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A‘.i|a!...)>e,.t3h| :39... L93...r:.) 153.1...) 3.1.... i); .n.\f .3)! & Iiiy‘.’vbbxléhbi .xciztlfxlllil’f. n ‘ 1;} (ix-ILQS}¢||:I‘ :r‘1- iltlltri“ $.11 ‘5 5 y.0¢.§lrt.‘nr€l\‘|r mam: ‘ LIBRARY ' Michigan State University This is to certify that the thesis entitled Hormonal Requirements for Mammary Growth and Lactation in Several Species of Animals presented by Subhash C. Sud has been accepted towards fulfillment of the requirements for __Rh_._D_ degree in _Bh¥siology Date 7—9—68 0-169 / [HES ‘ Tues, ABSTRACT HORMONAL REQUIREMENTS FOR MAMMARY GROWTH AND LACTATION IN SEVERAL SPECIES OF ANIMALS by Subhash c. Sud 1. Estrogen-progesterone requirements for udder growth in ovariectomized heifers“ Various combinations of estrogen and progesterone were injected into ovariectomized heifers for 20 weeks. Evaluation of whole udder preparations and histological and biochemical measurements indicated that optimal udder development, equivalent to that seen in pregnant 20-21 week heifer; was achieved when the doses given were 100 mg progesterone plus 400 ug estradiol or 200 mg progesterone plus 800 ug estradiol. It was concluded that the absolute amounts of hormones given are probably as important as their ratios. There were also indications that more than one criterion for udder development should be used since very few valid correlations between histological ratings and biochemical estimates were noted. 29 Udder development in heifers treated with an estrogen-progesterone combination or Melengstrol Acetate (MGA). Ovariectomized heifers were injected with 100 mg progesterone plus 400 ug estradiol and intact heifers were fed either 0.5 mg or 1.0 mg MGA daily for 20 weeks. Evaluation of whole udder preparations, and histological and biochemical measurements indicated that feeding 005 mg MGA gave significantly better scores on histological estimates than feeding 1.0 mg MGA or injections of progesterone and estradiolo THESI‘ Subhash C. Sud None of the groups differed from each other when measured by DNA and RNA° It was concluded that feeding the lower level of MBA was more beneficial for udder development than any of the other treatments. The indication that more than one criterion should be used for udder development was reinforced. 3. Milk yield in intact and ovariectomized heifers treated with estrogenfiprogesteronecombinations followed by 9-fluoroprednisolone acetate (Predef). Eight groups of ovariectomized or intact heifers were injected with either 100 mg progesterone plus 400 pg estradiol or 200 mg progesterone plus 800 pg estradiol for 20 weeks. At the end of 20 weeks, half were injected with Predef to initiate lactation. In the 100 mg progesterone + 400 ug estradiol group, two out of three ovarie- ctomized heifers gave more milk than intact heifers. No milk was obtained from non-Predef treated groups. The latter came into milk when injected with Predef, but the amount of milk was low. In the 200 mg progesterone + 800 pg estradiol groups, intact heifers gave more milk than ovariectomized heifers. The non-Predef treated animals came into milk only when injected with Predef. The variations seen in these animals may be due to differences in treatment or to indi- vidual responses of the heifers. 4. Effect of Melengstrol Acetate (MGA) on organ weight and mammary lobulo-alveolar development in Sprague-Dawley rats. MGA given by feeding or by injection, decreased the weight of the pituitary, ovaries, uterus and adrenals, but increased mammary Subhash C. Sud development of the intact rat. In ovariectomized rats, no effect on mammary development was noticed although uterine and adrenal weights were reduced. Supplementing MGA with estradiol injections in spayed rats caused significantly better mammary development than estradiol or MGA alone. Pituitary prolactin and hypothalamic PIF levels did not Show any change after MGA injections for 10 days. It was con- cluded that the dose of MBA employed stimulated mammary growth only in the presence of estrogen. 5, Effect of median eminence lesions on mammary lobulo-alveolar development in hypophysectomized rats bearing one pituitary transplant. Two groups of female hypophysectomized rats were injected with PMS and HCG to induce mammary development, and implanted with one anterior pituitary under the kidney capsule. A week later one group was given bilateral median eminence lesions and the other group was kept as a control. The lesioned rats showed significantly better mammary development and increased ovarian weight due mainly to the presence of large corpora lutea. It was concluded that in the lesioned rats, PIF in the plasma was decreased or abolished and this resulted in lesser inhibition of prolactin release from the pituitary transplant. 6. Hormonal requirements for initiation of lactation in the guinea- pig. In the castrated or castrated-adrenalectomized guinea-pig, the mammary gland was developed by a combination of estradiol and and progesterone. Later, the groups were injected with saline, 4 Subhash C. Sud prolactin, hydrocortisone or prolactin plus hydrocortisone. From studies of mammary histology, it was noted that injection of either hydrocortisone, or prolactin and hydrocortisone, led to the appear- ance of secretory material in the alveoli. No secretory material was found in the saline or prolactin treated guinea-pigs. It was concluded that adrenal corticoids were necessary for initiation of lactation in the guinea-pig. 7. Importance of insulin for mammary growth in female rats. Mature female adreno-ovariectomized rats were injected with alloxan, prolactin and growth hormone. In another group of intact or adreno-ovariectomized rats insulin was supplemented with prolactin and growth hormone. Neither the presence nor absence of insulin produced any significant change in mammary development from their respective controls. Insulin was also found to be without effect on pituitary prolactin or hypothalamic PIF levels in the intact rat. It was concluded that insulin has no role in mammary development in the rat under in vivo conditions. HORMONAL REQUIREMENTS FOR MAMMARY GROWTH AND LACTATION IN SEVERAL SPECIES OF ANIMALS by Subhash C. Sud A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1968 Dedicated to My Mother and My Brother "‘1 ES“ ACKNOWLEDGEMENTS The beginnings of this dissertation are rooted in the establishment of a U.S.A.I.D. project at U.P. Agricultural Univer- sity, Pant Nagar, India. Without the three and half year of study leave and financial support that led to this dissertation, it is unlikely that the author would have achieved this end. The author wishes to express his sincere appreciation to Dr. J. Meites, who advised, encouraged and inspired the author not only in these investigations but throughout his doctoral program. Other members of the Guidance Committee who offered helpful suggestions were Drs. E.P. Reineke, W.D. Collings, A.J. Morris, E.M. Rivera and H.A. Tucker. Special thanks are due to Mrs. Claire P. Twohy, Miss Elizabeth Bartels and Miss Ann Chapman for their technical assistance. The author has relied heavily on students and staff of the Neuroendocrine Research group for their assistance from time to time, and they deserve his deep appreciation. Thanks are also due to The Endocrinology Study Section, National Institutes of Health, Bethesda, Maryland, for the supply of pituitary hormones, and to Dr. R.G. Zimbelman, The Upjohn Company, Kalamazoo, Michigan, for the supply of ovarian hormones and heifers used in these studies. ii II. II. III. TABLE OF CONTENTS INTRODUCTION .................. . ................... REVIEW OF LITERATURE .............................. Development of the Mammary Gland .................. A. Embryonic and Fetal Development ............... B. Birth to Puberty .............................. C. Pregnancy, Lactation and Involution ........... Analysis of Hormonal Influences ................... A. Anterior Pituitary Hormones ................... B. Ovarian Hormones .............................. C. Insulin .... ....................... .., ......... D Placental Hormones ............................ E. Adrenal Corticoids ...... .......... . ........... F. Thyroid Hormones ...... . ........ . .............. G. Neurohypophysial Hormone ............. . ........ EXPERIMENTAL ...................................... Estrogen - Progesterone Requirements for Udder Growth in Ovariectomized Heifers ....., ..... ...... Udder Development in Heifers Treated with an Estrogen-Progesterone Combination or l7-Acet0xy- 6-Methyl-l6 Methylenepregna-4,6-Diene-3,20 Dione (Melengestrol Acetate, MGA) ..... . ....... . ........ Milk Yield in Intact and Ovariectomized Heifers Treated with Estrogen-Progesterone Combinations Followed by 9 Fluoroprednisolone Acetate (Predef). iii 11 l3 14 15 l6 18 20 20 37 TABLE OF CONTENTS (CONT.) VI. Effect of Melengestrol Acetate (MGA) on Organ Weight and Mammary Lobulo-alveolar Development in Sprague-Dawley Rats ........... . ............... 68 1. Effect of Feeding MGA on Mammary Growth in Intact and Ovariectomized Rats ....... ....... 68 2. Effect of Injecting MGA on Mammary Growth in Intact and Ovariectomized Rats ..... ...... 76 3. Effect; of a Combination of MGA and Estradiol on Mammary Growth in Ovariectomized Rats .... 83 V. Effect of Median Eminence Lesions on the Mammary Lobulo-alveolar Development in Hypophysectomized Rats Bearing one Pituitary Transplant .. ......... 92 VI. Hormonal Requirements for the Initiation of Lactation in the Guinea-Pig ..................... 103 1. Effect of Prolactin and Glucocorticoid on Initiation of Lactation in Guinea-Pig ....... 104 2. Effect of Prolactin and Glucocorticoid on Initi- ation of Lactation in Adrenalectomized Guinea-Pig105 VII. Importance of Insulin for Mammary Growth in Female Rats ..................................... 115 A. Effect of STH and Prolactin in Adreno- ovariectomized and Alloxan Treated Rats ..... 115 B. Effect of Insulin, STH and Prolactin on Mammary Growth in Intact and Adreno- ovariectomized Rats ......................... 116 GENERAL DISCUSSION .............................. 127 REFERENCES ................................. ..... 130 APPENDIX A. Milk Yield in Heifers Treated with 200 mg Progesterone + 800 pg Estradiol-17B .. ....... 151 B. Milk Yield in Heifers Treated with 100 mg Progesterone + 400 ug Estradiol-17B ......... 153 iv Table 10. ll. 12. LIST OF TABLES Mammary development and secretory activity of heifers treated with various levels of Progesterone (P) and Estradiol 17B(E) ......... ........ ................... Mammary development and secretory activity of heifers treated with various levels of Progesterone (P) and Estradiol 17B(E) .. ............... . ......... . ........ Correlation coefficients between biochemical and morphological estimates of mammary gland develop- ment and secretory activity .... ..................... Mammary development and secretory activity of heifers treated with Progesterone (P) and Estradiol l7B(E) or MGA .. ..................................... . ...... DNA and RNA content in mammary gland of heifers treated with Progesterone (P) and Estradiol l7B(E) or MGA .............................. . ......... . ..... Comparison of mammary development of heifers treated with 100 mg Progesterone (P) + 400 Mg Estradiol 178 (E) from Exp. 1 and Exp. 2 ..... . ............ . ....... Induction of lactation in heifers subjected to various experimental conditions. Experimental procedure Effect of feeding Melengestrol Acetate (MGA) on organ weights of adult female Sprague-Dawley rats ......... Effect of feeding Melengestrol Acetate (MGA) on mammary lobulo-alveolar growth in female rats ....... Effect of MGA injections on organ weights of adult female Sprague-Dawley rats ... ............... . ....... Effect of Melengestrol Acetate(MGA)injections on mammary lobulo-alveolar growth in intact female rats. Prolactdrlconcentration of anterior pituitaries (AP) from control and MGA treated intact rats ... ......... HypothalamicPIF content of control and MGA treated intact rats .................... . .................... Page 25 26 27 4O 41 46 54 71 72 79 80 81 82 14. 15. 16. 17. 18. 19. 20. 21. 22. Effect of MGA injections on ovariectomized female Sprague-Dawley rats ......... . ........ . .............. Effectof estradiol (E) and Melengestrol Acetate (MGA) on mammary lobulo-alveolar growth in ovariectomized rats 0000000000000 OQ 00000000000000000000000000000 .0000 Effect of median eminence lesions on organ weight in hypophysectomized rats with one AP transplant ....... Effect of ME lesions on mammary lobulo-alveolar growth in hypophysectomized rat with one AP transplant ...... Effect of alloxan, STH and prolactin on mammary growth in adreno-ovariectomized rats ....................... Effect of insulin, STH and prolactin on mammary growth in adreno-ovariectomized rats ................... .... Effect of insulin, STH and prolactin on mammary growth in intact rats ................. . ...... , ............. PIF content of hypothalami from control and insulin injected female rats (8/group) ...................... Prolactin content of anterior pituitaries from control and insulin injected female rats .......... .......... vi 84 85 95 97 117 121 122 123 124 —— TH 63“ Figure 10. 11. 12. LIST OF FIGURES Representative whole mount sagittal sections of udders from ovariectomized heifers treated with hormone combinations indicated 3 x weekly for 20 weeks ...... Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 200 pg E, 3 x weekly for 20 weeks. 70X ................. . ................ ..... Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 400 pg E, 3 x weekly for 20 weeks. 70X ...................................... Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 800 pg E, 3 x weekly for 20 weeks. 70X ...................................... Photomicrograph of the udder of ovariectomized heifer injected with 50 mg P and 400 pg E, 3 x weekly for 20 weeks. 70X ...................................... Photomicrograph of the udder of ovariectomized heifer injected with 100 mg P and 400 pg E, 3 x weekly for 20 weeks. 70X ...................................... Photomicrograph of the udder of ovariectomized heifer injected with 400 mg P and 400 pg E, 3 x weekly for 20 weeks. 70X .. ......... . .......................... Photomicrograph of the udder of a 20-21 week pregnant heifer. 70X ........................ ........ ........ Representative whole mount sagittal sections of udder from intact (MGA fed 20 weeks) and ovariectomized (hormone injected-3 x weekly for 20 weeks) heifers Photomicrogrgfllof the udder of an ovariectomized heifer injected with 100 mg P and 400 pg E, 3 x weekly for 20 weeks. 210X ..................................... Photomicrograph of the udder of intact heifer fed 1.0 mg MGA daily for 20 weeks. 210x ........... . ........ Photomicrograph of the udder of intact heifer fed 0.5 mg MGA daily for 20 weeks. 210x .................... vii Page 29 30 3O 31 31 32 32 33 43 44 44 45 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Graphic representation of milk yield in heifers treated with 100 mg P + 400 pg E and Predef .. ........ Graphic representation of milk yield in heifers treated with 200 mg P + 800 pg E and Predef ......... Graphic representation of milk yield in heifers treated with 100 mg P + 400 pg E. No Predef ........ Graphic representation of milk yield in heifers treated with 200 mg P + 800 pg E. No Predef ........ Overal average milk yield in heifers treated with 200 mg P + 800 pg E, with or without Predef ......... Overal average milk yield in heifers treated with 100 mg P + 800 pg E, with or without Predef .......... Whole gland Whole gland Whole gland Whole gland from normal diet ovariectomized rat. 7X ...... Ovary from an intact, MGA fed rat. 25X ............. Ovary from an intact, normal diet rat. 25X ......... Whole mount preparation of right inguinal mammary gland from hypophysectomized rat bearing AP transplant and ME lesions. 10X ................. . .............. Whole mount preparation of right inguinal mammary gland from hypophysectomized rat bearing AP transplant. No ME lesions. 10X ...., ...... ..... .............. ... mount preparation of right inguinal mammary from MGA fed intact rat. 7X ............ . ..... mount preparation of right inguinal mammary from normal diet intact rat. 7X ..... . ........ mount preparation of right inguinal mammary from MGA fed ovariectomized rat. 7X .... ...... mount preparation of right inguinal mammary Section of mammary gland from hypophysectomized rat bearing AP transplant and ME lesions. 70X... H & E Stain. 000000 oooooooooooooooooooooooooooooooooo 0090000 Section of mammary gland from hypophysectomized rat bearing AP transplant. 70X .. H & E Stain. ............................... ......OOOOOOOOOO No ME lesions. viii 55 56 57 58 62 63 73 73 74 74 75 75 96 96 98 98 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. Section of the ovary from hypophysectomized rat bear- ing AP tranSplant and ME lesions. H & E Stain. 25X. Section of the ovary from hypophysectomized rat bear- ing AP transplant without ME lesions. H & E Stain. 25X 0 O O O O O OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Section of mammary gland from castrated guinea-pig injected with 50 pg EB + 4 mg P. H & E Stain. 70X .. Section of mammary gland from castrated guinea-pig treated with 50 pg EB + 4 mg P and 1.5 mg prolactin. H & E Stain. 70X .................. . ................ Section of mammary gland from castrated guinea-pig treated with 50 pg EB + 4 mg P and 3 mg HCA. H & E Stain. 70X ......................................... Section of a mammary gland from castrated guinea-pig treated with 50 pg EB + 4 mg P and 3 mg HCA + 1.5 mg prolactin. H & E Stain. 70X ....... .. .............. Section of mammary gland from castrated-adrenalectomized guinea-pig treated with 50 pg EB + 4 mg P and saline. H & E Stain. 