, 1.0 and 0.30 ng/ml for single male fetuses and 0.70, 1.1 and 0.20 ng/ml for single female fetuses, respectively. Thus, the sex differences in serum LH levels for each age group are qualitatively similar for sheep and bovine fetuses. However, the sheep fetal serum LH appeared to peak near the end of the second trimester of gestation while the highest serum LH for the bovine fetuses was found in the first trimester. I found no significant difference in fetal arterial and venous serum LH (Table 17). These results suggest that placental transfer, production or uptake of LH is minimal, and the circulating LH serum levels remain rela- tively constant. TABLE l7.--Average fetal serum LH in the umbilical artery and vein. Sex Days Gestation Source 180 260 ---------- (ng/m1)----------- Male Artery l.1210.35a 0.8710.18 Vein 1.1410.34 0.8810.17 Female Artery l.4710.29 0.8410.26 Vein l.4710.26 0.8210.27 Average Artery 1.2810.23 0.8510.14 Vein 1.2910.22 0.8510.15 aMean 1 standard error, n = 5 to 7. A comparison of fetal pituitary LH to serum LH (Figure 6) revealed that pituitary LH increased with fetal age while the serum LH declined. Therefore, fetal pitui- tary LH was correlated (r = 0.41, P < 0.05) with the C-R lengths while the serum LH was inversely related to C-R length (r = -0.58, P < 0.05). b. Growth Hormone (GH).—-Growth hormone in fetal serum increased 140% (P < 0.01) from 90 days to 260 days. Serum GH was 42 ng/ml for 90-day fetuses and increased to 65 and 103 ng/ml for 180- and 260-day fetuses, respectively (Table 18). Although the 90-day female fetuses averaged 70% more serum GH than comparable males, the sex difference was not significant over all three stages of gestation. Differences in fetal arterial and venous GH levels were insignificant, and only fetal age differences were apparent (P < 0.01, Table 19). Bassett et a1. (1970) 84 7 4 ’00 - Pituitary - Serum — 3 600 l- ------ ...... ....... IIIIII eeeeee eeeeee o ....... . . ...... .... ...... . .. O . . ...... .. ...... .... .. . CCCCCCC .. . .. ...... 0°C.. a e eeeeeeee ... 000000 ......... eeeeeee ....... ...... ...... OOOOOO OOOOOO eeeeeeee . 000000 ... eeeeeeee . . .... ....... ..... 000000 ....... ... ‘ .."‘ .... | eeeeeee O. .. ....., . .. OOOOOOOO ... . . .' .| .. ....... ..... .sun.. ............ at... ”H” 0.... -..... eooo .. i ...... 0.. ..... ....... .. 0.... Ian... . ..... 0.. nnnnnn Pituitary Lit (ng ling ) ............. .:.CO vneetls : ....... ....... """"""" .......... ....... ...... ....... ....... ,,,,,,, ........ 90 too 260 Days Gestation 6 Levels of LH in fetal pituitary and Figure w blood serum. .-.'-;~;t;;;-;; 7 '00 2 ° ' Ell “WW" 323533333332; 5 3 m 5"“ 33:32:33: . 75 g o .a.e.o.°. -.t.o.o:n:0:0 E | 5 - 3.3.3} ::;I;::.:;:;: s g ........................ 5 .r: 3 3:32:33; -.'-f:§'.-’, 3:333:33} ‘ 50 z, e- .................... : E '0 7 35:35:33 . .-;-:-r-:-:-: ° 5 Z 53233135353: :2: . 1;; =.';.'-{:, 35:53:}th g h " ' :5} 5:33:33: ............. 2 5 3 g 5 333533;; e 0...: 35:55:. 3.10.: :‘:’:::::’:' a E 5.5.53 é.“ 3:5. E :°:°:':'- """"""""" 6 O 90 I80 2 60 Days Gestation no -i n fetal Levels of growth hormo Figure 7 pituitary and blood serum. Serum Ll-t (no/ml) (no/ml) 85 TABLE l8.--Average fetal serum growth hormone.a Days Gestation Sex 90 180 260 ------------ (ng/ml)---------------- Male 29:4b 64:7 109:8 Female 50:12 65:7 97:15 Average 42:8 65:5 103:8 a180 and 260 day values represent the average of the umbilical artery and vein. bMean : standard error, n = 5 to 8. TABLE 19.--Average fetal serum growth hormone in the umbili- cal artery and vein. Days Gestation Sex Source 180 260 -------- (ng/ml)----------- Male Artery 65:10a 109:11 Vein 64:11 109:13 Female Artery 66:9 93:20 Vein 65:12 100:26 Average Artery 65:7 102:10 Vein 64:8 105:13 aMean : standard error, n = 5 to 7. reported serum GH levels of 40 to 50 ng/ml and 110 to 120 ng/ml for 110 and 135-day fetal sheep, respectively. Bassett et_al, (1970) also found that serum GH levels decreased rapidly after fetal hypophysectomy; exo- genous and endogenous GH in the serum decreased at similar rates. Furthermore infusion of Is0prenaline (iSOprOpyl 86 nonadrenaline hydrochloride, Sigma) depressed fetal sheep serum GH levels, but GH quickly returned to normal after cessation of isoprenaline infusion. Thus fetal serum GH levels apparently are controlled by the fetus. Both fetal pituitary and serum concentrations of GH increase with fetal age (r = 0.41, P < 0.01, Figure 7). The correlations of C-R length with pituitary GH levels (r = 0.60) and of C-R length with serum GH levels (r = 0.57) were significant. (P < 0.01). Therefore the observed in- creases in serum GH levels do not arise from decreased fetal pituitary GH. The physiological significance of GH in the fetus has not been determined, although the high GH levels occur during the period of most rapid relative growth of the fetus. c. Prolactin.