70X ........... . ................... .... Section of mammary gland from castrated adrenalectomized guinea-pig treated with 50 pg EB + 4 mg P and 1.5 mg prolactin. H & E Stain. 70X ..... ...... . ............ Section of mammary gland from castrated adrenalectomized guinea-pig treated with 50 pg EB + 4 mg P and 3 mg HCA. H & E Stain. 70X .................. . .......... Section of mammary gland from castrated-adrenalectomized guinea-pig treated with 50 pg EB + 4 mg P and 3 mg HCA + 1.5 mg prolactin. H & E Stain. 70X .......... Whole mount preparation of right inguinal mammary gland from adreno-ovariectomized rat injected with saline. 20X ... ..................................... Whole mount preparation of right inguinal mammary gland from alloxanized-adreno-ovariectomized rat in- jected with saline. 20X ............................ ix 99 99 106 106 107 108 109 109 110 110 118 118 41. 42. Whole mount preparation of right inguinal mammary gland from adreno-ovariectomized rat injected with STH and prolactin. 20X .... ..... .... .......... . ..... 119 Whole mount preparation of right inguinal mammary gland from alloxanized-adreno-ovariectomized rat injected with STH and prolactin. 20X ... ............ 119 INTRODUCTION The mammary glands are unique structures common to all mammals, and serve as an integral part of the female reproductive system. Just as the maternal placenta serves to nourish the fetus during intra- uterine life, so the mammary glands provide for the nourishment of the young for a varying period during extra-uterine life. Apparently the newly born guinea-pig is the only mammal which can make extra- uterine growth on food other than milk. The preparation for the change from intra-uterine to extra-uterine nutrition requires a high degree of coordination and synchronization between the hypothalamus, pituitary, ovaries, uterus and mammary gland. Milk production of dairy cattle is in part dependent upon the number of milk secreting cells in the udder and, in part, upon hor- mones controlling the intensity of milk secretion and its maintenance. Any variability in total gland growth in cattle at the end of preg- nancy may be manifested in variability of milk production. Thus, if the total number of milk secreting cells is low, no matter how in- tense the milking stimulus may be as a result of suckling, milking or galactagogues, total production will not improve very much. In the study of the factors involved in the inheritance of milk pro- duction, the genetic-endocrine factors influencing normal growth of the mammary glands in dairy cattle becomes of special significance. Knowledge gained from studies on mammary growth of laboratory animals may be extended to dairy cattle. Like other structures of the reproductive system, growth and development of the mammary gland is primarily controlled by the pituitary and ovarian hormones. The varying secretion of the ovarian hormones during the estrous cycle is reflected in morphological and histological variations in growth of the mammary glands. Admin- istration of ovarian hormones stimulates mammary growth which later can respond with secretion. Dairy endocrinologists are faced with the problem of deter- mining which hormones directly or indirectly stimulate the growth of the udder. The levels of hormones which will produce growth of the udder equal to that produced by normal pregnancy have been studied to some extent. It was of interest to establish the op- timal dosages of ovarian hormones which would give the best udder growth and apply this knowledge for milk production in heifers. Related studies were conducted in laboratory animals to gain further knowledge of mammary development and lactational per- formances. REVIEW OF LITERATURE I. Development of the Mammary Gland A. Embryonic and fetal development Mammary glands are ectodermal in origin and appear as a mammary line on either side of the ventral midline. At the site of the future teat, a mammary bud appears which mainly grows downward into the mesenchymal tissue, while the teat itself is marked by a slight elevation at the bud. Primary sprouts lead- ing to secondary and tertiary Sprouts appear in a rather re- stricted area. At a later date, a lumen appears which connects the primary sprout with the teat. In males of some species such as the rat, there is no connection of the primary Sprout with the teat. This lack of connection is probably due to the action of fetal androgens (Raynaud, 1961). The number of teat canals varies with the species. As the fetus grows a few ducts might appear, otherwise very little growth takes place. B. From birth to puberty During the prepubertal period, there is a general increase in mammary ductal growth in some Species like the rat, mouse, goat, bovine, monkey, girl, etc. In other Species like the cat, rabbit, guinea-pig very scanty growth of the duct system takes place (for referencessee Folley, 1952). However, no lobulo-alveolar (LA) development is noted. Sinha and Tucker (1966) found increased DNA and total mammary area with advancing age in prepubertal rats. The greatest change took place during 20-40 days of age. The mammary gland, in some species seems to undergo cyclic changes with the ovarian cycle, regression and growth, and the net effect of estrous cycle is cumulative. Growth occurs during estrus and regression occurs during metestrus (Sinha and Tucker, 1967). In Species like the dog and fox (seasonal breeders), there is more mammary growth during the "off season" period (luteal phase) than during the "in season" period (Turner and Gomez, 1934). In species undergoing periods of psuedopregnancy, considerable mammary growth occurs. Wrenn _£ 11. (1966) reported that in rats a period of psuedopregnancy gave no advantage in lactation to the dams. C. Pregnancy, lactation and involution Qualitative changes in the mammary gland during pregnancy, lactation and involution have been described by Mayer and Klein (1961). Major growth of the mammary gland takes place during pregnancy. There is a gradual hyperplasia as well as hypertrophy of the cells. Tucker and Reece (1963a) and Munford (1964) showed that DNA levels did not Show much increase until day 4-5 of preg- nancy in the rat, but began to increase by the 8th day and con- tinued increasing throughout pregnancy. Recently, with auto- radiographic studies, Traurig (1967a,b) found that in the mouse marked mitosis of cells took place on day 6 and 12 of pregnancy and later there was relatively less growth. Cell proliferation occurred just before parturition and during early lactation. Histological observations of the mammary gland of the cow, goat and guineaepig showed that the involution process is char- acterized by a decrease in size of alveoli (Naito 35 _l., 1955; Naito, 1958; Turner and Reineke, 1936). The number of alveoli per lobule, and total number of alveoli and lobular volume is also decreased. The connective tissue becomes more obvious (Turner and Reineke, 1936). Schmidt §£_gl. (1962) found that ovariectomy or sham-ovariectomy caused a drop in milk yield in goats but it did not retard mammary involution. The total area decreased in proportion to milk produced but no change in the connective tissue was found during the involutory process. Tucker and Reece (1963c) found that DNA and RNA values fell during involution and reached the level of virgin rats by the 12th day post lactation. Involution of the mammary gland can be retarded by the milking or suckling stimulus (Turner and Reineke, 1936; Moon, 1962; Grosvenor, 1961), by the oxytocin injection (Benson and Folley, 1957; Meites and Nicoll, 1959; Meites and Hopkins, 1961) and by several neurohumoral agents (Meites, Talwalker and Nicoll, 1960). The DNA values were less in mammary glands of the rats with ligated teats than in nonligated glands in suckling rats (Tucker and Reece, 1963d). II. Analysis of Hormonal Influences A. Anterior pituitary hormones In the absence of anterior pituitary (AP) hormones, the responsiveness of mammary gland to the action of ovarian hormones is greatly reduced. Lyons in his extensive studies (see Lyons st 31., 1958) on Long-Evans rats found that full mammary lobulo-alveolar (LA) growth could be achieved in triply operated rats (hypophy- sectomized-ovariectomized-adrenalectomized) by the exogenenous injection of estrogen, progesterone, corticoids, growth hormone (STH) and prolactin. In doubly operated rats (hypophysectomized- ovariectomized) STH and estrogen caused normal duct growth. If the adrenals were also removed, corticoid administration became necessary for duct growth. However none of these treatments pro- duced LA development. For LA growth progesterone and prolactin were required. Nandi (1958, 1959) suggested strain differences in the response of mice to hypophysial hormones. He was of the opinion that the prolactin could be replaced by STH for LA development. Cowie and Lyons (1959) found similar results in hooded Norway rats. Recently Talwalker and Meites (1961, 1964) were able to achieve complete mammary growth with relatively higher dosage of prolactin and STH in doubly or triply operated animals suggesting that these rather than ovarian hormones are of primary importance in the mammary growth. Clifton and Furth (1960) also found that rats implanted with a ”mammotropic" pituitary tumor which secrete large quantity of STH and prolactin, had extensive LA development. In the goat (Cowie, Tindal and Yokoyama, 1966) found that estrogens and progesterone were ineffective in stimulating mammo- genesis after hypophysectomy, but considerable LA growth could be induced in hypophysectomized-ovariectomized goats with estrogen, progesterone, prolactin, STH and ACTH in combination. Ovariectomy (Bardin st 31., 1962) or hypophysectomy (Bardin _£'_1., 1964) prevented complete LA development in mice with tranSplanted pituitaries. It is probable the ovarian secretions in the graft-bearing host may have a dual role: (1) to stimulate growth and secretion of the hypophysial grafts and (2) to stimulate mammary duct and alveolar growth. Thus Browning and White (1965) found that with local grafts of pituitary or ovary at the mammary area in the ovariectomized or intact mice with an in gitu pituitary, the mammary gland showed a zone of alveolar proliferation around the ovarian tissue and a similar zone, with secretion, around the pituitary graft. Stimulation was appreciably greater when both ovarian and pituitary grafts were on the same side. In contrast, intact animals (without grafts) showed virtu- ally no alveoli in their glands and the intact animals with pituitary grafts showed general alveolar proliferation and secretion throughout the gland concerned. Strain differences in mice were also noted in the response. Different theories have been advanced to explain the mode of action of AP hormones on the mammary gland (Folley and Malpress, 1948; Lyons gt_al., 1958). Turner (1939) suggested that ovarian hormones do not act directly on the mammary gland but act through the mediation of AP and secretion of mammogens. It is difficult to explain by this theory, the observations which showed that local parcutaneous application of estrogen stimulated growth of a single mammary gland without affecting neighboring glands (Lyons and Sako, 1940; Lyons and coworkers, 1958). Turner (1950) suggested that estrogen increased local circulation of the mammary gland, thus making AP hormones available to the mammary gland in greater quantity. Astwood 25 31. (1937) suggested that with restricted food intake (”approximately that consumed by hypophysectomized rats”), rats failed to respond to the injection of estrone. This is con- trary to the observations by Reece (1950) that different estrogens, including estrone, could stimulate mammary duct growth in young male rats on severely restricted food intake, also by work (Samuels 35 31., 1941; Ahren, 1959c) which suggested that forced feeding of hypophysectomized rats given gonadal hormones did not stimulate mammary development. An interesting postulate was put forward by Turner (1939), according to which gonadal hormones produce their effect via AP. It was held that estrogen and progesterone stimulate secretion of specific ”mammogens” from AP which act directly on the mammary gland. The estrogen was thought to stimulate ”duct growth factor" and progesterone ”lobulo-alveolar growth factor”. Later on, these two mammogens were thought to be the same (Turner, 1950). Although the work of Lyons and coworkers (1958) and Nandi (1959) cast serious doubt on this factor,Damm and Turner (1958, 1960) again renewed the mammogenic hypothesis and put forward new arguments. Mammogenic fractions (A and B) were obtained. Fraction A containing less than 0.03% prolactin when injected with estradiol benzoate into estrogen- primed male mice was found to be 223 times as potent as progesterone in eliciting mammary growth whereas prolactin was only 1.6% times as potent as progesterone. Another mammogen C was prepared. How- ever, mammogen A and C proved ineffective in promoting the mammary development in rats. More work is needed, however, to show spec- ificity and activity of mammogenic factors as a separate constituent from prolactin and STH. Prolactin has been considered to be one of the two principal hormones in lactogenesis, the other being adreno-corticotrophic hormone (ACTH). Their significance in the initiation of lactation has been dealt with by Meites and his school in a series of papers (Meites and Sgouris, 1953; Sgouris and Meites, 1953; Meites, 1961; Meites and Nicoll, 1966). The manner in which prolactin brings about secretion has been investigated by Williams and Turner (1954, 1955), who have shown that in rabbits labelled 1131 prolactin be- came associated with the nucleoprotein of the cytoplasm and not with lipids. Topper and his group (Stockdale _£.El°’ 1966; Lock- wood §£_al., 1966; Turkington gt 31., 1965, 1967), in tissue culture studies on the mouse mammary gland indicated that prolactin increased the production of casein and other whey proteins Such as a lactal- bumin and a lactoglobulin. Hypophysectomy causes a dramatic depression in lactation (Cowie, 1957; Lyons t 1., 1958) in rats. In the goat the effects 10 are rather less immediate (Cowie and Tindal, 1960, 1961; Cowie £5 31., 1964), while in sheep, the effects are more rapid than in the goat (Denamur and Martinet, 1961). To initiate milk secretion in hypophysectomized animals the general concept is in favor of the Meites theory (1966) that both prolactin and adrenal corticoids are required for initiation of lactation. In the rabbit it is possible to initiate lactation by prolactin alone (Cowie and Watson, 1966) but this animal appears to be an exception. Main- tenancetxf lactation in hypophysectomized rats could be achieved by prolactin and ACTH injections, or by prolactin, ACTH, STH and thyroid stimulating hormone (TSH) (Cowie and Tindal, 1961). In hypophysectomized sheep and goats,reports (Cowie and Tindal, 1961; Cowie, Knaggs and Tindal, 1963) indicated that partial restoration of milk is possible with prolactin, STH, corticoids, tri-iodo-L -thyronine (T3) and insulin. The most effective hormones were pro- lactin and STH. Similar observations on goats were reported by Gale et 31. (1962) and Gale and Larsson (1963). It has been confirmed that STH increased milk yield in ruminants (Cotes _£._l°: 1949; Shaw _£__l., 1955). Prolactin injection had little or moderate galactopoietic activity in cows and goats (Brumby and Hancock, 1955). ACTH injections usually cause a prompt fall in milk yield in ruminants (Cotes 25 al., ——— 1949; Shaw t 1., 1955). Other pituitary hormones play little or no direct role. 11 B. Ovarian hormones The role of ovarian hormones in the mammary development of various species has been fairly well established. In the intact rat and mouse estrogen alone is mainly responsible for duct growth (Astwood _£.al., 1937; Silver, 1953; Flux, 1954). In the guinea- pig, estrogens were reported to cause full LA development (Folley, 1956) but later work (Benson et al., l957)indicated that progesterone is necessary for optimal growth. This was confirmed by Hohn (1957), who found that estrone alone caused only mammary duct growth in adrenalectomized guinea-pigs. Similarly in dogs (Turner and Gomez, 1934) and rabbits (Scharf and Lyons, 1941), estrogen alone caused mammary duct growth only. Studies in cows indicate that both estrogens and progesterone are necessary for complete normal udder growth (Sykes and Wrenn, 1950, 1951; Reineke gt al., 1952). In goats and cows, estrogens or synthetic estrogen (diethylstilbestrol, DES) given by injection, by implantation or by feeding, stimulated udder development and lactation. However, the udder showed various morphological abnormalities and there was wide fluctuation in milk yield (Mixner and Turner, 1943; Sykes and Wrenn, 1951; Cowie _£Hal., 1952; Benson 25.31;, 1955). When progesterone was also administered in combination with estrogen, the udder development appeared normal in Structure and milk yields were much less variable (Cowie gt al., 1952; Benson 35 al., 1955). Optimal ratios of progesterone and estrogen for mammary growth have been reported for the rat (Reece, 1950; Elliott and Turner, 1953; Smith, T.C., 1955) the mouse (Elliott and Turner, 12 1953) and the rabbit (Scharf and Lyons, 1941). Cowie et 31. (1952) reported that progesterone and estrogen given in the ratio of 4:1, 40:1, 160:1, and 400:1 produce no higher milk yield than hexoestrol given alone to goats. Reineke gt _1. (1952) found satisfactory Z lactation with a progesterone-estrogen combination in the ratio of 2:1 to 30:1 in cows. Turner 35 El: (1956) and Yamamoto and Turner (1955) found a 1000:l ratio of progesterone and estrogen to be optimal and milk yield closely approached the normal cows in a few of the treated animals. Nellor and Reineke (1958) used ratio of 40:1; 80:1; and 1000:1 of progesterone and estrogen, and could not find any clear cut indication of optimal doses for induction of mammary growth and lactation in goats. We (Sud gt _l., 1968) have tried to establish optimal dosage of estradiol and progesterone for udder growth in cattle and found that either 100 mg progesterone and 400 pg of estradiol-178 (ratio 250:1) or 200 mg progesterone and 800 pg of estradiol-17B (ratio 250:1) gave udder development in cattle which was equivalent to pregnant animals after the same duration of pregnancy as that of treatment. Williams 25 31. (1955) induced mammary development in cattle by injections of progesterone and estrogen and later initiated lactation by higher doses of estrogen, but did not find any difference in production due to different ratios of estrogen and progesterone given earlier. Benson 35 a1. (1955) found that 40 mg of progesterone and 1 mg of hexoestrol daily (ratio 40:1) caused abnormal mammary growth in the goat, while 70 mg progesterone and 0.5 mg hexoestrol (ratio of 140:1) gave normal udder development. Reece (1955) failed to stim- THESI 13 ulate udder development in sexually immature heifer calves with estrogen alone, although teat growth occurred. Udder growth also seems to be dependent upon the type of hormones used, method of injection and duration of treatment. Thus Benson gt _1. (1965) found that administration of hexoestrol and progesterone in crystalline suspension at infrequent intervals was less effective in inducing mammary development in the goat than the same amount of hormones given daily in oily solution. Cowie g£__1. (1965) found that suspensions of estradiol mono- benzoate crystals in combination with suspension of crystals of progesterone at infrequent intervals, were more effective than suspensions of hexoesterol and progesterone in goats and cows. C. Insulin The work of Salter and Best (1953) indicated that hypophy- sectomized rats could be made to resume body growth by insulin in- jections. A few reports from Sweden (Ahren and Jacobsohn, 1956; Ahren and Etienne, 1958; Ahren, 1959a,b) have tried to elucidate the effect of insulin on mammary growth. It was found that in hypophysectomized and gonadectomized rats, estrogen and progesterone and long acting insulin elicited considerable mammary duct growth. This growth promoting effect of insulin was enhanced by thyroxine (Jacobsohn, 1959), but nullified by cortisone (Ahren and Jacobsohn, 1957). In male hypophysectomized rats testosterone propionate produced a thickening of the mammary ducts and addition of insulin did not produce any greater mammary development than testosterone. 