--Fetal serum prolactin was low in 90-day fetuses (3.6 ng/ml) but increased over lO-fold at 180 days of gestation (43 ng/ml) and rose to 61 mg/ml at 260 days (Table 20). These prolactin changes (P < 0.01) TABLE 20.--Average fetal serum prolactin levels.a Days Gestation Sex 90 I80 260 ------------- (ng/ml)---------------- Male 3.5:0.4b. 30:5 60:17 Female 3.7:0.6 58:12 63:11 Average 3.6:0.4 43:7 61:10 'fl‘“ a180 and 260 day values represent the average of umbilical artery and vein. bMean : standard error, n = 5 to 8. 87 resemble those for GH, and the fetal serum prolactin and GH were correlated (r = 0.45, P < 0.01). Sex differences in serum prolactin levels were not detected, but the inter- action (P N 0.09) of sex and age was observed. This rela- tionship was produced primarily by a 50% lower prolactin in serum from 180-day male fetuses as compared to the females. Serum prolactin in l80- and 260-day fetuses was higher in umbilical arterial blood (Table 21) than in venous blood (P W 0.20), suggesting placental uptake or transfer but not production of fetal prolactin. TABLE 21.--Average fetal serum prolactin in the umbilical artery and vein. Days Gestation Sex Source 180 260 -------- (ng/m1)--------- Male Artery 34:8a 59:22 Vein 26:6 61:27 Female Artery 58:20 68:19 Vein 57:16 57:14 Average Artery 45:10 63:14 Vein 40:9 59:15 aMean : standard error, n = 5 to 7. When fetal pituitary and serum prolactin concentra- tions are compared (r 0.62, Figure 8), a parallel in- crease was evident during gestation. The pituitary pro- lactin (r = 0.78) and the serum prolactin (r = 0.57) levels appear to increase with fetal age, At the present 88 3 ....... q so ; Pituitary E MSerurn f; \ E O \ C c = = .3 2 ° 8 a I 20 a t’ E 8 3 E. :3 O. o ....... 0 Days Gestation Figures Levels of prolactin in fetal pituitary and serum. IOO F 80 - E a 60 - C .5 ‘6 2 4o - 2 a. 20 l- o l l 1 j l l I O I 2 3 4 5 6 Age (days) Figure. Neonatal calf jugular serum prolactin. 89 time the significance of the prolactin in the fetus also remains obscure, but the A-V difference across the placenta suggests fetal prolactin may aid placental function. D. Neonatal Serum Hormones The serum hormone data for neonates was treated statistically by analysis of variance with sex and age as variables. Age-changes were compared by orthogonal con- trast. l. Luteinizing Hormone (LH) Since sex differences for serum LH levels were not observed, the values for males and females were combined in Table 22. The average serum LH level ranges from 0.36 to 0.49 ng/ml for calves from birth to 6 days of age; there were no significant changes in serum LH during the first 6 days. TABLE 22.--Neonatal jugular serum growth hormone, LH and prolactin. Age Growth LH Prolactin Hormone (days) --------------------- (ng/hflJ ----------------- 0 36:9 0.36:0.11 101124 1 28:4 0.49:0.06 95:25 2 28:8 0.46:0.05 42:8 3 24:4 0.46:0.06 34:10 4 18:3 0.48:0.08 43:10 5 24:6 0.49:0.06 30:5 6 32:5 0.40i0.06 30:6 aMean : standard error, n = 9 or 10. 90 Macmillan and Hafs (1968) reported that plasma LH levels averaged 0.48 ng/ml in bulls between 1 and 7 days old. The serum LH in lambs l to 6 days old averaged from 0.20 to 0.60 ng/ml (Foster, 1971). Values of neonatal serum LH in other species were not available. 2. Growth Hormone (GH) Serum GH levels for male and female calves were similar, therefore the data were combined in Table 22. Daily averages ranged from 18 to 36 ng/ml of serum but no significant variation with age was detected. The growth hormone levels shown in Table 22 agree with previously reported data by Purchas gt_§l, (1970). Grumbach gt_§1. (1968) reported that human fetal umbilical cord blood contained 33.5 ng/ml of GH. 3. Prolactin Daily average serum prolactin levels are reported in Table 22 for calves from birth to 6 days of age. The values for males and females were again combined since no sex difference was observed. A significant and rapid de- crease in serum prolactin level occurred between day l and day 2 (P < 0.01). This decrease is shown graphically in Figure 9 (p. 88). Both the day of birth and day of birth plus day l were significantly higher (P < 0.01) than the averages for the remaining days. Other reports on neonatal prolactin levels were unavailable. GENERAL DISCUSSION A. Maternal-Fetal Hormone Interactions 1. Growth Hormone (GH) When the average GH in serum from the three blood sources taken from cows was compared with the average GH in fetal serum (Figure 10), fetal GH averaged 10 to 20 times higher than maternal GH (P < 0.01). Human fetal serum GH has been found to be six times higher than maternal levels at birth (Hutchinson et;al., 1962). While Bassett gt_al, (1970) only compared the fetal sheep-maternal serum GH during the third trimester, they also reported fetal levels ten times higher than maternal levels. In at least the cow, sheep and human, fetal serum GH levels are much higher than for