14 In alloxan diabetic rats, injections of estrogen and proges- terone produced mammary duct growth and this was approximately the same as in non-diabetic animals. However less LA development was seen in experimental rats as compared to controls (Ahren and Angervall, 1963), suggesting either lack of prolactin or insulin effect on the LA development (Ahren st 31., 1965). It was concluded by the authors that insulin has no effect on mammary development in intact animals. However, Kumaresan and Turner (1965) found increased mammary DNA content in the insulin treated rats, Suggesting that - ..., - .. ”as. 3 ~.~' insulin increased mammary growth. Studies on mammary tissue culture have indicated that insulin is essential for cellular maintenance (Rivera, 1964a,b; Barnawell, 1965, 1967) and increases DNA synthesis (Turkington, 1968). D. Placental hormones The importance of the placenta as an endocrine organ par— ticipating actively in mammary development has been discussed in the review (Jacobsohn, 1961) of the hormonal milieu regulating growth of the gland. However definite proof that mammogenic or lactogenic hormones are produced in the placenta has been difficult to acquire. Moreover, this field has remained neglected, since complete mammary development including lactation could be accom- plished artificially in non-pregnant animals by purified hormones from sources other than placenta and, in many species, marked mammary development occurred during pseudopregnancy, a condition in which there are no placental influences Selye st 31. (1935) observed that when the embryos and g L .4 15 ovaries were removed from rats in the middle of pregnancy, the mammary glands remained in the developed but non-secretory con- dition, provided the placentae were retained intact. Newton and Beck (1939) and Newton and Richardson (1940) showed that removal of fetuses coupled with hypophysectomy at mid-pregnancy, was only followed by involution of the mammary glands of mice if the placentae were also lost. Leonard (1945) wrote that his results ”indicate that the placenta of the rat is an endocrine organ and the active princip1e(s) work synergistically with hormones of the hypophysis and ovaries to control mammary growth.” Ray gt 31. (1955) and Cerruti and Lyons (1960) provided evidences implicating mammotropic and lactogenic properties to the placenta from rats and mice. However, Wrenn _£ _1. (1966) could not alter the decline seen in the mammary gland development of pseudopregnant rats with various placental suspensions. E. Adrenal corticoids The effect of adrenal steroids on mammary gland growth has been studied in rats and mice (Johnson and Meites, 1955; Selye, 1954; Ahren and Jacobsohn, 1957). There was marked branching and LA development in treated animals. Studies by Lyons and coworkers (1958) and Nandi (1959) have indicated that in certain strains of rats and mice glucocorticoids may help in the duct growth of the mammary gland. However full LA growth could be achieved by in- jection of estrogen, progesterone, STH, and prolactin. According to Talwalker and Meites (1961), in the triply operated rats, full % .. ._- 16 LA growth could be achieved by injection of STH and prolactin, indicating that adrenal hormones may be dispensed with. Adrenal corticoids and prolactin are essential for initiation of lactation. Thus lactation has been induced in pregnant animals by injection of cortisone acetate (Talwalker gt 31., 1961; Tucker and Meites, 1965; Delouis and Denamur, 1967). Glucocorticoids can depress established lactation in cows (Cotes gt g1., 1949; Shaw gt gl., 1955; Flux gt gl., 1954). In goats sags:- Meites (1955) found no depression in milk yield with cortisol in- jections. In rats Johnson and Meites (1958) and Talwalker, Meites and Nicoll (1960) found increased litter weight gains with cortisone acetate injections. Campbell gt gt. (1964) concluded that single injections of ACTH had no effect, while long acting ACTH or repeated doses of ACTH reduced milk yields in cows. Adrenalectomy in rats caused a sharp fall in milk yield, which can be increased by in— jection of cortisol or its 9—halo-derivatives (Cowie and Tindal, 1955). F. Thyroid hormones In laboratory animals conflicting results have been reported on the effects of thyroidal hormones on mammary growth and lactation. Reviews by Folley (1956) and Meites (1961) indicate that in rats some degree of hypothyroidism enhances alveolar development, while in the mouse hypothyroidism seems to inhibit mammary development. Cohen (1953) showed that thiouracil given to pregnant guinea-pigs had no effect on pregnancy but brought about a failure of lactation. 17 One to three days after the normal onset of lactation, withdrawal of thiouracil resultedin recovery of lactation. Chen _t gl. (1955) found the thyroid to be essential for the growth of mammary gland and lactation in triply operated and thyroidectomized rats. Meites and Kragt (1964) found that in hypophysectomized rats or in hypophysectomized rats bearing anterior pituitary-transplants, thyroxine injections resulted in reduced mammary growth. Schmidt and Moger (1967) indicated that feeding high levels of thyroxine during pregnancy in rats resulted in marked reduction in survival of litters, but when feeding was withdrawn on day 19 of pregnancy, the litters grew normally. During lactation thyroid feeding at higher doses also caused a reduction in survival of litters. In cows, Spielman gt g1. (1941) found decreased udder growth when thyroidectomy was performed during gestation. Thyroid hormones have a considerable effect on lactation. There is increased milk yield in cows fed thyroid, especially during the declining phases of lactation. Investigations have indicated that galatopoietic effects obtained with thyro-active substances are temporary in nature and are often correlated with a reduced length of lactation (see Leech and Bailey, 1953). Iodinated casein in cows induces a temporary increasesin milk yield (Sirry and Hassan, 1955; Swanson gt 31., 1954). According to Friedberg and Reineke (1956), thyroxine from iodoproteins is released in the body and the thyroidal action is then directly attributable to this free thyroxine. 1131 has been shown to be excreted in milk when injected into cows (Lengmann gt 31., 1955) 18 and goats (Wright gt 1., 1955). G. Neurohypophysial hormone Implication of the neurohypophysis has been mainly with the milk ejection from the mammary gland. Very early work (Ott and Scott, 1910), indicated that the ejection of milk could be elicited in the goat and cow by the administration of posterior pituitary extracts. Andersson (1951a,b,c) stimulated the anterior hypothalamic area in conscious sheep and goats and elicited milk ejection which persisted despite spinal anesthesia or denervation of the udder. Additionally, blood taken from the goat during electrical stimulation of the hypothalamus elicited milk ejection in a second goat when given intravenously. Cross and Harris (1952) showed that electrical stimulation of the supra-optico-hypophysial tract in anesthetized lactating rabbits, evoked milk ejection from cannulated teat, while its destruction by electrolytic lesions, interrupted milk ejection despite vigorous suckling by pups. The administration of oxytocin to a lactating animal helps in evacuating the udder completely, and may have a long term increase in the yield of both milk and butter fat (Denamur and Martinet, 1961). Petersen (1944) suggested that oxytocin stimulated prolactin release from the AP and thus played an important role in the initiation and maintenance of lactation. Similar ideas were reported by Benson and Folley (1957). But more recent work does not favor this view (see Meites and Nicoll, 1966). It is generally agreed that mammary involution in rats is !" l9 retarded by oxytocin administration (Benson and Folley, 1957). This ”is believed to reduce the pressure against epithelial cells and blood capillaries surrounding them, permitting more synthesis of milk” (Meites, Nicoll and Talwalker, 1960). Contrary to the above reports, Griffith and Turner (1962) were unable to detect any retardation of involution by oxytocin in rat mammary glands after weaning, while prolactin and hydrocortisone acetate were effective in this respect. Their estimations were based upon DNA estimates. Similarly Denamur (1962) also reported that oxytocin, unlike prolactin, failed to maintain the levels of DNA and RNA in the mammary glands of lactating rabbits after weaning. Intensity of suckling stimulus has a quantitative effect on the maintenance and proliferation of secretory epithelium (Tucker, 1966). Duration of lactation can be considerably extended by supplying successive fresh litters to dams (Tucker and Reece, 1963b). Yield of the milk can be increased by more frequent suckling and by an increase in the number of suckling young (Reddy and Donker, 1965). Donker _t gt. (1954) also found that oxytocin in- jections in cows increased total milk yield and butter fat. Does it mean that oxytocin has a galactopoietic effect by itself or through the secretion of some other hormone(s)? This remains to be seen. EXPERIMENTAL I. Estrogen-Progesterone Requirements for Udder Growth in Ovariectomized Heifers. Objective As a result of extensive research in experimental animals, it has been shown that the ovarian hormones — estrogen and proges- terone -can stimulate mammary gland growth in ovariectomized animals comparable to that observed in mature animals. Ratios of the two hormones for optimal synergism for mammary growth have also been suggested. Thus 1 part of estrogen (E) and 1000 or more parts of progesterone (P) has been shown to be optimal for the mouse (Mixner and Turner, 1943; Yamada gt _t., 1954), rat (Elliott and Turner, 1953) and dog (Trentin gt gt., 1952). In the rabbit (Scharf and Lyons, 1941) 24—96 pg of E and 1 mg P (ratio 1:11 to 1:42) were required for optimal lobulo-alveolar development. Mixner and Turner (1943) used a daily injection of 20-30 mg of progesterone with 100-150 pg of DES (200:1 ratio) for 60 days, and observed development of tightly packed alveoli comparable to the glands at mid-pregnancy. Cowie _t _t. (1952) used a ratio of 1:40 (1 mg hexostrol and 40 mg P), Benson gt gt. (1955), Cowie gt gt. (1965a,b) and Schmidt gt gt. (1964), used 1:140 ratio of hexoestrol or estradiol and P in goat for mammary development. Nellor and Reineke (1958) used 1:40, 1:80 and 1:1000 ratios of E to P in goats but could not come to any definite conclusion as to which ratio was superior. Turner gt gt. (1956) used a ratio of 1:1000 in cows 20 g 21 for optimal udder development. Despite extensive studies in laboratory animals showing synergism between E and P for mammary growth, comparable studies in goats and cows have lead to varieties of conclusions and inter- pretations. With administration of E alone, the udder may show abnormalities, mainly cystic alveoli (Mixner and Turner, 1943; Sykes and Wrenn, 1951). Addition of P alleviates this condition, and it seems to be necessary for normal lobulo-alveolar growth of the mammary gland (Meites, 1961). Absolute amounts of hormones used may also be as important as ratios of the two hormones. It should also be considered that the estrogens used do not all have the same activity, and hence ratios by themselves are inSufficient to evaluate hormones used to stimulate mammary growth. Since there is wide difference in dosage and ratios used by different investigators, the purpose of this study was to determine the best combination of estrogen-progesterone for optimal udder growth in ovariectomized heifers. Materials and Methods Thirty mature Holstein heifers of approximately 14 months of age and weighing between 270-320 kg. were obtained.* They were housed in a dairy barn of Michigan State University during the period of study, with free access to water and fed standard feed as practiced in the barn. After the 4th week of ovariectomy, the * Dr. Robert Zimbelman, The Upjohn Co., Kalamazoo, Michigan pro- vided the ovariectomized heifers and the hormones used in this study. 22 heifers were divided into six groups of five animals each by random selection and predetermined doses of estradiol-178(E) and progesterone (P) dissolved in corn oil were injected into each heifer three times a week (usually Monday, Wednesday, Fri- day) for 20 weeks. Two or 3 days after the end of the treatment period, the entire udder was removed from each heifer. In addition, the udders from three Holstein heifers pregnant for about 20-21 weeks were also removed and used as controls. The udders were separated antero-posteriorly into two halves along the median suspensory ligament. One half was immediately frozen on dry ice and stored at -200C for biochemical analysis. At a convenient time later, the udder half was thawed, trimmed of extraparenchymal fat tissue, weighed, ground in a meat grinder and its nucleic acid content meaSured according to the method of Schmidt and Thannhuser (1945), with slight modifications. The other half-udder was placed in Zenker formol fixative. About 100 ml. of the fixative was injected into each teat for speedy fixation. About 3-4 weeks later, the udder was cut medially above the teat into 2 halves. One half was Stained in Mayer's hemalum for gross whole mount study and stored in 70% alcohol. Maximum length and width of parenchyma was measured in each half udder from the gross mount section, while the whole udder half was used to measure thickness. Approximately 6 blocks of tissue were re- moved between the top of the gland cistern to the most distal portion of the parenchyma, from the other half. Thin sections i¥—_‘ ‘ 23 about 10 p thick were cut from each block and stained with hematoxylineosin using standard methods of staining. The histological sections from the six areas of the udder of each heifer were examined under the microscope by two different observers and rated for lobulo-alveolar growth and secretory activity. The growth ratings were based upon the following considerations: a) amount of connective and fatty tissue relative to lobulo- alveolar area b) degree of alveolar development - partially or fully differentiated c) relative number of epithelial cells in the alveolar area The sections were rated for growth as follows: 1 = Mammary ducts and few alveolar elements interspersed among much fat and connective tissue. 2 - Moderate lobulo-alveolar development in most sections. 3 = Extensive lobulo-alveolar development throughout section. 4 = Compact alveoli with fully differentiated epithelial cells throughout section. The sections were rated for secretroy activity as follows: a) presence of secretory activity in the alveoli and ducts b) distension of alveoli and ducts with secretory materials. The scale for secretory ratings were as follows: ,_. ll Alveoli showing little or no secretion; more in the ducts. 2 = Many alveoli showing secretion, some secretion in the ducts. U.) H Alveoli moderately distended with secretion. 4 = Extensive secretory activity in alveoli. h+~ J 24 Tables 1,2 and 3 summarize the histological ratings, bio- chemical analysis and statistics on the biochemical and histological ratings of the udders of these heifers. As can be seen from Table 1, the heifers in group 5 treated with 100 mg P + 400 pg E gave the best rating for growth as well as secretory activity. These ratings were similar to those found in pregnant heifers and there was no significant difference between the two. The second best treatment appeared to be 200 mg P + 800 pg E (group 3) which though significantly different from both pregnant heifers (group 7) and group 5 for growth ratings, is not different from the pregnant heifers on secretory ratings. Group 3 is different from group 5 on the secretory ratings. None of the other treatments (group l,2,4,6) differ significantly from each other on the growth rating scale. Study of the biochemical estimates indicated (Table 2) that, as far as RNA is concerned the pregnant animals and group 3 animals (200 mg P + 800 pg E) show no significant difference from each other, while they differ from other groups significantly. Also, there is no significant difference between groups 1,2,4,5 and 6. DNA when used as a basis of cell growth showed no significant difference among any of the groups. Thus we see that histological estimates and biochemical estimates do not give the same results. The correlation coefficients among the various growth and secretory activity measurements are given in Table 3. The cor- 25 mN.N ow.a Hm.a oa.a mm.o ow.o Ns.o mcaumm emu: m a m N o a a .oz macro o a m a o m a .02 Hmaumm wmswumm %Houopomm %HNEENZ .N ma.N ms.N Hm.a wq.a mm.a mm.a as o maauma cams m a m N a o a .oz macro u a m n o m a .oz Hmapmm mwcaumm Luzopo mumeamz .H Amo.o V H mv ume mwcmm mamwuasz 3oz m_cmocsn ow.o H ow.a 0N.H H ma.~ --- Asoaumase mausoe mv muowwmfi ucmcwmpm m n am.o H ma.o Nm.o H mm.a oooa m mi cos + a we cos n o wN.o H MN.N aw.o H ma.N omN a mi ooq + a we 00H m m No.0 H Na.o ao.o H ao.o mNH m m: cos + m we om m a ow.o H Hm.a om.o H am.a com a w: cow + a we oom m m Hm.o H ea.a aa.o H we.a com a mi ooq + a we oom m N HN.o H ow.o Hw.o H mm.a oooH a mi cow + m we ooN m a mxme ow pow wswumu wcaump oflump %mem3 x m mpmwwm: kHOOmpoom LUBOMO m\m quEumwuH mo .02 asouu Amvmma HOHUmuumm ram Amv osopwumwwopm mo me>mA wsowum> pups pwumeH mummfimm mo >uw>fluo< >u0umpowm was unmEmoam>mn hudEEmz .H maan 26 0N.0 A.mHm aoz ommm.N u m >o¢v zaamm boa moon "mwumaHumm <29 >H 00.N 0H.0 0N.0 N0 0 00.0 NN 0 00.N 0=Hm> 000: N m 0 N 0 0 a .oz asopu 0 a m 0 0 m 0 .oz ashram 000050000 azm HHH Amo.o V n mv umoH wmcmm oHaHuasz 3oz m.cmossn .m.m USN mdmmz an N0.0 H.0H.H 0H.N H.00.N 0N.N H 00.0 --- 0.N0N H 0.00NH coaumps0 wrucoa 0 uwwwom uswcwwum m 00.0 H N0.0 00.0 H 0N.0 H0.0 H H0.m 000 H.NNNN 0.00 H.0.000 m 0: 000 + a ms 000 0 No.0 H 00.H 00.0 H NN.0 00.0 H 0N.0 H00 H 0N00 0.0Ha H 0.NH0 m 01 000 + m 05 00a 0 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00H H 0HNH 0.0a H 0.000 m 01 000 + a we 00 0 00.0 H 0H.H N0.H H 0H.0 00.H H 00.0 000 H 0000 0.00H H 0.000H m 01 000 + a we 00N 0 00.0 H 00.0 00.0 H N0.0 HN.0 H N0.0 H0N H 00HN 0.00 H 0.000 0 mi 000 + 0 we 00N N 00.0 H 00.0 0N.0 H.00.N 00.0 H 0N.N maN H 000N «0.00 H 0.00s 0 0: 00N + a we 00N H A00 A00 as A00 wEDHo> mwoum Apcmfiw manslmsov ¢za\mA msowum> SUHB pmumopH mpmeom mo zuH>Huo< mucuouowm ppm unwanHo>mn kHMEEmZ .N maamp 27 .suaaapmposa 00 H0>0a NH 00 0 Soup 0G0000000 sauemoaaaemam «A .suaaanmnoua mo Hm>0a N0 00 0 Beam 000000000 saucmoawacwam A akaw.o wcHumu kHOumpomm fluHB ¢ZQ\ Esapxma :uHB mdflump Luonw aro¢.o wcHumu :uBonw fiqu uanoB «kgm.o «EDH0> EDEmeE EUH3 uanmz #mm.o mcHumu :u30pw suH3 EdemeE nuH3 Huo< %H0uwpomm was ucoanHo>wQ vcwau humEEmz mo mmumEHumm HauwmoH0£apoz was HmoHEmsoon domBOmm mquHonwwoo cowumaouuoo .m wHDMH 28 relation coefficient between DNA content and weight was much greater (r = 0.93) than either with maximum volume (r = 0.41) or histological growth rating (r = 0.35). Similarly there was a significant correlation between weight and maximum volume (r = 0.54) or histological growth rating (r = 0.46) and between growth and maximum volume (r = 0.64). Growth rating and secretory rating were also highly correlated (r = 0.81). Surprisingly there was no correlation between RNA and secretory rating (r = 0.17), while the correlation between RNA/DNA ratio and secretory ratings (r = 0.38) was low. Figure 1 represents a cross section of a representative udder from 4 groups of heifers. As can be seen, the two best hormone combinations, group 3 and 5, produced udder development most nearly approaching the pregnant state. The poorest development occurred when 50 mg P + 400 pg E were given. The remaining groups had glands which lie between these two extreme degrees of development. However some of the individual hormone-treated heifers in the poor groups had udders as well developed as the pregnant controls. Representative histological sections from udders of the various groups of heifers are shown in Figures 2—8. It can be seen that udders from heifers given the two best combinations of hormones show closely packed alveoli with slight secretory activity (Fig. 2,6) comparable to the udders of pregnant controls (Fig. 8), whereas the poorest hormone combination resulted in limited lobulo-alveolar growth interspersed amidst much connective and fat tissue (Fig. 5). The other groups showed moderate development. 29 Representative whole mount sagittal sections of udders from ovariectomized heifers treated with hormone combinations indicated 3 x weekly for 20 weeks. 30 Fig. 2. Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 200 pg E, 3 x weekly for 20 weeks. H and E stain. 70X. Fig. 3. Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 400 pg E, 3 x weekly for 20 weeks. H and E stain. 70X. 31 Fig. 4. Photomicrograph of the udder of ovariectomized heifer injected with 200 mg P and 800 pg E, 3 x weekly for 20 weeks. H and E stain. 70X. Fig. 5. Photomicrograph of the udder of ovariectomized heifer injected with 50 mg P and 400 pg E, 3 x weekly for 20 weeks. H and E stain. 70x. 32 Fig. 6. Photomicrograph of the udder of ovariectomized heifer injected with 100 mg P and 400 pg E, 3 x weekly for 20 weeks. H and E stain. 70X. Fig. 7. Photomicrograph of the udder of ovariectomized heifer injected with 400 mg P and 400 pg E, 3 x weekly for 20 weeks. H and E stain. 70X. 33 Fig. 8. Photomicrograph of the udder of a 20-21 week pregnant heifer. H and E stain. 70X. 34 Discussion Early experiments (Folley and Malpress, 1944a,b; Hammond and Day, 1944) showed that considerable udder development could be induced in cows, heifers and goats by synthetic estrogens. Since there were great individual variations in milk yield with some undesirable effects like nyphomania and pelvic fracture (Cowie, 1944), a combination of E and P was considered as more likely to produce good results. A combination of E and P pro- duced not only normal mammary growth but little or no persistent estrus (Sykes and Wrenn, 1950, 1951). In 1941 Gardner noted in- hibition of mammary growth in various species of animals given high doses of E. Optimal mammary alveolar development in rabbit appeared to depend on a delicate balance between E and P, as demonstrated by Lyons and McGinty (1941) and Scharf and Lyons (1941). Later, optimal doses of these two ovarian hormones were also demonstrated for the mouse (Mixner and Turner, 1943), rat (Smith, 1955), goat (Benson gt 1., 1955) and cow (Turner t al., 1956). Benson _t _t. (1957) indicated that optimal mammary growth responses in guinea—pig were obtained with dose levels of 10-15 pg estrone and 1000 pg progesterone (ratio of 1:100, 1:10). They also noted that ”within a species the absolute quantities of proges- terone and estrogens are the factors of real moment in controlling the growth of the mammary gland. The ratio of progesterone to estrogen pgt gg is of no importance ..” This study shows the quantitative and qualitative develop- mental responses of the mammary gland of ovariectomized heifers to 35 to varying doses of estrogen and progesterone. The optimal doses appeared to be 100 mg P and 400 pg E or 200 mg P and 800 pg E. These doses produced alveolar development very similar to that of normally developed udders from pregnant cows. The 200 mg P and 800 pg E combination produced the most DNA, second highest RNA per udder half and second highest histological growth and secretory ratings of the experimental groups. The 100 mg P and 400 pg E group showed the highest histological growth rating, second high- est secretory rating and ranked fifth and sixth in total DNA and RNA respectively. When all the groups were rated by all parameters of growth, they ranked in descending order, as follows: 200 mg P and 800 pg E, 100 mg P and 400 pg E, 400 mg P and 400 pg E, 200 mg P and 400 pg E, 50 mg P and 400 pg E and 200 mg P and 200 pg E. On the basis of estrogen-progesterone ratio the order would be: 1:250, 1:250, 1:1000, 1:500, 1:125 and 1:1000. The present study would seem to support the idea (Benson gt _t., 1957) that the ratio of E to P is of lesser importance in mammary development than the actual doses of hormones given, since the poorest development occurred when either P or E were given in low doses even though the ratios varied from 1:125 to 1:1000. 0n the other hand, the estrogen to progesterone ratios of the two optimal combinations were identical, 1:250. Whether this indicates that E to P ratios are of importance only within a certain range of absolute doses of the hormones remains to be tested. None of the combinations administered in our study stimulated precocious milk secretion or abnormal mammary growth. Whether milk 36 yields from heifers receiving estrogen and progesterone will match those from pregnant controls in unknown. The high correlation between mammary DNA and mammary weight of the heifers is similar in magnitude to the correlations between mammary surface area and total DNA of pubertal rats reported by Sinha and Tucker (1966). On the other hand, the correlations be— tween DNA, mammary weight and gross volume with histological criteria were relatively low. Although quantitative measurements probably indicate that a certain combination of progesterone and estrogen result in more total cell numbers in the udder, it does not necessarily reveal whether the cells consist primarily of ductal or alveolar elements, or indicate the distribution of these cells types within the gland. Whether prolongation of the treatment to nine months would promote further development and differentiation remains to be tested. Although DNA has been used extensively as a measure of mammary development since the mid-1950's (Kirkham and Turner, 1953; Sinha and Tucker, 1966; Tucker, 1964), it is clear from the present study that DNA measurements alone do not necessarily reveal the character of the parenchymal development. The results of this study indicate that a discrepancy may be present when mammary development is compared by DNA and histological methods. It is believed to be important, therefore, that several criteria be used to measure hormone-induced mammary development. II. Udder Development in Heifers Treated with an Estrogen- Progesterone Combination or 17—Acetoxy-6-Methyl-l6- Methylenepregna-4,6-Diene-3,20-Dione (Melengestrol Acetate MGA) Objective It is established that the udder can be developed in rumi- nants by the administration of ovarian hormones. For this purpose E alone or in combination with P have been used (for review, see Meites, 1961). Reports have been published indicating that suit- able doses of P given alone to ovariectomized guinea-pigs, rats, mice and monkeys (see Folley, 1952) can produce some mammary growth. This does not mean that estrogens are not playing any role at all, since in the absence of ovaries, E and P produced by adrenal glands may be active. Schmidt gt _t. (1964) noted that some of their goats produced considerable mammary gland growth when fed 6-methyl-17 acetoxy progesterone (MAP) alone without any major source of E. More recently The Upjohn Company, Kalamazoo, Michigan has marketed a new progestational compound named Melengestrol Acetate (MGA) which on the basis of biological activities (Duncan gt _t., 1964) seems to be more potent than MAP or P. Zimbeleman and Smith (1966a,b) reported that certain dosage of MGA not only produce progestational effects (inhibition of ovulation, maintenance of pregnancy in ovariectomized heifers), but also increase endogenous estrogen 37 38 secretion during treatment. These observations suggest that proper doses of MGA may produce mammary development in intact heifers. Therefore the purpose of this study was to measure the efficiency of MGA at two different doses on udder development, and to compare it with the mammary growth produced by an estrogen-progesterone combination. Materials and Methods Fourteen Holstein heifers, approximately 14 months of age, were purchased from dairy farms in Wisconsin. One heifer of the Brown Swiss breed was obtained from the Michigan State University dairy farm. They were randomly divided into three groups of five each. One group, including the BrOWn Swiss heifer, was ovari- ectomized while the rest of the animals remained intact. After 4 weeks of ovariectomy, the heifers received injection of 100 mg P and 400 pg E dissolved in corn oil three times a week (usually Monday, Wednesday and Friday) for 20 weeks. The second group of heifers received 0.5 mg MGA/day (Melengestrol acetate-MGA Premix- 100, The Upjohn Company, Kalamazoo, Michigan*) orally in the diet for 20 weeks. The third group of heifers were fed 1.0 mg MGA/day orally for 20 weeks. Two to 3 days after the end of the treatment period, the mammary glands of all the heifers were removed and processed as described in Experiment 1. Both the histological and biochemical estimates were made on the udders by the same criteria as used in Experiment I. * Dr. Robert Zimbelman, The Upjohn Co., Kalamazoo, Michigan kindly provided the hormones and MGA Premix-lOO used in this study. %7 39 Results Table 4 summarizes the histological ratings, biochemical analysis and statistics on these heifers. It can be seen that the lower level of MGA (0.5 mg/day) gave better responses on histological estimates than the higher dose. Both growth ratings and secretory ratings of the group fed 0.5 mg MGA/day had signif- icantly greater scores than the injected group or the group fed 1.0 MGA. There was no significant difference between the 1.0 mg MGA group and the hormonally injected group in growth or secretory NE; ratings. - On the other hand the biochemical measures i.e. DNA and RNA, in all three groups did not show any significant differences. 0n the basis of biochemical secretory activity (RNA/DNA), there was a difference between the injected animals and animals fed MGA at both levels. There was a greater ratio of RNA/DNA in the MGA fed animals (1.17 i 0.03 and 1.15 i 0.08 with low and high doses of MGA respectively) than in hormonally injected animals (0.86 i 0.07), but there was no difference between low and high MGA fed animals. Since there were variable weights of the udders in all three groups, it is possible that any differences which might have existed were masked. Therefore, the values of DNA and RNA for all the heifers were reduced to amounts per half udder weight. The results are tabulated in Table 5. No statistical difference between the three groups were noted. 40 kkmw.o n wcHumu %HOumHowm squ mcHumm £03000 mH.o u wcHumH kuououoow zuHB N 00. v u 0 00 0 0. v u aN 0> 00 m N “.m.z u m m> H mHo. V n N m> Hv m msHumm kHOumuoom 00. v u m 0> N ..m.z u m 0> H 000. v u AN 0> 00 m 000000 003000 0 .0 z .0.2 A %mu\we o.H wo.o + mH.H o¢.o + ON.N oq.o + Hm.N wH.o + H.H NH.o + H.m mm + wmm <02 m I I | >mw\wfi m.o no.0 + NH.H mn.o + oo.q Ho.H + Mn.q om.o + w.N mo.o + H.¢ HmH + ONHH ¢UZ N I | l I | I m mi 00¢ + 00.0 + ow.o No.0 + «H.d mw.o + mo.m mN.o + H.H mH.o + N.m qu + MHMH m we OOH H va va va ¢Zo\Huo< kHOOwHowm was udwEm0H0>wn xmeEmE .q mHan 41 G665 MO .m.m.mvfi.m 05Hm> Gmwz H. D. ufimeB Hove: N\H mo wx H 0u cmNHvumwdem mwsHm> ¢zm tam mQ xmeEmz Ho acmemQEOU .o anmH 47 Discussion To make use of sterile cows or cows with breeding failures, for milk production, the first step would be to develop their udders experimentally. Three major methods have been extensively studied, namely (1) implanting hormones under the skin (Folley and Malpress, 1944; Meites gt gt., 1950, 1951; Cowie gt_gt., 1952), (2) single or periodic injections of hormones (Nellor and Reineke, 1958; Turner gt gt., 1956; Schmidt gt gt., 1964), (3) oral feeding (Folley and Malpress, 1944; Sykes and Wrenn, 1950, 1951; Turner and coworkers, 1956). Of all these methods, the simplest one under field appli- cation is oral feeding. Oral feeding of estrogenic compounds like DES has been used by a number of workers with variable results. The amount of estrogenic activity entering the blood stream from DES feeding is probably much less than from subcutaneous injection. Mixner, Meites and Turner (1944) found that DES administered orally was about 1% as effective in inhibiting lactation in goats as by in- jection. Lewis and Turner (1941) found that the dose of DES needed to produce mammary development in male mice was about 6 times higher than when given subcutaneously. Folley and Malpress (1944) reported that less than 10% of orally administered estrogens were utilized by the body. In addition, estrogen alone may cause abnormal behav- iour and/or abnormal mammary development. Therefore, reliance must be placed on other compounds which not only Would show better udder growth potential but minimize abnormalities. These compounds could be either progestational or a combination of estrogen and progesterone. 