any period after birth. The physiological significance of high fetal serum GH re- mains obscure since fetal growth appears to proceed almost normally in hypophysectomized lamb fetuses (Liggins and Kennedy, 1968). Placental transfer of GH appears minimal, because a large concentration gradient was maintained across the placenta throughout pregnancy. Gitlin gt_al. (1965) in- jected radioactive GH into pregnant women and could not detect placental transfer of labeled GH to the fetus. If 91 92 I00 E: Cow a Fetus 80 60 40 20 Growth hormone (no/Int) so too 260 Days Gestation “3"“ 1" Levels of growth hormone in cow and fetal blood sera during gestation. Ea Cow E Fetus Ll-l‘ (no/ml) O O 90 IGO 260 Days Gestation Figure 11 Levels of LH in cow and fetal blood sera during gestation. .. is 93 the bovine placenta is equally impermeable to GH as the data in Figure 10 suggest, then the high circulating fetal levels must arise from the fetal pituitary. As further» evidence to support this hypothesis, Bassett §t_al. (1970) observed that sheep fetal GH levels rapidly decreased after fetal hypOphysectomy. That I found no AV differences in GH across the placenta also supports the suggestion that GH is neither taken up, produced nor transferred by the placenta. 2. Luteinizing Hormone (LH) A comparison of the maternal serum LH (average of jugular vein, uterine artery and uterine vein) to the average in fetal serum LH is shown in Figure 11. The 90- day fetal serum LH is three times higher than maternal serum LH, but this difference in serum LH levels decreased by 180 days and nearly disappeared at 260 days of gestation. As was found for GH, the placenta maintains serum LH con- centration differences between the fetal and maternal sys- tems. Foster (1971) injected exogenous LH to raise the level of either the fetal or the maternal serum LH of sheep and showed that the sheep placenta does not transfer LH in either direction. If the same is true in the cow, as the data in Figure ll suggest, then bovine fetal serum LH must arise from the fetal pituitary. Absence of AV differences in LH across the placenta also supports this suggestion. 94 3. Prolactin Serum prolactin in the cow (average of jugular vein, uterine artery and uterine vein) is compared With average fetal serum prolactin levels in Figure 12. In contrast to the results for LH and GH, fetal serum prolactin levels were lower than the maternal levels during all three tri- mesters of gestation, and maternal serum prolactin averaged from 3 to 30 times higher than fetal serum levels. But as for GH and LH, placental transfer of prolactin appeared to be minimal. Three different patterns are observed for these three hormones. Fetal GH and LH levels were higher than maternal levels Iduring'gestation, but GH increased while the LH decreased. Serumprolactin which was higher in maternal serum than in fetal serum increased during gestation. The above variations in the ratios of the fetal to the maternal serum levels of GH, LH and prolactin all support the hypothesis that the bovine placenta does not transfer, take up, or produce these hormones. 4. Effect of Fetal Sex Another interesting fetal-maternal relationship in this study was the effect of the sex of the fetus on maternal GH and the prolactin (Table 6). Cows carrying male fetuses averaged nearly twice as high GH levels in the jugular vein as cows with female fetuses at both 180 95 260 . . 0 . . . .. . . 0 . . 0 . . . . 0 . 0 0 0 0 0 . 0 . 0 . . . . . 0 . . . . 0 . . . 0 0 0 0 . ’ ? ’ , ’ ’ ’ ’ ’ ’ ’ , ’ ’ ’ ’ ’ ’ ’ } ’ ? E3 Cow E Fetus IOO Days Gestation Levels of prolactin in cow and fetal ”’b”bp”” 90 ’-’?bP”””” 250 - 200 r- p 0 Ru IOO l- so #- o 23.x 9: 5322.. Figure 12 during gestation. blood sera .95 33 2.25.2. 5329 O. 8 O. . RV z z ' ' It d Cl 0.5 Gfié LHE FED ‘ “ ‘14 ‘ ‘ ‘ ‘l‘A‘ ‘ 4‘ ‘ ‘ 1‘ 0.. 0 0 00 0.0 0 0 0 0.0.00000000000... . 0 0 0 0 0 0 0 0 0 0 0.. 0 0 0 . 0 0 0 . . 0 . . 0 . 0 0 0 . 0 .0. 000 0 0000 0 0 0 0......0000000. 00 .0 00 .0 0 0 000.00 0000...... 000.0. 00 .- . Q ~ . O ............ . ............. .oo...... ., . .............. u ................ ................... ‘1‘“““““‘ O 2500 p p O O 0 0‘ I500 - IOOO - 500 - .95 051.. .8 £822“. , Days Gestation Fetal anterior pituitary growth hormone, LH and prolactin. Figure 13 96 and 260 days of gestation (P W 0.11). The difference in maternal serum prolactin levels due to the sex of the fetus (Table 6) was greater for prolactin (P < 0.05) than for GH. Furthermore, it was evident early in gestation (90 days), because cows with male fetuses averaged more than three times higher serum prolactin levels than cows with female fetuses at 90 days of gestation. At 260 days of gestation cows carrying male fetuses were more than six times higher. Sex of the develOping fetus previously was reported to alter maternal endocrine and metabolic functions. Pri- mates carrying male fetuses had higher blood testosterone levels (Resko, 1970). Women carrying male babies averaged higher HCG levels (Brody and Carlstrom, 1965) and also ex- creted more pregnanediol (Rawlings and Krieger, 1964). A possible mechanism for fetal control of maternal hormone levels may be androgen transfer from the fetal to the maternal system. The fetal androgen then may act on the maternal hypothalamus or pituitary. An important aspect of maternal-fetal relationships has been reported by MacMillan (1970). He found that women who delivered abnormally small babies had significantly lower serum human chorionic somatomammotropin levels. An understanding of the basis for these observations is de- sirable to prevent abnormal develOpments. 97 Shane gt_§l. (1969) caused pseudohermaphroditism in female dog fetuses when the pregnant dog was fed methyl testosterone. Coxofemoral hypoplasia and premature epi- physeal ossification was reported by Gustafsson and Beling (1969) in pups from mothers that received estradiol during gestation. Both of these reports demonstrated maternal transfer of steroid hormones to the fetus. Medical implica— tions of developmental defects due to maternal medication are obvious. Clinical problems such as coxofemoral hypo- plasia are not uncommon. B. Fetal Pituitary and Serum Hormone Comparisons l. Pituitary Content Comparisons of fetal pituitary hormone levels at all three trimesters of gestation are shown in Figure 13. The pituitary concentration of GH, LH and prolactin all increased with fetal age. GH was approximately ten times higher than LH or prolactin. GH apparently is one of the first hormones that can be identified in quantity in the fetal pituitary. Gitlin and Biasucci (1969) reported 60-day human fetal pituitaries can synthesize GH. Acidophils containing GH were found in fe- tal pituitaries from the 43-day sheep (Stokes and Boda, 1968) and the 18-cm C-R bovine (Meneghelli and Scapinelli, 1962). These GH cells increased in number as the fetal age ad- vanced. Because GH cells in the pituitary were most numer- ous early in fetal life, it was not surprising that GH 98 levels in the 90-day pituitary were higher than for LH and prolactin (Figure 13). 0 Fetal pituitary LH increased from 90 to 180 days (P < 0.01), but little change was evident between 180 and 260 days. The LH level was approximately five times higher than the prolactin level in the 90-day fetal pituitary. However, prolactin levels were two to four times higher than LH levels in l80- and 260-day fetal pituitaries. Cells containing LH have been identified in the 49-day fetal sheep pituitary (Dubois and Mauleon, 1969). Biological LH activity has also been reported by Mauleon and Reviers (1969) in 60-day fetal sheep pituitaries. Foster (1971), using RIA, quantified LH in 55-day fetal sheep pituitaries and demonstrated the biological activity of fetal LH. The above reports on fetal sheep pituitary LH correspond to fetal ages approximately equal to the 90-day bovine fetus. The pituitary LH concentrations was more than 300 ng/mg at 90 days in the bovine fetus. How soon after conception LH may be detected by RIA remains to be tested. Although Reece and Turner (1937) found prolactin in bovine fetal pituitaries, they only studied fetuses in late gestation. Stokes and Boda(l968) found prolactin con- taining cells in 80-day sheep fetuses. Measureable (RIA) quantities of prolactin were also present in the 90-day bovine fetal pituitaries (Figure 13). Of the three hor- mones studied (GH, LH and prolactin), prolactin may be the last to appear in the bovine fetal pituitary. 99 2. In Vitro Synthesis Comparative synthesis of GH, LH and prolactin by anterior pituitary slices in vitro is shown in Figure 14. A significant quantity of GH, LH and prolactin was synthesized in vitro. The correlation between pituitary GH content and in vitro synthesis (r = -0.09) was not sig- nificant. The only correlation that was significant (P < 0.05) between GH content and in vitro synthesis was the lBO-day male fetal pituitaries (r = -0.78, Table 23). Biological and immunological (RIA) activity of GH produced by incubation of human fetal pituitary tissues was re- ported by Gailani, gt_al. (1970). TABLE 23.--Some correlations between fetal pituitary hor- mone concentrations and net in vitro hormone synthesis. Days of Sex ‘Pituitary vs.'net synthesis in vitro gestation GH LH Prolactin 130 Male -o.78a 0.17 0.55 Female 0.41 0.15 0.48 260 Male 0.01 0.00 -0.28 Female -0.42 _-0.70 0.89a 5‘1? < 0.05. In vitro synthesis of LH also is compared with the pituitary LH content in Figure 14. Correlations, within sex and age, of LH synthesis compared with pituitary LH con- tent are shown in Table 23 and were not statistically sig- nificant. Previous studies with tissue cultures of human 100 A 25F .Gontent DNetSynthesls F '1 g .._ i °\ \\\ § ~° l0- s o; \ h c" § § : \ \ s \ s ‘6 5* s A at s \ \ s s o» S s l600 2606 l60¢ 2604 l60¢ 2604 Growth hormone L H Prolactin “8“,. 