48 During the last decade various progestational compounds have been used for control of estrus in animals and as oral contraceptives in human beings. On screening large number of 19-norsteroids, it was found that some of them like norethynodrel and its related compounds can inhibit human ovulation and menstruation (Rock gt gt., 1957). Changes in breast size have been observed by the users but the available data Show that there is no significant general in- crease in breast size, although some individuals claim a milk hypertrophy or an increase in breast sensitivity (see Pincus, 1965). This occurs mainly during the first cycle of use and the frequency of such complaints declined markedly thereafter (Mears, 1963). High doses of Enovid, however not only may bring about an increase in breast size, but may inhibit lactation (Pincus, 1965) in women. In women, Enovid has been reported to decrease gonadotropin secretion, to prevent ovulation and formulation of corpora lutea, to inhibit development of large follicles and to stimulate uterine endometrial growth (Gracia gt gt., 1965). In the rat it was found to decrease pituitary FSH and LH and increase prolactin content; decrease FSH-RF, LRF and PIF content in the hypothalamus; and induced mammary LA development and secretory activity (Minaguchi and Meites, 1967). MAP caused mammary growth in ewes without exogenous estrogen therapy (Schmidt gt gt., 1964). Studies with MGA have suggested that daily doses of 4 mg were adequate to maintain pregnancy in 7 out of 8 ovariectomized heifers. A lower dose (1 mg/day) maintained pregnancy for the first thirty days in 9 of 13 heifers. Lower than 1 mg dose terminated pregnancy shortly after ovariectomy (Zimbelman 49 and Smith, 1966a). Further studies (Zimbelman and Smith, 1966b,c) indicated that MGA in cattle given orally has more potency than MAP in inhibiting ovulation and is equally or more potent when given orally than by intravenous injection. The minimal effective dose of MGA to prevent ovulation in dairy heifers has been determined to be in the range of 0.2 to 0.5 mg daily. Another study (Zimbelman, 1966) Suggested that in intact heifers MGA caused increase in pituitary LH, but not in bilaterally ovariectomized heifers. In the absence of the corpus luteum, follicular weight increased up to 3 fold. No consistent effect on pituitary FSH was seen. Thus MGA has progestational activity and in the absence of the corpus luteum can increase follicular growth. The ovaries of MGA treated heifers had large follicles and practically no corpora lutea. This suggests that probably there was enough endogenous estrogen. Thus the mammary development in cattle appears to be due to endogenous secretion of estrogen and exogenously fed MGA. The present author has used 0.5 mg and 1.0 mg MGA daily for udder development and found that mammary development is almost similar to that found by treatment with 100 mg P and 400 pg E by injection. Since a discrepency was found between histological and biochemical estimates, it reinforces our previous conclusion (Sud _t _t., 1968) that more than one estimate should be utilized for quantitative and qualitative analysis of bovine mammary glands. The present study confirms the results reported in the first experiment, when comparison is made between the results obtained by histological or biochemical estimates in the groups treated 50 with 100 mg P and 400 pg E. This consistency is much more apparent when DNA and RNA values are reduced to a basis of 1 kg of half the udder weight. In conclusion, therefore, it may be said that MGA can be utilized in cows for udder development by the oral route, and this can replace the tedious and time-consuming method of in- jecting hormones. The long term effects on the breeding efficiency of treated animals remains to be tested. III. Milk Yield in Intact and Ovariectomized Heifers Treated with Estrogen-Progesterone Combinations Followed by 9-f1uoropredni- solone Acetate (Predef). Objective Mammary gland development can be achieved in dairy cattle, goats and sheep by oral feeding, implantation or injection of ovarian hormones (see Meites, 1961). There is a high correlation between the total alveolar area and milk yield in goats treated with estrogen or with a combination of estrogen-progesterone (Benson gt gt., 1955). The first encouraging application of the E treatment to the bovine was made by Walker and Stanley (1941) who obtained 14 and 16 lbs. of milk daily from tow heifers whose udders had been developed by a prolonged course of injection of diethylstilbestrol (DES). These results were confirmed by Reece (1943) who obtained peak daily yields of roughly 3 gallons of milk daily from each of the two heifers. Folley and Malpress (1944a,b) used implantations of DES or hexoestrol to develop the mammary glands of heifers and bring them into lactation. The milk yield was variable. Most of the later studies used estrogens for initiating lactation in animals. Tucker and Meites (1965) used injections of an adrenal corticoid hormone (Predef = 9-f1uoroprednisolone acetate) to initiate lactation in heifers pregnant 2, 5 or 7 months. Delouis and Denamur (1967) confirmed the above finding by initiating lactation in pregnant 51 52 goats with hydrocortisone acetate. We (Sud gt _t., 1968) attempted to establish the optimal doses of E and P for mammary development in ovariectomized heifers. The optimal combinations appeared to be either 200 mg P + 800 pg E or 100 mg P + 400 pg E. The purpose of the present study was to use the above optimal estrogen-progesterone treatment in both intact and ovariectomized heifers and subsequently bring these animals into lactation with Predef. We also wished to determine which combination of hormones and status of heifers resulted in highest milk yield. Materials and Methods Twenty-four Holstein heifers approximately 14 monthsof age were purchased from dairy farms in Wisconsin and maintained in The Michigan State University dairy barn. They were fed a standard dairy ration. Half were ovariectomized and half remained intact. They were divided into six groups of three each. These heifers were injected with either 200 mg P + 800 pg E or 100 mg P + 400 pg E for a period of 20 weeks. The ovariectomized animals received their first injection of hormones after 4 weeks of ovariectomy. At the end of the injection period one group of the intact animals and one group of ovariectomized animals was injected with 15 mg Predef/day for 7 days (9-f1uoroprednisolone, The Upjohn Company, Kalamazoo, Michigan) (hereafter termed Predef-treated groups), while the control heifers in each of the hormone treated groups were not injected (non-Predef—treated groups). At the end I” 53 of Predef treatment, all the heifers were machine-milked twice a day and daily milk yields were recorded. After 15 days of milking, the Predef treated heifers were injected with 10 mg estradiol-17B and 15 mg of Predef for another period of 6 days. Milking, however, was continued during this in- jection period and later as well. The non-Predef treated heifers were initially milked for 15 days. Since these animals barely gave any milk (most of them none at all), they were injected with 15 mg of Predef daily for 9 days (groups 2 and 4) or for 11 days (groups 6 and 8). These animals were not milked during the injection period. At the end of Predef treatment, they were milked again twice daily for a varying length of time. Results The experimental treatments are indicated in Table 7. Since the milking period varied in different groups, and within each group the heifers gave variable amounts of milk, 4 to 6 days milk yield of each individual animal is graphed in Figures l3, 14, 15 and 16. It can be seen from Figure 13, that the milk yield varied con- siderably in all the animals. Two of the three ovariectomized heifers (no. 82, 83) had an upward trend of milk production while the third heifer (no. 61) showed a consistently low yield. Even the "booster" dose of E and Predef did not cause any appreciable effect on lactation. The effect of the booster dose is difficult to interpret in the other two animals. The trend of milk yield 54 .GOHuommcH wowopm Ho onuw@ 0:0 mchsw wwaHE 00: @003 m .0 .0 .N @5000 .cOHuowde mmwmum umpr Hmuwm woxHHE mesosaHudoo 0003 m 0m .m .H @3000 00 . 00 - 0 oz 0 01000+0 0000N .00000>0 00.00.00 0 0N - 0 00 00» m m300$ 0000N .00000>0 00.00.00 N 00 00 - 0 oz 0 0:000+0 00000 .00000>0 00.00.00 0 N0 - 0 00 000 m 01000+0 00000 .00000>0 00.N0.00 0 N0 0 - 0 oz 0 01000+0 0000N 000000 00.00.00 0 N0 - 0 00 000 0 01000+0 0000N 000000 00.00.00 0 N0 0 - 0 oz 0 01000+0 00000 000000 00.00.00 N N0 - 0 00 00N m 01000+0 00000 000000 N0.00.00 0 Am>Mpv mmmw n mzmw >m0\mE mH wamvv mmc\mE mH mxmwS ON 000 mHmMHom wcHxHHZ unweummuH zmv\mmvop@ UmxHHZ ucwEummHH xmoB H x m mo .05 HeuoH movmpm we mH + m we oH m%mo wowou@ quEuMmpH mauMum HMMHom @3000 mpswmooum HeucmEHHume mdoHuHUcoo HaucoEHHw@xm msoHHm> 00 wmuommnsm muwMHmm CH :oHumuUMH wo GOHuUSUCH .n 0HQMH 55 00000.0 000 0 0a 000 + .0 00 000 0003 0000.00.00 000M005 :0 300% 0:05 mo 0.000000000000500 00:00.00 .MH .m0m Amaaouw 033.5 «000.05 00000000; 00000 0.0000 :0 060.30 M05020”: m o _ _ q — _ _ _ fi I \ IE I I I 00 ox 00 l m.N mm I 0.0 No 1 m0 0000.096 I I l | 00000: I . \o\ o OH \ \ \ 0 \ \ 00.700 00 K \ \o 003000.00 \ 0000.5 + m l . \ m NH \\ \ \ \ 00 o\ L 'sqI uI HUI“ mm 56 .000000 000 0 0.0000 + 0 00 000 0003 0300.3 0.000000: :0 0.7.00.0 0:05 we =00umuawmou@wu 0003000 :1 .000— Amazon» woulmuu vacuum.» 0 0;.000 + 0 0a 000 0010 00 0.0000 000000: 0.0 mm 00 3 ON ma ca m o — _ A q _ \ — III|%IIIIIMIIUII III T\\\ mm‘nll II J \lnl'll. \ \x m.~ ‘\ \\ \\. \ \ \ ‘ 00.w\ oom m.m .0000uw>0 I. 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Illl 03 uumucH IL 'sqI “I pIaIK mm 59 was already upward, and there did not seem to be any sharp in— crease in slope after the booster dose. The break in continuity of the graph for heifer no. 61 is due to the fact that this animal was off feed and was in Veterinary Clinic from day 10 to day 15 for treatment. No apparent pathological condition could be diagnosed. The heifer was not milked while undergoing inspection in the Veterinary Clinic. On the last day of milking, heifers no. 83 and no. 82 gave 16.50 lbs. and 12.00 lbs. of milk, respectively. The booster dose of E + Predef seemed to have a beneficial effect on the intact animals. Two of the three animals showed an upward trend in milk yield (no. 53 & 62) but the other heifer (no. 60) did not show any effect except that the downward trend of milk yield was checked. Whether the upward trend would have continued cannot be ascertained. Figure 14 represents milk yields of groups treated with 200 mg P + 800 pg E followed by Predef. The intact animals gave more milk and showed more consistency in their milk yield than the ovariectomized animals. The intact animals (no. 57, 58, 59) showed an upward trend of milk production. Was the booster dose of E and Predef beneficial to the these animals? The data seem to be against this. All these animals showed lower milk yields during the in- jection period, and later the slope of milk yield was almost the same as it was before the injection phase. On the last day of lactation (day 32) these animals gave 15.20, 16.45 and 14.90 lbs. of milk, respectively. IF 60 The results in the ovariectomized animals are difficult to interpret. Heifers no. 77, 78 gave low milk yields while heifer no. 81 gave much more milk, in fact almost as much milk as the intact animals. As in the ovariectomized animals given 100 mg P and 400 ug E (Fig. 13) the booster dose did not seem to influence milk yield or at the most, it had a slightly beneficial effect. Figures 15 and 16 represent graphically the milk yield of animals given low or high dose of ovarian hormones without Predef. As shown in Figure 15, none of the intact or ovariectomized heifers raj gave any milk before injection with Predef after 9 days of milking. After injecting Predef for 9 days to the intact animals, some ”bagging” of udders took place. The injections were stopped and milking was begun. All of the animals yielded variable amounts of milk. Heifer no. 64 gave 8 lbs. of milk only 14 days after the last Predef injection and judging from the slope, might have given more milk if milking had been continued. The other two animals in this group gave less than 2 lbs. of milk. In the ovariectomized group, two animals (no. 84, 87) gave only a small amount of milk and the third animal (no. 86) gave no milk. Figure 16 represents the milk yield of non-Predef treated heifers, both intad: and ovariectomized, given 200 mg P and 800 ug E dose. Heifer no. 54 gave no milk throughout the period of experi- ment. Heifer no. 55 gave about 1.25 lbs. of milk before Predef injection, probably due to the milking stimulus, but after Predef injection, the milk yield improved. The maximum yield was 9 lbs. of milk. Heifer no. 56 did not produce any milk before Predef in- 61 jections, but after injections milk yield began to improve and on the last day (day 32) she gave 13.40 lbs. of milk. In the ovariectomized group two heifers went off feed during the Predef injection and were sent to the Veterinary Clinic. They never recovered. The autopsy did not show any gross morbid changes and the cause of death could not be ascertained. The third animal improved its milk yield after Predef injection , but it never went beyond 6.50 lbs. daily. Figures 17 and 18 summarize the overall mean milk yields per day from both Predef and non~treated groups, in both high and low gonadal hormone treatment groups. The figures also show milk yield before, during, and after E and Predef treatment in both intact and ovariectomized animals. They also show before and after Predef treatment yields. In all cases the averages represent the mean of all three animals in each group, and no consideration has been given as to whether the animal was sick or had died during the treatment period. At least two items are very clear from these figures, namely (1) Predef treated animals gave more milk than non-Predef treated animals, and (2) Predef treatment was effective at least to a limited extent in promoting lactogenesis, even when the treat- ment was started 9-10 days after the last ovarian hormone injection. Discussion Many dairy animals are discarded from herds each year due to breeding failures. Some of these have good milk producing potentials 62 does—m 2.233 .8 5:. .m 3 8+ + .— ml 2: :33 cause: 93“qu cu vawuz Anal value?- udahos 2 .wum m maoce+m waoS peanut. menu; :02 ton-noun. wuvoum .uUthflsJO UUQUr—w .UUU“H.>O UUIUBM O ~.\\\\\\\\\ L O.m .. mg cozusooum xdaoo S 1 m A: :3.uu:13»m xuu—wa 8 agoth + m h0uw< N 3?...» .73: E ..oooum + m 95.25 D more: «Cor—um I vacuum 4 m uncuun I i n.- was awn. aqua.» v3:— moém>< 'sq1 ”I pleIA nIIH [£113.31 L' ‘ "Q IWI— I W, J .9 I rte-w qu- a~ 4.1.11- , m.- Iran-Inc - n ,I / rum \L'u--'t".f}f.“ 55"1‘Vl‘n‘: I ‘1 . I‘ c ; '_, \ .---;.o ' .‘ it t 3‘ PI .... ‘.r-'- c \ 1‘ 4 ¥ . "a. .u- nu - "fa; :; 1‘ a ‘5 ‘ E.‘Vur an - I. .—- i‘fi-{Q 'nx- . 63 C25; 32:: .8 53 .m ma oom + m mE ooN cuwz owumouo muowwo: :« vflwwz Jags ummuw>m Hamho>o m .3 com + a we oo~ commons woven. coz .u .40; ~C>Z ....Zoa..fi.on,_l ...HL : B c.3495 pouhi .0297” b _b . ..c I >1: cowuosooum xfidmo ...qufim L m nwuw< wwkum + m weauao Museum - m ouowwn mum CAMHV XJHZ m0< PHI" mu I: l ' (:(Il 64 and could be utilized for this purpose, especially in underdeveloped countries where average milk production is very low. In addition, hormone treatment produces other benefits as well such as a moderate to considerable increase in body weight and increased efficiency for food utilization. Also some of these animals may be successfully bred later and may show regular breeding cycles subsequently. At suitable levels of E and P where the absolute amounts and ratios may be of importance, the udder can be developed, hopefully, to a state equivalent to that in pregnant animals (Sud g£_al., 1968). Subsequent administration of E alone for a brief period results in initiation of lactation. However the yields of milk have shown a great deal of variability. In heifers and cows, it has varied from a few ounces to as high as 80 lbs. per day, and in goats from a few ml. to 3-4 litres per day. Periods of time required to reach the peak of lactation have also varied extensively. There is no adequate explanation at this time for such wide variabilities (see Meites, 1961). Very recently Tucker and Meites (1965) were able to initiate lactation in pregnant heifers by administration of adrenal corticoids. This has a benefit over E therapy in that nymphomanaic symptoms and pelvic abnormalities are not exhibited by the treated animals. In laboratory animals the initiation of lactation has been achieved by either corticoids or prolactin or a combination of both. Since numerous species differences have been noticed, there is no general theory for the initiation of lactation that would apply to all species. In pregnant rats injection of cortisol acetate can induce 65 lactation (Talwalker gt al., 1961). In pregnant rabbits, cortisol or prolactin (Meites, Hopkin and Talwalker, 1963) can induce lactation. Cowie and Watson (1966) demonstrated a lactogenic response in pseudo- pregnant adrenalectomized rabbits by injecting 10 i.u. of prolactin twice daily. On the basis of in yitrg studies, it is suggested (Barnawell, 1967) that dogs probably behave like rabbits in that increased prolactin levels lead to an increased secretory response. Using 33 31539 organ culture techniques, Rivera (1964) demonstrated that corticosterone or cortisol added to cultured mouse mammary glands resulted in the initiation of secretory activity. In a series of papers Topper and his coworkers (1965, 1966, 1967) indicated that in cultures of mouse mammary gland, insulin is involved in the stimulation of mammary epithelial cell proliferation, hydrocortisone and prolactin for subsequent differentiation of cells and protein synthesis. In culture medium containing only insulin or insulin and hydrocortisone secretory material is usually absent. In an insulin- prolactin medium it is present to a minimum degree by 48 hours of incubation. The combination of the 3 hormones effects a progressive increase in the amount of stainable material in the lumen of the alveoli. Thus both prolactin and adrenal hormones are required for the lactogenic response. The present study confirms this view. Even the low levels of intrinsic prolactin