1,, Fetal pituitary content and in vitro synthesis of growth hormone, LH and prolactin. . Growth hormone IOO _ E Prolactin a LH 9:5 2:; j 3 so r. :35 0 2 1: 2 5 .._ g 0 g ‘ 60 i- : Z d 2 E " :E g ° '5 § 2; g .' = :: 8 E :2 5 3 ‘° ‘ =5. E . b = 5.: 2‘7 5 .— — .0 —- ° = :3 2° ' E f: 0‘ E “7'? ":' ,0: . :-:~:» :4; ’-:.:- .0 so l60 230 Days Gestation “gm 15 Fetal blood serum growth hormone,Ll-l and prolactin during gestation. Lit (no/ml) L__J . lOOO soo 3 600 g C 5 400 5 zoo o lOl fetal pituitary by Gey §t_al, (1938) indicated that LH may be synthesized in vitro. Also, Gitlin and Biasucci (1969) and Gailani gt_al, (1970) showed that LH was syn- thesized in human fetal tissue cultures. In vitro prolactin synthesis by fetal pituitary tissue (Figure 14) is much greater than the net synthesis of either LH or GH. Female pituitaries synthesized more prolactin during incubation than pituitaries from males (P m 0.06, Table 15). The correlations within sex and age groups are shown in Table 23. The 260-day female fetal pituitary content was correlated to in vitro prolactin production (r = 0.89, P < 0.05). Brauman gt_3l. (1964) reported increasing synthesis of prolactin by fetal pitui- tary cells up to 33 days of incubation. Fetal sex and age appear to influence the synthetic capacity of pituitary tissue. The relatively low (Table 14, Appendix VIII) net synthesis of GH during incubation may have resulted from feed-back inhibition, because the medium levels of GH became very high. Another explanation for low net synthesis of GH and LH compared with prolactin may be the requirement for hypothalamic stimulation for the synthesis of GH and LH. Generally, hypothalamic action on pituitary GH and LH is stimulatory, while it inhibits prolactin synthesis and release. In vitro prolactin syn- thesis appears to be enhanced because hypothamic inhibition has been removed. Because in vitro synthesis of hormones 102 in fetal pituitaries differed in males and females, the fetal gonads apparently influenced pituitary function be- fore l80 days. At 180 days the female pituitaries syn- thesized 3-fold more prolactin than pituitaries from males. 3. Serum Levels Fetal serum LH decreases (Figure 15) with increas- ing fetal age (P < 0.05), while both GH and prolactin serum levels increase as the gestation period lengthens (P < 0.01, P m 0.08, respectively). These differential age-related changes indicate a fetal control system functions during develOpment. In support of this hypothesis, sex of fetus caused significant (P < 0.01) differences in fetal serum LH. Foster (1971) reported similar sex and age differences in fetal sheep serum LH levels. Foster injected ex0genous LH releasing factor to determine if the fetal pituitary would respond by releas- ing LH. Although the variation in response to LH releasing factor was large, evidence of LH release was reported. Table 24 lists coefficients of correlation between fetal pituitary hormone concentrations and fetal serum hormones within sex and age groups. Overall, pituitary and the serum levels of GH (r = 0.41) and prolactin (r = 0.62) were significantly correlated (P < 0.01). But within age and sex groups, only those between GH in the pituitary and serum of the 90-day males (r = 0.79, P < 0.05) and between pituitary and serum prolactin for 180-day males 103 TABLE 24.--Some correlations between fetal pituitary and fetal seruma concentrations of hormones. Days of Sex Pituitary vs. Serum Concentration gestation GH LH Prolactin 90 Male 0.79b 0.08 0.14 Female 0.11 0.59 0.26 180 Male -0.11 0.21 0.98C Female 0.33 0.40 0.72 260 Male -0.69 0.29 -0.05 Female 0.57 -0.32 0.50 aAverage of umbilical artery and umbilical vein. bP < 0.05. CP < 0.01. were significant (r = 0.98, P < 0.01, Table 24). These latter two groups are small numbers (N = 5) and caution should be used in interpreting the results. The overall correlations are probably meaningless, since they are com- posed of significantly different components. Correlations between pituitary synthesis in vitro and fetal serum hormone levels are listed in Table 25. Overall, none of the correlations between in vitro and fetal serum hormone levels was significant. However, pi- tuitary synthesis and serum levels were significantly cor- related within certain sex and age groups. Differences among correlations for the three hormones within age and sex groups may indicate fetal pituitary hormone synthesis and release are controlled differently for the three hor- mones s 104 TABLE 25.