are sufficient to sensitize the udders to corticoid action. Confirmation of this idea is provided by the observation that the milking stimulus (and other stimuli) which increases prolactin secretion is not able to 66 initiate lactation in most animals (Figures 15 and 16), but in- jection of Predef even after 9 days of last ovarian hormone injection, elicited appreciable milk yield in some heifers (no. 55, 56, 64) and none to negligible amounts in others (no. 54, 86, 87). This work is further Supported by the report of Delouis and Denamur (1967) who did not observe any appreciable lactation from prolactin injections in pregnant ewes, whereas hydrocortisone injections resulted in moderate milk secretion. No conclusion can be drawn from milk yields from the present study. It is unfortunate that due to space problems in the dairy barn, milking could not be continued beyond 4 to 5 weeks in these animals. The animals, particularly in group 3 and 5, were showing an increasing trend of milk production. As stated earlier the peak milk yield varies in different animals. In the Holstein cow, Turner (1959) reported that at 14 weeks the maximum yield was 33.3 lbs. per day. Would our heifers have continued giving increased amount of milk until a peak was reached? The milk yields reported here are quite comparable with some of the earlier work (Hammond and Day, 1944; Folley and Malpress, l944a,b; Turner gt _l., 1956). However these results can be compared only with some reservations, since mammary development was different depending upon the method of administration, absolute and relative doses of hormones used, and duration of treatment. Reineke g£_al. (1952) reported that one Holstein-Freisian cow (4 years old, 1 calf) produced a spectacular 80 lbs. of milk at the peak while another Holstein-Friesian cow (4 years old, 1 calf) gave 67 only a maximum of 45 lbs. of milk. In both animals, udder develop- ment and lactation had been artificially induced. The author has no explanation as to why there was more milk yield in intact than in ovariectomized animals given the 200:800 dose, while heifers given the 100:400 dose, the ovariectomized animals gave more milk than the intact animals. Heifer no. 81 (Ovariect., 200:800) had almost the same yield as the intact animals of that group and heifer no. 61 (ovariect., 100:400) had an even lower yield than the intact animals of this group. Are individual variations a sufficient explanation for this observation? Or does pituitary secretion of hormones necessary for maintenance of lactation vary regardless of the ovarian hormone status in the body, and is this an explanation? Increase in milk production in ovariectomized animals certainly indicates that galactopoietic responses came mainly from the hypophysis. Either increased secretory activity per cell had taken place or an increase in actual number of secretory cells had resulted. This would indicate further mammary growth under the influences of pituitary hormones. IV. Effect of Melengestrol Acetate (MGA) on Organ Weight and Mammary Lobulo-alveolar Development in Sprague-Dawley Rats Objective The biological activity of MGA has been described by Duncan gt 3}. (1964). It was found to be more potent than MAP or proges- terone as a progestational compound. It has been shown to inhibit ovulation (Zimbelman and Smith, l966a,b) and maintain pregnancy in ovariectomized heifers (Zimbelman and Smith, 1966a). Our study (Experiment 2) indicated that MGA can be used for udder develop- ment in heifers and is apparently of equivalent potency to in- jections of estradiol benzoate and progesterone. Owing to the limited reports dealing with the biological activity of MGA in laboratory animals, it was considered that some insight should be sought on its mechanism of action. Therefore, the object of the present study was to study the effects of MGA on mammary growth in rats in three separate experiments. Materials and Methods Experiment 1: Effects of feeding MGA on Mammary Growth in Intact and Ovariectomized Rats. Mature female Sprague—Dawley rats from Spartan Animal Farms (Haslett, Michigan) were used in this and all other rat experiments. The animals were housed in a temperature con- trolled (75 i 10F) and lighted (14 hrs/day) room. The animals 68 69 were maintained on a diet of Wayne Lab Blox QAllied Mills, Chicago, Illinois) and tap water. One group received the normal diet in ground form while the second group was fed MGA mixed into the diet. (MGA premix-100 mixed with ground pellets of rat diet to give final concentration of 5 ug MGA/g of feed). The third and fourth groups of rats were ovari- ectomized. One week after ovariectomy, the third group was given the normal diet and the fourth group the MGA mixed diet. After 30 days of feeding these diets, the rats were sacrificed and their organs were collected, weighed and processed for histological studies. Histological Preparation and Rating System The tissues including left inguinal mammary glands were fixed in Bouin's fluid and sectioned at 6-10 u for histological examination. The sections were stained with hematoxylin and eosin. The right inguinal mammary glands were removed and fixed in Bouin's fluid for whole mount preparations; The standard procedure for staining with Mayer's hemalum was followed and the glands were rated for degree of growth and secretion as described by Talwalker and Meites (1961) with slight modification. Briefly the rating scale was as follows: 1. Few ducts; few or no end buds. 2. Moderate duct growth; moderate number of end buds. 3. Numerous ducts and branches; many end buds. 4. Numerous ducts and branches; moderate lobulo-alveolar growth. 70 5. Numerous ducts and branches with dense lobulo-alveolar growth as in late pregnancy. 6. Numerous ducts and branches with extensive lobulo- alveolar growth. Secretory activity always present. Results The results of this experiment are given in Tables 8 and 9. As can be seen from Table 8, MGA feeding significantly reduced the weight of the anterior pituitary, ovaries, uterus and adrenals in the intact animals. The reduction of adrenal weight was also noted in spayed rats, however in this case pituitary weight was not significantly affected. Table 9 describes the mammary ratings of these animals. As can be seen, MGA in the intact animals produced a significantly higher rating than the control animals, with lobulo- alveolar growth present in all rats. On the other hand, in the castrated group MGA did not stimulate mammary growth and the ratings were not different from the castrated control group. Figures 19 and 20 show the whole mount of the mammary glands of intact MGA treated and intact control rats, while Figures 21 and 22 are the whole mount of mammary glands of ovariectomized rats fed MGA and normal diet, respectively. None of the groups showed any secretory activity in the mammary gland except for one intact rat given MGA. Ovarian histology indicated the presence of healthy corpora lutea in the MGA treated groups with a fair number of follicles as well (Fig. 23). Control rats showed atretic and fresh corpora lutea (Fig. 24). The adrenal histology suggested a re- Q'JK'-’ .LL . I Ho. V n m u «r q m> m m u m N m> H m u N Bum Ho 3m: m u 83. 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The pro- lactin (Table 12) and PIF assays (Table 13) indicated that MGA did not alter either of these parameters significantly. Experiment 3: Effects of a Combination of MGA and Estradiol on Mammary Growth in Ovariectomized Rats In this experiment 21 primiparous female rats were ovari- ectomized. Ten days after ovariectomy, one group of rats was in- jected with 2 ug daily with estradiol in 0.1 ml of corn oil for 10 days. The second group was injected with 50 ug of MGA daily and the third group with a combination of estradiol and MGA in the above doses. After 10 days of injection, the animals were sacrificed and their organs processed as described above. Statis- tical analysis was done according to methods described by Kramer (1956). Results The results of this experiment are presented in Tables 14 and 15. When the estradiol treated rats are considered as the control group, it is clearly indicated in Table 14 that MGA alone reduced AP, ovarian and adrenal weights. Treatment with MGA and estradiol returned AP weight to the control level, but adrenal weight was significantly reduced. However there was no significant difference between adrenal weights of the MGA and MBA and estradiol treated rats. Uterine weight was higher in the MGA and estradiol treated rats than in the MGA treated rats, but was still lower H m N u museum H m N u HmsoH0< H m N u uHm ud¢ Ho. V n m umwu m.d«ocsn .m.m H coax u m Amomov o>onmv 0.0m + 0.0mq 0m.m + 0.00 Nm.o + o.mN OH + Nom 0 + HwN HonmHumm + 002 m I | I I I %M0\umu\m1 Om w.oN + w.NON mq.N + H.¢0 50.0 + o.0H 0H + oom 0H + mmN .002 N I | l I I %mv\umu\m1 N ¢.Nm + 0.0m0 HO.H + m.mn mwm.H + w.0N 0H + mom 0H + mmN .HOHomnowm H AwEv Amav AmEV m w U3 HB 03 03 uB monou: HmcoH0< .uHm .ud< Avon Hmch >000 HmHuHcH unwEumeH maoouo mama %wH3mQIw:meQm mHmamm 00NH80uooHHm>O co mGOHuoomcH ¢OZ wo uowwwm .qH oHQMH 85 HO. V n m m> N HO. V n m w> H HO. V n N m> H u m whmp OH How momov NH.o+N.s I m N I I I N m>ofi5$z+m m w%00 OH Mom NH.o+N.H I I I I s m N N23620:? N m%m0 OH How 0N.0+NN I I H e N I N NHHmemmiN H A.m.m H CNOZV O m ...N M N H wHMEHC/w moHumu mmmuw>< mwcHumn mumesmz wo .oz ucmeumouH wadouo mama OoNHEOHUwHHm>O EH SHBOHU HmHow>HHMEEMZ co A¢UEV oumuwo< HouumomaoHoz 0cm Amv HoHpoHumm mo uoowwm .mH oHan 86 than in the estradiol treated rats. There was no significant changes in the body weights of all the three groups either before or at the conclusion of the study. One unuSual aspect of this experiment was that AP weight was much higher than normally seen. The histology of the AP did not show any apparent change, although the AP sections were only stained with eosin and hematoxylin. The mammary gland ratings are presented in Table 15. It is clear that the combined treatment of estrogen and MGA is nec- essary for maximal lobulo-alveolar development. Estradiol alone stimulated the mammary development whereas the mammary glands of the MGA treated rats were almost rudimentary. Discussion During the past several years a great number of synthetic analogs of gonadal hormones bearing unnatural substitutes on ring A or B, or a side chain usually on the 16 or 17 position, have become available for biological and clinical evaluation. Some of these compounds have led to a dramatic increase or decrease in their hormonal activities. Studies by Ringold(1964) indicated that electro-negative substituted derivatives of testosterone were metabolized more rapidly in the body than the parent compound it- self, whereas the methyl-substituted derivatives were very slowly reduced. Similarly Glenn _3 _l. (1959) found that Zy-methylcortisol, GI-methylcortisol and 61-methy1progesterone were reduced more slowly than 6B-fluoro-cortisol. Moreover introduction of a methyl group 87 into progesterone made otherwise "inactive" progesterone capable of causing glycogenesis and act as anti-inflammatory compound. It has also been demonstrated that biological activity can be greatly changed by the addition of a hydroxyl group at position 16 or 17 (Kessler and Borman, 1958) or by methylation at position 2 or 6 (Babcock st 31., 1958) and finally by an enolic ether in- volving position 3 (Ercoli _E _l., 1960). Thus Cooke and Vallance (1964) found that with 17a-acetate group and 6-methy1 group, the rate of metabolism of the progesterone structure was reduced. With the methyl group at C 6 with B configuration i.e. 6B-methyl- l7a-acetoxyprogesterone, the compound is less potent than its 6a methyl isomer, although it is still more active than progesterone. A double bond at C 6 with or without an additional methyl group at C 16, caused a slight increase in oral potency by the McPhail assay, whereas introduction of a methylene group at C 16 and a double bond at C 6 (MGA) caused a fourfold increase in endometrium stim— ulatory activity by both parenteral and oral routes (Duncan 25 al., 1964). Since the synthetic steroidal ovulation inhibitors are replications of endogenous ovarian hormones, it is expected that they will exhibit biological activities characteristic of these hormones. Even so the predictions are not always true and some- times wide variations have been noted. Most of the drugs used for oral contraception contain both estrogen and progesterone components. It is therefore reasonable to believe that the target tiSSues for both progesterone and estrogen will be affected, depending upon 88 factors such as dosage, estrogen-progesterone interaction and probably individual response variations. It has been shown by Piacsek and Meites (1966) that proges- terone (4 mg dose) failed to alter hypothalamic LH-RF content, whereas Nallar gt a1. (1966) suggested that progesterone can stim- ulate LH release in rats only under certain conditions. It ele- vated plasma LH after ovariectomy and also stimulated LH release when given during the pre-ovulatory phase of the estrous cycle. Mangili E£.El' (1965) found that progesterone induced a hypophysial block and lowered urinary gonadotropin. There was a "rebound" effect after the withdrawal of progesterone and urinary gonadotropin became higher than the control level, suggesting that progesterone interferred with the process of liberation of LH rather than its synthesis. In spayed rats progesterone led to a clear cut drop in the elimination of urinary gonadotropin. This drop was reversible and in fact, after the withdrawal of treatment, there was a distinct rise in the urinary gonadotropin which after 4 to 10 days had reached much higher levels than basal levels (Mangili gt 31., 1965). It is difficult to draw clear cut conclusions from these studies. Urinary gonadotropin studies are vague since there may be differential changes in FSH or LH secretion. Studies with derivatives of progesterone have similarly resulted in some controversy. Investigations by Kincl and Dorfman (1963) revealed that Enovid (98.5% northynodrel and 1.5% mestranol) decreased ovarian weight and caused an apparent reduction in pitu- itary LH content. Minaguchi and Meites (1967) in a study using 89 different doses of Enovid in rats found that it decreased pituitary FSH and LH content but increased pituitary prolactin concentration. Concomitantly it decreased FSH-RF, LH-RF and PIF activity of the hypothalamus. The pituitary, uterine and adrenal weights showed an increase, whereas ovarian weight was decreased. They suggested that Enovid was predominantly estrogenic in action. This is not surprising since both components of Enovid have estrogenic activity. In clinical studies Venning (1962) found evidence for the suppression of follicular growth and ovulation i.e. both FSH and LH were suppressed by MAP. Brown gt gt. (1962) believed that L;‘ northynodrel and its acetate depressed the urinary output of estrogen and pregnandiol without significantly affecting total urinary gonadotropins, suggesting a dual action on the ovary. Similar results were reported by Loraine gt gt. (1963) on Enovid and Anovlar users. Since ”total" gonadotropin does not indicate variations in its component hormones, Stevens and Vorys (1965) used FSH and LH assays to study their content in the urine. Their results indicated that the mid-cycle peak of LH was abolished whereas early and late cycle FSH were unchanged. MAP does not have any apparent estrogen but has been found to depress adrenal weight in both male and female rats. The effect is more pronounced in the castrated female rat (Glenn gt gt., 1959), this suggests that its effect is predominately progestational. MGA, an analog of MAP, has potent progestational activity and is devoid of estrogenic and androgenic activity (Duncan gt gt., 1964). It depressed the adrenal weight in intact and spayed female 90 rats when given either orally or subcutaneously. Whether this effect is operative via the hypothalamus or directly on the adrenal gland, is not knOWn, although progesterone has been reported to de- crease pituitary ACTH secretion. Credence to this view comes from the studies of Pozzanza _t _l. (1964) and Glenn gt gl. (1959) who found that MAP exerted a blocking action on ACTH secretion and lowered plasma corticosterone levels. Injections of ACTH con- comitantly in MAP treated rats did not produce adrenal atrophy. In addition, the adrenal glands from rats treated with MAP synthesized less steroid per unit weight than the contrOl rats, although after introduction of ACTH in an ER gtttg medium, corticoid production by the adrenal glands showed no significant change. This suggests that MAP does not reduce the adrenal response to ACTH. As shown in the present study, the MGA reduced ovarian and uterian weights. The ovaries showed large number of follicles and a few corpora lutea. . When estradiol was given concomitantly, the weight changes in the uterus were less pronounced but still were lower than uterine weight due to estrogen treatment alone. Its effects on the pituitary weight were inconsistent. It did not cause any apparent change in the hypothalamic PIF content or pituitary prolactin concentration. In cows, MGA injections have been reported to increase pituitary LH content, but not in ovariectomized heifers where pituitary LH was already low (Zimbelman, 1966). It was also found that effects of MGA on pituitary weight were inconsistent. The basophils appeared to be degranulated. Suppression of ACTH and some component of the gonadotropic hormone 91 complex was also suggested. However, the acidophils did not show any change (Zimbelman, 1966). In earlier studies Nicoll and Meites (1964) reported that progesterone had no effect on pituitary pro- lactin release i2 3tttg but increased pituitary prolactin content lg 3t3g by acting either on the hypothalamus or through some other mechanism (Meites and Nicoll, 1966). Sar and Meites (1968) using higher doses of progesterone, reported that progesterone reduced hypothalamic PIF content and increased pituitary prolactin con- centration. However, this does not exclude the possibility that progesterone may have been converted to estrogen lg 3339 before acting on the hypothalamus (Villee, 1961). The mammary glands are a prime target for ovarian hormones. Both mammary growth and milk secretion are estrogen-progesterone conditioned. Users of Enovid and other contraceptive pills have given varied responses. Breast discomfort has been noted in a few'women (Mears, 1963). Some investigators have noted changes in breast size but others have not (Pincus, 1965). Enovid treat- ment in rats (Minaguchi and Meites, 1967), markedly stimulated LA growth in intact rats with evidence of secretory activity. In the present study with MGA, there was marked LA development in the intact female or ovariectomized rat given estrogen concomitantly, but not in the ovariectomized female. No secretory material was noted, however. The combined action of estrogen and MGA on the mammary glandsis believed to have made them refractory to the action of prolactin, and hence no milk secretion was seen. The absence of mammary growth in spayed rats after MGA treatment further indicates that it is a progestational compound without any estrogenic activity. V. Effect of Median Eminence Lesions on the Mammary Lobulo- Alveolar Development in Hypophysectomized Rats Bearing One Pituitary Transplant 9213M Several reports have indicated that following hypophysectomy, hypothalamic neurohumors can be detected in the plasma of Such animals. Otherwise they are not detectable in the plasma of normal animals (CRF-Brodish and Long, 1962;LH-RFLNallar and McCann, 1965; FSH-RF - Negro-Villar _t _t., 1968). Whether PIF is also increased in the plasma after hypophysectomy is unknown. If it were present in the plasma, it should inhibit prolactin secretion by ectopic pituitaries. Indirect evidence for this possibility was presented by Meites gt _l. (1963), who demonstrated that hypophysectomized rats bearing pituitary transplants had well developed mammary glands when injected with reserpine, formalin or serotonin. These drugs have been shown to decrease the PIF content of the rat hypothalamus, thereby increasing prolactin secretion from the pituitary, but have no direct effect on pituitary prolactin release (for refs. see Meites and Nicoll, 1966). In order to provide further evidence for the presence or absence of PIF in the blood, the effect of median eminence (ME) lesions were studied on mammary growth in hypophysectomized rats bearing one pituitary transplant. Materials and Methods Mature female virgin hypophysectomized rats were purchased 92 93 from Charles River Breeding Lab., Brookline, Massachusetts. The animals were fed gg libitum with Lab Blox pellets and their diet was supplemented with sugar cubes, oranges and carrots. The rats were housed in a constant temperature room (76 : 10F) with auto- matic controlled lighting (14 hours light daily). Ten days post- hypophysectomy (with no appreciable body weight gain) they were transplanted with one anterior pituitary (AP) underneath the kidney capSule with reasonable aseptic precautions. The AP's were obtained from mature male Sprague-Dawley rats killed by guillotine. Twenty- three days post-hypophysectomy each rat was injected with 50 IU Pregnant Mare Serum (PMS) and 60 hours later was injected with 25 IU Human Chorionic Gonadotropin (HCG). The idea was to induce follicular growth and ovulation, thus minimizing some of the variables influencing mammary growth. A week later, bilateral ME lesions were placed in one group of rats. Bilateral ME lesions were produced by passing a direct current of 1.5 ma for 8 seconds through an epoxylite coated number 1 insect pin electrode. The ME was located by the use of deGroot coordinates (deGroot, 1959). In the control group, the electrode was similarly lowered but only into the cortical area, without reaching the ME lest any of the hypothalamic pathway should be disturbed. Ten days later the rats were killed. The ovaries, uteri and mammary glands were processed as described previously. The sella turcica of each rat was examined with a magnifying glass for remnants of pituitary tiSSUe. The kidney was also examined for successful 'take' of the transplant. Only those rats which showed 94 good pituitary 'take' and no remnant of pituitary tissue in the brain were included in the study. At the outset the experiment was started with groups of 15 rats each which were randomly distributed with approximately equal body weights. Some animals died during the course of the study and some had to be discarded for lack of good pituitary 'take'. This drastically reduced the total number of animals especially in the control group, and produced discrepancies in the differences in initial body weights in the control and the experimental group (Table 16). This table also indicates that there was no significant change in the body weight of the animals during the course of the experiment. The ovaries of the experimental animals were heavier than the controls even on a per 100 g b.w. (P = < .05), whereas there was no significant difference in uterine weight. Whole mounts of mammary glands (Figs. 25, 26) indicated that the mammary glands in the experimental group were better developed than in the control group (Table 17). Histologically, there was secretory activity in both experimental and control mammary glands (Figs. 27, 28). Ovarian histology indicated that ovaries of lesioned rats had bigger and more numerous corpora lutea (Figs. 29, 30). Discussion The present study supports the concept that PIF is present in the plasma of hypophysectomized rats, although it is not known whether this represents any change from intact rats. ME lesions 95 NO. A mo. V n m u as dams wo .m.m H some n H stH + NNNH $.an + me N06 + N03 sz + 0.me 0 Houston SHN H 0.me NmNH H 3.9.. 0m.N H H.HNH Hens H HENH a meoHBH m2 a5 35 30 as us 03 03 %won H3 %000 mHmEHcm odHuqu cmHHm>O Hmch HmHuHGH mo .oz macho usmHmmamufi m< mco HHHB mumm OwNHEOHUomxfiaoazm CH uLmHm3 Gmwuo co mdonoH wococHEm cmemz mo uowmwm .0H oHLMH Fig. 25. Fig. 26. 96 Whole mount preparation of right inguinal mammary gland from hypophysectomized rat bearing AP transplant and ME lesions. 10X. -\ Whole mount preparation of right inguinal mammary gland from hypophysectomized rat bearing AP transplant. No ME lesions. 10X. 97 humHHsuHm HOHuouc< u m< N mosoaHEm amHooz u m2 H NN.on.N I I I m m I £828 N Ho.v 0H.oH0.m I I a m I I .8onng H o w> H mm H.comz 0 m e m N H m onuou owwuo>< wwHumm JHBOHO hpoaemz unoEuooHH .axm ucmHawcmuH Nm¢ woo suHs mama OoNHEoHoomzfimomzm CH Huaouo HmHom>HE stain. 25x. Fig. 30. Section of the ovary from hypophysectomized rat bearing AP transplant without ME lesions. H & E stain. 25x. 100 in the hypophysectomized rat led to a decrease in PIF concentration in the plasma. This is indicated by the observation that rats bear- ing ME lesions had better developed mammary glands than those with- out lesions. No attempt was made to detect the presence of PIF in the plasma but it is reasonable to assume that PIF content in the plasma of non-lesioned rats was responsible for an inhibitory effect on pituitary prolactin release. The control of hypothalamic releasing factors, in addition to external environmental factors, depends upon the hormones released by the target organ of pituitary hormones (long feedback), or by the pituitary hormones themselves (short feedback). Thus, gonadal hormones depress hypothalamic content of FSH-RF and LH-RF (Meites E£.El" 1966), whereas gonadectomy increases hypothalamic FSH-RF and LH-RF and plasma levels of FSH and LH in rats and mice (Parlow, 1964) and goats (McDonald and Clegg, 1966). Adrenal corticoids depress CRF and pituitary ACTH release (Vernikos-Danellis, 1965). Thyroxine decreases TSH in the pituitary but not hypothalamic TRF (Sinha and Meites, 1965). Prolactin release can be induced by certain hypothalamic lesions in rats and rabbits (Cross and Harris, 1952; Donovan and van der Werff den Bosch, 1957; Yokoyama and Ota, 1959). ME lesions resulted in retardation of mammary involution following litter re- moval from lactating rats, indicating increased prolactin secretion (McCann and Friedman, 1960). Gale (1963) and Gale and Larrson (1963) reported that in goats with ME lesions, prolactin is not necessary to check the decline in milk production but ACTH, STH, T3 and insulin 101 are required. Transplantation studies where one or more AP's were transplanted either subcutaneously or underneath the kidney capsule (Everett, 1954), have generally led to the conclusion that prolactin secretion is continued at an accelerated rate while the secretion of other AP hormones is considerably reduced. ACTH placed in the ME area (but not into the pituitary) of the rat would lead to a reduction in plasma corticosterone levels (Motta _t__l., 1965). FSH implants in the ME area reduce hypothalamic FSH-RF and pituitary FSH content in the rat (Corbin and Story, 1967). Prolactin implants in the hypothalamic area (Clemens and Meites, 1968) or trans lantation of ”mammotropic” pituitar tumors (MtTW or MtTW P y 5 15) (Chen gt. 1., 1967) resulted in high PIF content in the hypothalamus and low pituitary prolactin concentration. It appears, therefore, that the removal of the pituitary gland itself can lead to an increased secretion of hypothalamic releasing factors, presumably due to removal of the inhibition by the 'short' feedback component. The presence of CRF (Brodish and Long, 1962), LH-RF (Nallar and McCann, 1965), FSH-RF (Negro-Villar gt gl,, 1968) activity in the plasma of hypophysectomized rats supports this concept. Prolactin is one of the important hormones for mammary function. Prolactin implants in the ME of lactating rats have re- sulted in regression of the mammary gland and loss in litter weight gain (Clemens, Sar and Meites, 1968). Prolactin implants in ME also increased hypothalamic PIF activity (Clemens and Meites, 1968). It is, therefore, reasonable to believe that lesions in the ME 102 region would reduce or eliminate the PIF concentration in the general circulation, and less of it if any, would reach the ectopic pituitary to exert its inhibitory effect. Consequently more pro- lactin would be released and be available for enhanced mammary development. The present study supports this concept. The increased ovarian weight in the experimental animals is believed to be due to the fact that higher prolactin concen- tration was able to maintain most of the corpora lutea in a vigorous functional state, while in the control group the amount of circulating prolactin was lower and less effective in main- taining high luteal tissue activity. This has previously been observed by Meites gt gt. (1963). VI. Hormonal Requirements for the Initiation oeractation.in the Guinea-Pig Objective The mechanism for the initiation of lactation shortly after parturition has been a matter of controversy for a long time (Cowie and Folley, 1961; Meites, 1966). It has been shown that in pregnant rats or in rats with suitably developed mammary glands, injection of glucocorticoids stimulates secretory activity in the mammary gland (Talwalker gt gt., 1961). In the rabbit HCA alone (Talwalker gt_gl., 1961) or prolactin alone (Cowie and Watson, 1966) stimulated secretory activity. In the mouse, Nandi and Bern (1960) reported that inadequate secretion of glucocorticoids was the limit- ing factor in initiating mammary secretion during pregnancy. In the cow and goat the initiation of lactation has been achieved by high doses of estrogens (Turner gt gt., 1956; Schmidt gt gt., 1964), probably by stimulating increased release of prolactin and ACTH from the AP. Tucker and Meites (1965) and Delouis and Denamur (1967) used adrenal corticoids to initiate milk secretion in preg- nant cows and pregnant ewes, respectively. The French workers also noted that prolactin injections alone resulted in limited milk secretion in a few ewes (4 out of 9 animals). The requirements of the guinea-pig are not clear. It is possible that guinea-pigs behave like rats and mice and require glucocorticoids, or like rabbits in which either prolactin or 103 104 glucocorticoids can initiate mammary secretion. In order to further elucidate the control of lactation in guinea—pigs, the present study was undertaken. Experiment 1: Effect of Prolactin and Glucocorticoid on Initiation of Lactation in Guinea-pigs. Materials and Methods Forty male guinea-pigs weighing approximately 350-500 g. body weight were purchased from a Supplier in Madison, Wisconsin. They were housed in groups of 5 per cage in a constant temperature room (75 i 10F) with automatic controlled lighting (14 hours light daily), and were fed Wayne Rabbit Ration (Bement Feed and Supplies, Mason, Michigan) supplemented with fresh cabbage gg_ltt. A week later, all the animals were castrated under ether anesthesia with the necessary asceptic precautions. Two days post-castration, each animal was in- jected with 50 ug estradiol benzoate and 4 mg progesterone dissolved in 0.2 ml of corn oil daily for 20 days. Following this treatment they were divided into 4 groups and injected for the next 5 days as follows: group 1, saline (0.85% NaCl); group 2, 0.75 mg prolactin twice daily; group 3, 3 mg hydrocortisone acetate (HCA) per day; group 4, 1.5 mg prolactin and 3 mg HCA daily. At the end of the treatment period, the animals were sacrificed with an overdose of ether and the mammary glands were stained for histological examination with hematoxylin and eosin. PW— ragw- . I. 105 Results The results of this experiment are shown in Figures 31 to 34. It can be seen that the mammary glands in the saline injected control group (Fig. 31) did not show any secretory activity. In the prolactin injected group, there was limited secretory activity (Fig. 32). Out of 10 animals in this group, only one had relatively abundant stain- able material in the alveoli which was not as abundant as in the HCA treated group (Fig. 33) or in the prolactin and HCA treated group (Fig. 34). Both of the latter groups had alveoli which were di3h tended with stainable material and there was relatively little histological difference in the degree of distention in these two groups. Experiment 3: Effect of Prolactin and Glucocorticoid on Initiation of Lactation in Adrenalectomized Guinea-Pigs. Thirty-five male guinea-pigs were used. They were fed and housed as stated above. A week later they were castrated under ether anesthesia (8 or 9 animals per day) and injected with 50 ug estradiol benzoate and 4 mg progesterone daily for 20 days. One day after the last injection of gonadal hormones, bilateral adrenalectomy was performed on each group, according to the method suggested by Hoar (1966) with minor modifications. Following adrenalectomy all the animals were placed on .9% saline water and the diet was supplemented with sugar cubes and bread. At the same time, they were divided into 4 groups and injected similarly as in the previous experiment for the next 4 days. At the end of the 106 Fig. 31. Section of mammary gland from castrated guinea-pig injected with 50 Mg EB + 4 mg P. H & E stain. 70X. Fig. 32. Section of mammary gland from castrated guinea-pig treated with 50 ug EB + 4 mg P and 1.5 mg prolactin. H & E Stain. 70X. 107 Fig. 33. Section of a mammary gland from castrated guinea-pig treated with 50 ug EB +.4 mg P and 3 mg HCA. H & E stain. 70X. Fig. 34. Section of a mammary gland from castrated guinea-pig treated with 50 pg EB + 4 mg P and 3 mg HCA + 1.5 mg prolactin. H & E stain. 70X. 108 treatment period, the animals were sacrificed with an overdose of ether and their mammary glands were studied histologically. Only those animals which had no remnant of adrenal tiSSue were included in this study. Results The mortality in guinea-pigs after adrenalectomy was very high. Only two animals remained in the saline treated group, two animals in the prolactin treated group and three each in the HCA and HCA + prolactin treated groups at the end of the injection period. Histological pictures of the mammary glands of the surviving animals are shown in the Figures 35 to 38. It can be seen that in the saline treated control group (Fig. 35) or in the prolactin treated group (Fig. 36), there was no evidence of secretory activity. On the other hand treatment with HCA alone (Fig. 37) or in combination with prolactin (Fig. 38) showed large amounts of stainable material. No difference was observed between the latter two groups. Discussion The mechanism for the initiation of lactation has not been completely clarified. Two major theories as to why lactation is withheld during pregnancy and begins shortly after parturition, have been advanced. Folley and his colleagues (Folley and Malpress, 1948; Folley, 1956; Cowie and Folley, 1961) believe that low levels of estrogens are stimulatory but higher levels are inhibitory for the lactogenic function of the pituitary. Progesterone at certain levels is I HI fan -r-«L-.I!;A'_s-i 109 Fig. 35. Section of mammary gland from castrated-adrenalectomized guinea-pig treated with 50 ug EB + 4 mg P'and saline. H 6: E stain. 70X. Fig. 36. Section of mammary gland from castrated-adrenalectomized guinea-pig treated with 50 ug EB + 4 mg P and 1.5 mg prolactin. H & E stain. 70X. 110 Fig. 37. Segtion of mammary gland from castraced-adrenalectomized guinea-pig treated with 50 ug EB + 4 mg P and 3 mg HCA. H 6: E stain. 70X. Fig. 38. Section of mammary gland from castrated-adrenalectomized guinea-pig treated with 50 ug EB + 4“mg P and 3 mg HCA + 1.5 mg prolactin. H & E stain. 