-~Some correlations between fetal seruma hormones and pituitary net in Vitro synthesis. Serum hormone levels vs. Days of net synthesiS'in'vitrO' gestation Sex GH LH Prolactin 180 Male 0.08 -o.7i 0.65 Female 0.97c -0.02 0.46 260 Male 0.39 0.910 -0.31 Female 0.10 0.71 0.80b A aAverage of umbilical artery and umbilical vein. bp < 0.05. CP < 0.01. To my knowledge, all hormones found in adults also have been found in the fetuses of one or more Species of animals. Whether the hormones function in the developing fetus in the same manner as the adult has not been estab- lished. A few functions of fetal hormones, however, appear well documented and are reviewed by Jost and Picon (1970). Fetal testicular stimulation by fetal pituitary gonado- trOpin (LH) during development apparently occurs in most Species, and liver glycogen synthesis in the fetus appears to require fetal GH. No function requiring fetal prolactin has been reported. Although I did not study fetal adrenal hormones, others showed that fetal pituitary ACTH controls fetal serum adrenal corticoid levels (Kitchell and Wells, 1952) and that fetal corticoids participate in the initia- tion of parturition (Drost and Holm, 1968). 105 C. 'Fetal-Neonatal‘Serum'Hormones Fetal endocrine develOpment is a gradual process that continues up to birth (Figure 16). LH was highest at 90 days and gradually decreased until birth. The LH level at birth did not change significantly during the first 6 days of life in the calf. These data agree well with the serum LH levels Foster (1971) reported for fetal and neo- natal sheep. Serum prolactin levels gradually rose from 90 days to the highest level at birth (>100 ng/ml, Figure 16), and then decreased sharply to 30-40 ng/ml by day 2 of life. More frequent antenatal sampling would be necessary to determine if the serum prolactin increases as rapidly prior to birth as the postnatal decrease. No function of the relatively high prolactin levels at birth has been determined. High serum prolactin levels may be caused by stresses to the calf during parturition, since Tucker (1971) and Raud gt_§l. (1971) showed that stress can cause release of prolactin in cows. ‘ Fetal serum GH increased from 40 ng/ml at 90 days to a peak of 100 ng/ml at 260 days. But, at birth, GH dropped to an average of 36 ng/ml and remained relatively constant during the first week following birth. Since intermediate samples between 260 days and birth were not taken, when GH drOpped during this time is not known. The sharp changes in GH and prolactin near birth while LH 106 3:333 23 I4 .2363... £233 :33» .303 .2232. E3 .63... 38:3... 3.333 225 5L5 com om. cm 0 cu m _ w m a \r O¢ He. 0 u n \I m I .. m i u om M n TEE 5329:. mm 00. 2353.. 539.9. m JO euouuoq umoaa 107 remained relatively constant strongly indicate that differ- ential hormone control mechanisms are Operative during this important period in the life of the fetus. Knowledge of the hormonal levels during fetal development established by this study pave the way for further studies to determine more precisely the role of these hormones in the develOp- ing fetus. Information from this study will serve as the beginning of further studies. Each of us may see new horizons from the platform of knowledge built by preceding researchers (for example Lillie, Jost and Wells). At present, the effect of hormones on the metabolic activity of various tissues and cells are of great interest. The present study should provide a platform to build future studies of fetal hormone action. SUMMARY AND CONCLUS IONS Growth hormone (GH), luteinizing hormone (LH) and prolactin were quantified by radioimmunoassay in umbilical artery and umbilical vein at 90, 180 and 260 days of de- velopment of 37 bovine fetuses and through the first 6 days after birth of 10 calves. Also blood samples were taken from the jugular vein, uterine artery and vein of each pregnant cow. Average fetal body weights were 0.5, 6.4 and 26.7 kg for 90, 180 and 260 days of gestation, respectively, and body weight was correlated with fetal age (r = 0.86, (P < 0.01). Crown-rump length averaged 22.5, 54.0 and 84.4 cm, respectively. The crown-rump lengths were corre- lated with fetal age and with fetal body weights (r = 0.97, r = 0.93, respectively, P < 0.01). The adrenals, ovaries, uteri, testes and seminal vesicles all increased in weight as gestation lengthened and were correlated with crown- rump lengths (r = 0.96, r = 0.84, r = 0.95, r = 0.91, r = 0.80, respectively, P < 0.01). Maternal serum GH levels increased from 5.7 ng/ml at 90 days to 10.0 ng/ml at 260 (not significant), but averaged from two to four times higher in the jugular vein than in either of the uterine vessels (P < 0.01). At 90, 108 109 180 and 260 days, the uterine arterial blood levels of GH were higher than those of the uterine vein, but these dif- ferences were not significant. The jugular blood sample from cows with male fetuses averaged significantly higher GH (12.3 ng/ml) at 260 days than the cows with female fetuses (7.3 ng/ml, P N 0.11). This is the first report of a fetal sex effect on maternal pituitary secretion to my knowledge. The fetal anterior pituitary GH concentration in- creased during gestation (4.2, 8.9 and 18.1 ug/mg at 90, 180 and 260 