70X. 111 believed to antagonize the action of estrogen. Thus combinations of estrogen and progesterone during pregnancy have an inhibitory effect. At the time of parturition, there is believed to be a decrease in the progesterone-estrogen ratio which replaces the inhibitory effect by a positive lactogenic effect. Meites and his colleagues (Meites, 1961; Meties, Nicoll and Talwalker, 1963; Meites, Hopkins and Talwalker, 1963) have a somewhat different explanation for the onset of lactation. According to Meites (1966) the major hormones in lactogenesis, prolactin and glucocorticoids, are low during pregnancy. The actions of prolactin and glucocorticoids are further reduced owing to refractoriness of the mammary gland under the influence of gonadal hormones. Shortly before parturition, progesterone level falls while that of estrogen is increased. Concomitantly prolactin and adrenocorticoid levels are increased, and this results in lactogenesis. The dual theory of estrogenic action has neither been proved or disproved conclusively, but most of the recent work from EB 3139 and lfl:¥l££2 studies leans heavily towards Meites' theory. Meites and Sgouris (1953, 1954) showed the refractoriness of the mammary glands of rabbits under gonadal hormone influence, where either an increase in prolactin levels or a lowering of gonadal steroid levels brought about secretion in the mammary glands. Studies in mice, rats and rabbits from various labora- tories (Nandi and-Bern, 1960; Talwalker t al., 1961) have shown that injection of adrenal corticoids can initiate secretory 112 activity in the mammary gland. Intraductal injection of prolactin directly into the mammary galactophores in pseudo-pregnant or preg- nant rabbits (Meites and Turner, 1946) resulted in copious secretion of milk. In adrenalectomized pseudopregnant rabbits, high doses of prolactin alone resulted in lactational activity (Cowie and Watson, 1966). In tissue culture studies, Rivera (1964a,b) recognized the importance of corticoids and prolactin for the initiation of secre- tory activity in mammary gland explants of mice. Topper and his group (l966a,b; 1967) a150 working with pregnant mice mammary gland explants, further stressed this point. Barnawell (1967) came to similar conclusions based on studies of mammary explants of dogs. Adrenal corticoids seem to be the limiting factor for the initiation of lactation in pregnant heifers and ewes (Tucker and Meites, 1965; Delouis and Denamur, 1967). Thus we see that there is an apparent species difference in hormonal requirements for lactogenesis. With the exception of the rabbit, all other species studied require adrenal corticoids for the initiation of lactation. The mechanism of initiation of lactation in the guinea—pig has not been fully investigated. One of the reasons is that, as in the rabbit, adrenalectomy is a very difficult operation to perform in the guinea-pigs. Even with the best of care, the mortality rate in adrenalectomized guinea-pig is very high. Secondly, estrogen injections in adrenalectomized guinea-pigs is very toxic (Meites, Trentin and Turner, 1942). Hohn (1957) applied estrone on 113 the nipple of adrenalectomized male guinea-pigs which resulted in duct growth only. The average survival time of estrone-treated adrenalectomized castrated guinea-pigs was only 4 days, as compared with 16.6 days in similar animals not treated with estrone. Prolactin levels during pregnancy have been reported to be low (Meites and Turner, 1948). Gala and Westphal (1967) reported that corticoid-binding globulin activity was high during pregnancy while levels of free corticoids was low in the guinea-pigs. At parturition CBG activity decreased while unbound corticoid levels increased. This, together with increased prolactin levels in circulation at parturition could account for the initiation of lactation. In the present study with male guinea-pigs with in- tact adrenals, it was found that there was increased secretory activity in the mammary glands of guinea-pigs treated with HCA or HCA and prolactin, but animals receiving prolactin alone showed only limited secretion. This limited secretion may be due to the presence of endogenous adrenal corticoids. In the second experi- ment where the adrenals had been removed, there was almost no alveolar secretion in the saline or prolactin injected groups. HCA or HCA and prolactin administration resulted in abundant secretory activity. The results of the last experiment, however, should be evaluated with caution, since only a limited number of animals survived. From this limited study it may be concluded that adrenal corticoids as well as prolactin are of primary importance for the 114 initiation of lactation in the guinea-pig. Although no exogenous prolactin was administered to two of the groups, endogenous pro~ lactin was always present and might have shown an increased secre- tory rate since stress stimulates the release of prolactin from the pituitary (Meites and Nicoll, 1966; Grosvenor, 1965). VII. Importance of Insulin for Mammary Growth in Female Rats Objective Observations published in recent years suggest that treat- ment of hypophysectomized rats with ovarian hormones and long- acting insulin produces slight development of the mammary gland not equal to that seen in rats with an intact pituitary (Ahren and Jacobsohn, 1956). Injections of ovarian hormones or testosterone into alloxan-diabetic rats promoted marked growth of the mammary gland which was approximately the same as seen in non-diabetic rats (Ahren and Angervall, 1963; Ahren gt gt., 1965). On the other hand Kumaresan and Turner (1965) reported increased DNA content of the mammary gland when rats were injected with insulin. In view of the above discrepencies, the present study was under- taken to further elucidate the effect of insulin on mammary growth. Materials and Methods Experiment 1: Effect of STH and prolactin in adreno-ovariectomized and alloxan treated rats. Mature virgin female Sprague-Dawley rats were used in this experiment. Food was withheld for 72 hours. Half of them were injected subcutaneously once with alloxan (6 mg per 100 g.b.w.) and immediately placed on a normal diet. About 10 days later, one m1. of jugular blood was obtained from each rat and blood glucose was estimated according to the method of Somogyi (1945). Alloxanized rats which did not have at least 200 mg glucose/100 ml. of blood 115 116 were discarded. Non-alloxanized rats showed blood glucose levels of about 80-100 mg percent. A week later all rats were ovariectomized and adrenalectomized under ether anesthesia. The alloxanized and non-alloxanized rats were divided into two groups; one was given saline injections and the other STH (1.5 mg/day/rat) plus prolactin (l mg/day/rat) by subcutaneous injection for 10 days. On the day following the last injection, rats were killed and their mammary glands were removed for whole mount and histological study. Results The results of this experiment are given in Table 18. It can be seen that there was no significant differences in mammary develop- ment in the saline injected alloxanized or non-alloxanized rats. Similarly, there was no difference in the hormone treated alloxanized or non-alloxanized rats, although both hormone injected groups had better mammary growth than the saline injected groups. This indicates that prolactin and STH can induce extensive mammary growth in the absence of insulin. Figures (39, 40, 41, 42) represent whole mount preparations of mammary gland from various groups. No secretion was noticed in any of these rats. Experiment 3: Effect of Insulin, STH and Prolactin on Mammary Growth in Intact and Adreno-ovariectomized Rats. Four groups each of intact and adreno-ovariectomized rats were injected as follows: group 1, saline; group 2, insulin (Protamine Zinc Insulin, Ely Lilly (2.5 i.u. daily)); group 3, STH (1.5 mg daily) 117 mWIIm. MIIIH Momma wuHHHHmnoum m.omousn .xmp you we H u oHuomHoum N .mmw you we m.H .oooauom zuaouw u mam H .woco cho dw>Hw .3.m OOHNwE0 u :mNOHH¢ % MH. + No.0 I N OH H I I mH :HuooHoum + mHm + coonH< q HN. H om...” I N N N I I HH NaHuomHSH + Ham m NH .0 H mmH I I I o m a a IamonHa + 2:3 N .NH .0 H mmH I I I o N m NH 3:3 H coo: Ho .m.m HUH3 0 0 0 m N H mums wcHuom owmuo>< mwcHumm mumEEmz Ho .oz unoauooHH asouo mumm poNHEouooHHm>OIoc6u0< CH nuaouo HHmEEmE co :HuomHoum 0am mam .omonH0 Ho uoomwm .wH MHcmH 118 Fig. 39. Whole mount preparation of right inguinal mammary gland from adreno-ovariectomized rat injected with saline. 20X. Fig. 40. Whole mount preparation of right inguinal mammary gland from alloxanized, adreno-ovariectomized rat injected with saline. 20X. 119 w - (I... Fig. 41. Whole mount preparation of right inguinal mammary gland from adreno-ovariectomized rat injected with STH and Prolactin. 20X. Fig. 42. Whole mount preparation of right inguinal mammary gland from alloxanized adreno—ovariectomized rat injected with STH and Prolactin. 20X. 120 and prolactin (1.0 mg daily); and group 4, insulin, STH and pro- lactin in the above doses. A day after the last injection the rats were killed and the mammary glands were processed as described previously. In addition, pituitary prolactin and hypothalamic PIF content were measured in the intact saline-injected and insulin-- injected rats by the methods described previously. Results Tables 19, 20, 21 and 22 show the results of the present experiment. It can be seen in Table 19 that insulin alone or in combination with the pituitary hormones did not produce any signi- ficant difference in mammary growth in intact rats when compared with their respective control groups. Similarly in Table 20, it is clearly indicated that in adreno-ovariectomized rats, insulin injections alone or together with STH and prolactin had no beneficial effect on mammary growth under the experimental conditions employed. Tables 21 and 22 show pituitary prolactin and hypothalamic PIF levels in the saline and insulin-injected intact rats. No difference in prolactin content or PIF content is revealed as a result of insulin injections. Discussion The results of the present study indicate that under the conditions employed, the presence or absence of insulin in the rat does not affect mammary growth. These results confirm the observa- tions of Ahren and Angervall (1963) and Ahren gt a1. (1965), who showed that extensive mammary growth could be achieved by injection 121 0 m N H Mummy HuHHHnmnoum m.cmoosm how Mom we O.H u oHuomHoum how you we O.H u 390 haw pom .D.H m.N u GHHsmaH 0H0 H 0.0 I m m I I I 0 EH35 + aHHomHotH + 090 0 0N0 H 0.0 I .0 m H I I 0 030280 + 00.0 m 0H0 H N.H I I I I 0 N 0 033.5 N 0H0 H 0H I I I I m m 0 2:3 H .00 H 982 0 m 0 m N H 33 onumm owmuo>< mmcHumm HHMEEMZ mo .02 udoEudeH masouo . mumm OoNHEOuomHHm>OIocouv¢ CH Huaouw xumEEmE co cHuomHoum cam mHm .GHHSmaH mo uoommm .OH wHHmH 122 0 m N H Mummy %uHHHHmnoum m.omocsn >00 you we H u oHuomHopm hop pom we m.H Amooauom Huaouwv u mHm %mw Hon .D.H m.N u GHHsmcH OH.O + 0.0 m m I I I w GHHSwGH + GHuomHoum + mHm q 0H .0 H 0.0 0 0 I I I 0 33305 + mam m 0H0 H 0.0 I H 0 H I 0 EH35 N NN.0 H 0N I H 0 N I 0 00:00 H .m.m H 0002 m 0 m N H 33 onuom owmuo>< mwcHumm humesmz 00 .oz HooEumoHH msouu mumm HomucH oH Luaouo kaEEmz co :HuomHoum pom mHm noHHHHmcH Ho Hoommm .ON mHan 123 HemoHHHamHm 002 « humuHSOHm pwumnsoaH hp OommoHou cHuomHoum Ho maumu :H uaoucoo mHm oHEmHmsuom%£ moumoncH N .m.m.+ new: H Amsme 0H I .80 B 0N0 0H.O + Nm.O m N CHHSmGH a.m.z 0H.0 H.H0.0 0 N Houseoo H N.HH0< we 00H\0H0 m SSHOoE OucH mcoome mHHmm .oz OommmHom cHuomHoum Ho .02 HmmHm Ho .02 ucoeummHH .axm AQSOHNNOV mumm mHMEmm OoHUOHGH GHHsmcH 0:0 Houucoo Eoum HEmHmzuomzm 00 0500500 mHm .HN mHan HomoHHHemeIdoz u .m.z N ....H.m H 0002 H Nmsme 0H I24 I .833 0N0 I. N.m.z 0N.0 + 0N.N 0 aHHsmaH 0 00.0 H.0m.N 0 mH8080 0 H L 0 Ama we 00HNOHO mcomem mums .oz H cHuomHoum Ho .02 HomaummHH Ho .02 .axm mumm onEmm wouommsH GHHDmcH 0cm Houuooo Eoum moHHmuHsuHm HoHHouc< Ho Homucou cHuomHoum .NN oHan 125 of gonadal hormones in alloxan diabetic rats, but are at variance with the observations of Kumaresan and Turner (1965) who reported increased DNA content in the rat mammary gland following insulin injections. Insulin has been shown to deplete pituitary STH, probably by discharging hypothalamic GRF (Muller and Pra, 1968). It is therefore possible that this STH so released may stimulate mammary growth. Earlier studies by Talwalker and Meites (1961) indicated that STH (2.0 mg/day) given to adreno-ovariectomized rats stim- ulated mammary growth, although the growth was not equal to that seen when either prolactin was given alone or in combination with STH. Lyons E£.El~ (1958) indicated that small amounts of estrogen together with STH gave better mammary growth than STH alone. There- fore some mammary growth might possibly result after insulin admin- istration, which would be due to the discharged STH from pituitary. However no such growth was noticed in the present study. It is possible that STH released after insulin administration was not sufficient to cause any appreciable change in mammary growth. The absence of insulin in alloxanized rats had no deleterious effect on mammary growth. The mammary glands response was comparable to that seen with gonadal hormones (Ahren and Jacobsohn, 1957) or pituitary hormones in non-alloxanized rats. This reinforces the belief that insulin is not necessary for mammary growth in the rat. In tissue culture studies the presence of insulin has been shown to be essential for the maintenance and differentiation of 126 mammary tissue (Rivera, 1964). Recently it has also been reported to be involved in the stimulation of mammary epithelial cell pro- liferation and in increased DNA content of the mammary tissue (Lockwood E£.§l°’ 1967; Turkington, 1968). A mammary tissue culture apparently is similar to the "hypophysectomized" state. In hypophysectomized rats insulin has been shown to cause slight mammary growth when given with ovarian hormones, though this was not as good as in the rats with tg.gttg pituitaries. Therefore it seems that insulin may be a hormone of importance in maintaining a normal metabolic state of the system under 12.Xi££2 conditions or in hypophysectomized animals. Its importance under EB 3139 conditions and especially in animals with intact pituitaries remains doubtful. The absence of any change in pituitary prolactin or hypothalamic PIF content further reinforces the above view. Nicoll and Meites (1963) indicated that insulin did not cause any significant release or inhibition of release of pituitary prolactin from cultured rat pituitaries. Goodner and Freinkel (1961) observed that the AP is responsive to insulin $2_21E£2 and that carbohydrate metabolism of the AP of intact animals may be conditioned by the availability of insulin. General Discussion Udder development in the bovine has been achieved by in- jection, implantation or oral feeding of gonadal hormones. Estrogens alone usually cause abnormal udder growth, nymphomaniac symptoms and bone fractures. Supplementation with progesterone usually alleviates these symptoms. Great stress has been laid in the past on the ratio of estrogen to progesterone for udder growth, but pre- sent studies and those of Benson gt gt. (1957) have indicated that the absolute amounts of these hormones may be as important as their ratios. Furthermore, unlike laboratory animals, there was low correlation between histological findings and biochemical values. It was stressed therefore that in the bovine several criteria should be used for measuring and describing udder growth. Bio- chemical measurements do not definitely indicate the character of the mammary development, i.e., ducts, alveoli, lobules, secre- tory, non-secretory, etc. Oral feeding of a progestational compound, MGA, without any addition of exogeneous estrogen gave satisfactory udder growth in the bovine.l Histologically the alveoli were filled with secre- tory material. The ovaries of heifers treated with MGA had more follicles. Interestingly, in the rat, MGA when either fed or in- jected, required the presence of estrogen for mammary growth and no secretion was noted in the alveoli. It was also found to de- crease the weight of the ovaries, uterus and adrenal in rats. 127 128 Whether this decrease is seen in large animals remains to be determined. There were more follicles in the ovaries of rats treated with MGA than controls. It did not have any effect on pituitary prolactin or hypothalamic PIF content in the dose given. The role of insulin in mammary development is still unclear. Its presence or absence from the body did not affect the mammary growth in the rats used in the present study. STH and prolactin, with or without insulin, produced comparable mammary development in intact or alloxan-diabetic rats. No change in hypothalamic PIF or pituitary prolactin was noted, which is consistent with the observation that insulin had no effect on mammary growth. How- ever, 22 vitro studies have indicated that insulin is essential for maintenance and proliferation of mammary tissues under these conditions, perhaps reflecting a metabolic need of the tissue. The same may be true in hypophysectomized rats. Studies on the initiation of lactation in guinea-pig in- dicated that this Species requires adrenal corticoids as well as prolactin for initiation of lactation. So far, only the rabbit appears to be an exception to the general observation that both prolactin and adrenal cortical hormones are essential for the initiation of lactation. In the hypophysectomized or adrenalectomized rabbit, prolactin alone can initiate lactation. The presence of PIF in the plasma was indirectly demon- strated and earlier indications from this laboratory were con- firmed. ME lesions in hypophysectomized rats bearing one 129 pituitary transplant resulted in significantly better mammary development than in hypophysectomized rats not given an ME lesion. A radioimmunoassay for rat prolactin, currently being developed in our laboratory, should make it possible to deter- mine whether a ME lesion results in higher prolactin levels in the plasma. REFERENCES Ahren, K. 1959a. 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