days, respectively, P < 0.01). Male and fe- male fetuses had similar pituitary GH at 90 and 180 days, but males averaged 12.1 and females 25.3 ug/mg at 260 days (P < 0.01). Serum GH levels increased (P < 0.01) with fetal age from 42, to 65 and to 103 ng/ml for 90, 180 and 260 days, respectively, but male and female fetuses did not differ in serum GH. Umbilical arterial and venous serum GH levels were not significantly different. In the pregnant cows, serum LH levels did not differ in the three blood sources or with stage of gesta- tion, and averaged 0.74 to 0.89 ng/ml. Fetal pituitary LH concentration increased with fetal age from 323, to 474 and to 535 ng/mg for 90, 180 and 260 days, respectively (P m 0.06). In contrast, fetal serum LH levels decreased from 3.00, to 1.28 and to 0.85 ng/ml for 90, 180 and 260 days of fetal age, 110 respectively (P < 0.01). Female fetal serum LH averaged 3.90 ng/ml at 90 days, significantly higher than males (1.46 ng/ml) at the same age (P < 0.01). Female fetal serum LH also averaged higher at 180 days, but males were higher at 260 days causing a signifiCant sex-age interaction (P < 0.01). Levels of serum LH in the umbilical arterial samples were similar to those in the venous samples. Jugular serum prolactin in cows averaged 220, 145 and 365 ng/ml at 90, 180 and 260 days, respectively; these prolactin levels averaged slightly but not significantly higher than those in the uterine vessels. At 90, 180 and 260 days, cows carrying male fetuses had significantly more (3-, 2- and 7-fold respectively) jugular prolactin than cows carrying females (P < 0.05). Fetal pituitary prolactin concentration increased (P < 0.01) with fetal age; 72, 1150 and 2508 ng/mg for 90, 180 and 260 days, respectively, but sex of fetus did not influence fetal pituitary prolactin. The fetal pitui— taries synthesized large amounts of prolactin during in zitrg incubations; up to 23.6 ug/mg during the 72 hour incubation period in 180- and 260-day fetuses. Female pituitaries synthesized three times more prolactin than males at 180 and 260 days (P m 0.07). Prolactin levels in the fetal serum averaged 4, 43 and 61 ng/ml for 90, 180 and 260 days of gestation, 111 respectively (P < 0.05). As for GH, prolactin levels in sera from umbilical arteries averaged slightly higher than those from umbilical veins. At birth serum GH averaged 36 ng/ml and LH aver- aged 0.36 ng/ml, and neither GH nor LH changed signifi- cantly during the first week following birth. However, serum prolactin averaged 101 ng/ml at birth, decreased to an average of 42 ng/ml by the second day after birth (P < 0.01), and remained relatively constant to day 6. The serum hormone gradients maintained by the placenta between the fetus and the cow indicate minimal placental transfer of GH, LH and prolactin and that the fetal pituitary is the principal source of these hormones in fetal serum. Furthermore, age and sex differences in the fetal pituitary hormones, in vitro synthesis of hor- mones and in fetal serum hormones all indicated that the fetal endocrine system is at least in part controlled independently of the dam. In fact, fetal influences on the maternal endocrine system were also observed in this study. BIBLIOGRAPHY 112 BIBLIOGRAPHY Adams, W. M. and W. C. Wagner. 1970. The role of corticords in parturition. Bio. Reprod. 3: 223-228. Ainsworth, L. and K. J. Ryan. 1967. Steroid transformations by endocrine organs from pregnant mammals II. Forma- tion and metabolism of progesterone by bovine and sheep placental preparations in vitro. Endocrinol. 81: 1349-1356. If Ainsworth, L. and K. J. Ryan. 1970. 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Mix monobasic and dibasic to give pH 7.5. Dispense in 1 ml portions, store at -20°C. Store the monobasic and dibasic buffers at 4° C. 2. 0.05 M sodium phosphate buffer, pH 7.5 Solution A Merthiolate 0.01 g Dilute to 100 ml with distilled water. Solution B NaHPO4/7H20 26.825 g Merthiolate 0.05 g Dilute to 500 ml with distilled water. Use 16 m1 Solution A, 84 ml Solution B, dilute to 400 ml with distilled water. Adjust pH to 7.5 with NaOH, if necessary. Store at 4° C. 3. Chloramine-T Upon receiving chloramine—T, dispense into small, tightly sealed vials, cover with foil, and store at -20° C. Dilute 10 mg* chloramine-T to 10 ml with 0.05 M NaPOa, pH 7.5 buffer. Use within 30 minutes of preparation. Dis- card chloramine-T remaining in vial. *30 mg for CH 4. Sodium metabisulfite, 2.5 ng/u]_ Dilute 25 mg Na2S205 to 10 ml with 0.05 M NaPO4, pH 7.5 buffer. Use within 30 minutes of preparation. 5. Transfer solution Sucrose 1 6 3 K1 0.1 g Dilute to 10 ml with distilled water. Dispense in 1 ml portions, store at -20° C. 129 130 6. Rinse solution Sucrose —- 0.8 g KI 0.1 g Bromphenol blue 0 001 g Dilute to 10 ml with distilled water. Dispense in 1 m1 portions, store at -20° C. B. Reagents for Radioimmunoassay l. 0.01 M phosphate buffered saline, pH 7.0 (PBS) NaCl - — 143 g Monobasic phosphate 100 m1 (See Appendix A.1) Dibasic phosphate-— 260 m1 (See Appendix A.l) Merthiolate 1.75 g Dissolve in distilled water and transfer to a large container. Dilute to 17.5 liters with distilled water. Adjust pH to 7.0, if necessary, store at 4° C. 2. 0.05 M EDTA - PBS, pH 7.0 disodium EDTA 1 18.612 g Add approximately 950 m1 PBS. Adjust pH to 7.0 with 5 NaOH while stirring. Dilute to 1 liter, store at 4° C. 3. PBS - 1% egg white albumin (PBS - 1% EWA) or PBS - 1% bovine serum albumin (PBS - 1% BSA). Add 990 m1 PBS to beaker. Add 10 g EWA (Sigma Chemical Corp.) or 10 g BSA. Mix over magnetic mixer. Filter through Whatman No. 1 filter paper. Store at 4° C. 4. Hormone standards (LH, GH and prolactin) PBS - 1% EWA is used for LH and PBS - 1% BSA is used for CH and prolactin; hereafter they will be referred to as buffers. Rinse a small screw-cap vial with buffer, dry. Weigh 200—400 ug NIH-LH-BS, NIH-GH-BlZ or NIH-P-Bl on Cahn Electrobalance and transfer hormone to the screw- cap vial. Add 0.85% saline to 1 mg/ml. Add buffer to 9 volumetric flasks. Using Hamilton microliter syringes, add appropriate volumes of the lmg/ml stock hormone to volumetric flasks to obtain the following concentrations: LH - 0.16, 0.32, 0.64, 1.28, 2.56, 5.12, 10.24, 20.48 and 40.96 ng/ml. GH - 0.2, 0.6, 1.0, 1.6, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 ng/ml. Prolactin - 0.2, 0.4, 1.0, 1.6, 2.0, 3.0, 4.0, 6.0 and 8.0 ng/ml. 131 Add buffer to final volume in each volumetric flask. Dispense each standard in quantities suitable for one assay. Freeze in Dry Ice - ethanol, store at -20° C. For use, thaw rapidly with 38° C. water. 5. 1:400 normal guinea pig serum (NGPS). Obtain blood from guinea pig that has not been used to develop antibodies. Allow blood to clot, recover serum and store the serum in convenient quantities at -20° C. Add 2.5 ml of appropriate serum to a 1 liter volumetric flask, dilute to 1 liter with 0.05 M PBS—EDTA, pH 7.0 Divide into lOO-ml portions and store at -20° C. 6. Guinea pig anti-bovine LH (GPABLH, identified in our labora- tory as antibody 1), guinea pig anti-bovine GH (GPABGH), or guinea pig anti-bovine prolactin (GPABP). Dilute the antisera to 1:400 with 0.05 M PBS-EDTA, pH 7.0. Dispense in small quantities, store at -20° C. On day of use, dilute the 1:400 antisera to the required concentration using 1:400 NGPS as diluent. 7. Anti-gamma globulin Use goat anti-guinea pig gamma globulin (GAGPGG) in LH assay and sheep anti-guinea pig gamma globulin (SAGPGG) in CH and prolactin assay. Dilute antisera to required concentration with 0.05 M PBS-EDTA, pH 7.0. Store at 4° C. or at -20° C. C. Antibody and anti-gamma globulin production 1. Guinea pig anti-LH 0.5 or 1.0 mg NIH-LH-BS was dissolved in water and Freund's complete adjuvant added (1:1 ratio). 1.1 or 0.6 m1 of the emulsion per guinea pig was injected subcutaneously in 4 scapular region sites. The above procedure was repeated 15 and 30 days later sub- stituting Freund's incomplete adjuvant for adjuvant. Antisera was collected by cardic puncture 46 and 78 days after the initial injection. 2. Goat anti-guinea pig gamma globulin Guinea pig gamma globulin (Fraction ll, Pentex, Inc., Kankakee, Illinois) (40 mg), streptomycin (100 mg) and penicillin (1000 I.U.) was emulsified in 5 m1 of water plus 5 m1 Freund's complete adjuvant. 10 ml was subcutaneously injected in 8 scapular sites of a 75 kg goat. The above procedure repeated 15 days later substituting Freund's incomplete adjuvant for adjuvant. 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Sex of Cow Weight Pituitary explants Incubation media Age fetus (or fetal) of explant number CH LH Prolactin CH LH Prolactin (mg) (Ug/mg) (mg/mg) (pg/mg) (us)8 (as)3 ( g)8 180 Male 554 1.9 1.5 135 9.7 9.2 997 8.1 555 2.5 4.7 1042 2.5 5.6 70 16.5 556 3.2 0.8 308 0.9 3.1 424 4.6 558 4.3 1.9 129 1.2 12.3 497 10.8 559 4.6 1.6 925 0.4 5.5 817 2.1 Mean 2.1 508 2.9 7.1 561 8.4 S. E 0.7 197 1.7 1.6 161 2.5 n 5 5 5 5 5 5 Female 546 1.3 6.8 5 0.8 97.7 612 135.6 547 3.1 3.6 110 2.2 3.8 241 24.6 551 (2.7)b 1.4 245 1.9 5.4 542 14.9 557 3.2 1.0 187 0.9 6.3 370 11.8 567 2.7 1.2 514 0.2 3.2 906 2.0 Mean 2 8 212 1.2 23.3 534 37.8 S E 1.1 85 0.4 18 6 113 24.7 n 5 5 5 5 5 5 260 Male 515 1.3 0.9 243 0.4 4.1 516 11.6 524 1.8 1.2 649 0.2 0.5 1472 2.6 539 3.5 b 0.4 76 0.3 5.2 509 26.1 549 (2.7) 3.2 246 1.6 5.2 622 15.1 562 3.8 1.0 228 2.8 6.5 195 7.2 566 2.7 9.6 230 4.4 19.0 305 15.6 Mean 2 7 279 1 6.8 603 13.0 S E. 1 4 79 0.7 2.6 185 3.3 u 6 6 6 6 6 Female 525 2.3 0.4 90 3.8 5.8 475 65.2 550 (2.7)b 15.6 465 5.9 8.3 504 54.4 560 3.5 14.1 249 1.9 20.1 297 14.6 563 3.2 8.0 262 4.3 24.7 309 21.1 564 6.0 15.6 208 2.6 8.6 77 20.9 Mean 10.7 255 3.7 13 5 332 35.2 S E 2 9 61 0.7 3 7 76 10 2 n 5 5 5 5 S S a Per mg pituitary per 72 hr incubation. 9 Average of all slices used because weights of slices lost.