ENDOCRINE AND REPRODUCTIVE DEVELOPMENT OF THE BOVINE FEMALE FROM BIRTH THROUGH PUBERTY Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY Claude Desiardins 19.66 THESIS IWWWWW 00081 7282 S LIBRA R Y Michigan State University I I This is to certify that the thesis entitled Endocrine and Reproductive Development ‘ of the Bovine remale From Birth Through Puberty presented by Claude Desjardins has been accepted towards fulfillment of the requirements for ph o D 0 degree in Dal: 14‘] / "\ / f A -. :// A / \ IA, / A A A Z/ ZAAA Major professor Z ' [hm December 1M, 1966 (A- f. ‘ ~31 f; I E ' 9" i} U :3 I \;‘ I I]. ABSTRACT ENDOCRINE AND REPRODUCTIVE DEVELOPMENT OF THE BOVINE FEMALE FROM BIRTH THROUGH PUBERTY by Claude Desjardins A total of 65 Holstein calves were purchased be- tween 0 and 5 days of age to study endocrine and repro- ductive development from birth through puberty. These animals were slaughtered in 13 groups of five animals at 1-month age intervals. For the purposes of this experiment, puberty was defined as the onset of first estrus. The average age at first estrus was 29.7 i 1.3 weeks, and the average estrous cycle length was 20.5 i 0.6 days. The average weight of the whole pituitary gland increased 1.1g between birth and 12 months of age. Approximately 90 per cent of this increase was attri- butable to increases in the anterior lobes. The average concentration of pituitary lutein- izing hormone (LH), measured by ovarian ascorbic acid depletion assays, increased four-fold between birth and 3 months of age, did-not change significantly from 3 to 7 months, and declined from 10.3 pg at 7 months to U.8 ug at 12 months of age (P < 0.05). The average nrv'45”5“9‘. 4 O ‘ cJuv-M‘U“". ‘ cccurred aft «.u‘tarq E “I" '«aan T‘ A “d, a!” be.“ Claude Desjardins concentration of pituitary follicle stimulation hormone (FSH), measured by ovarian weight augmentation assays, was 1.67, 2.68 and 1.06 ug at 0, l and 2 months of age, respectively. These concentration differences were significantly different from each other (P < 0.05) while the differences in pituitary FSH among the remaining age groups were not significant, although a small decline occurred after puberty. Total pituitary LH and FSH con- tents paralleled the concentration values for these gonadotropins. The results were interpreted to indicate that pituitary LH and possibly FSH decreased beginning at the onset of puberty. Pituitary LH and FSH concentrations of the post- puberal heifers in this experiment were studied in separate analyses. LH concentration decreased 800 per cent Just before the time of ovulation and then increased about 300 to 400 per cent within the first six days after ovulation. Pituitary FSH concentration decreased less dramatically than LH, and somewhat before LH, during the last few days before estrus. Increases in ovarian weight due to age appeared to be linear. Average numbers of small (1 5 mm diameter) and large (> 5 mm diameter) ovarian follicles paralleled each other throughout. They increased up to 4 months of age, declined after A months of age and became relatively constant after 6 months of age. The relationships between organ weight, total deoxyribonucleic acid (DNA), the Claude Desjardins total ribonucleic acid (RNA) and the total protein con- tents of the uterus, cervix and vagina were determined between birth and 12 months of age. Increases in these four parameters between 0 and 12 months were best de- scribed by quadratic growth response curves whereas the increases in these same parameters between 0 and 6 months of age were linear. The results were interpreted to mean that reproductive growth was accelerated after the onset of first estrus. Oviduct, uterine, cervical and vaginal epithelial cell heights were always greater at 0 months than at 1 month of age, probably due to maternal or placental hormones prior to parturition. Increases in weight, total DNA, total RNA and total protein of adrenal glands between 0 and 12 months of age were linear (P < 0.01). The width of the zona glomerulosa remained relatively constant to 12 months of age, but the combined width of the zona-reticularis-fasciculata in- creased linearly between 2 and 12 months of age. Thyroidal development was measured in these heifers but large vari— ations in thyroid weights prevented meaningful interpre- tation of these data. Thymus weight and total DNA in- creased six-fold between birth and 1 year of age, but the RNA/DNA ratio, did not change appreciably (P > 0.25). These data revealed a marked increase in the rate of growth of the reporductive organs beginning at the time of puberty--changes which coincided with and were probably caused by the release of pituitary LH and Claude Desjardins possibly FSH into the blood. The data suggested that puberty was precipitated by a sudden release of pitui— tary LH and that this precipitous LH release apparently occurred regularly on the day of estrus of each estrous cycle for all postpuberal heifers. ENDOCRINE AND REPRODUCTIVE DEVELOPMENT OF THE BOVINE FEMALE FROM BIRTH THROUGH PUBERTY By Claude Desjardins A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1966 To Jane Elizabeth DesJardins ii BIOGRAPHICAL SKETCH of Claude Desjardins Born: June 13, 1938, Fall River, Massachusetts Education: a. Durfee High School, Fall River, Massachusetts from 1952-1956 (graduated) b. University of Rhode Island, Kingston, Rhode Island from 1956-1960 (8.8.) c. Michigan State University, East Lansing, Michigan from 1960-1966 (M.S. and Ph.D.) Employment: Research Instructor at Michigan State University from 1960 to 1966 iii ACKNOWLEDGMENTS The author gratefully appreciates the assistance provided by his major professor, Dr. Harold Hafs, during the course of this research as well as his guidance in preparing this manuscript. The help and advice provided by Drs. Allen Tucker, Joseph Meites and Lon McGilliard was greatly appreciated. Special thanks are due the authors colleagues Mr. Ed Convey, Drs. Kenneth Kirton, Jock Macmillan, Max Paape, Yogi Sinha for their help with the various techni- cal aspects of this work. The devoted and untiring assis- tance of Mrs. Helga Hulkonen during all phases of this research deserves gargantuan praise. The gifts of hormones from the Ayrst Laboratories, the Upjohn Company and the Endocrinology Study Section of the National Institutes of Health as well as the financial support provided by the National Institutes of Health (grant number HD0137A) are recognized and appreci— ated. iv TABLE OF CONTENTS Page DEDICATION. . . . . . . . . . . . . . ii BIOGRAPHICAL SKETCH. . . . . . . . . . . iii ACKNOWLEDGMENTS . . . . . . . . . . . . iv LIST OF TABLES . . . . . . . . . . . . viii LIST OF FIGURES . . . . . . . . . . . . ix LIST OF APPENDICES . . . . . . . . . . . xi Chapter I. INTRODUCTION. . . . . . . . . . . 1 II. GENERAL LITERATURE REVIEW . . . . . . A Definitions of Puberty . . A Parameters Used for Assessing Puberty in the Female . . . . 5 General Endocrine and Reproductive Organ Changes Occurring During Puberty . . . . . . . . . . 6 III. MATERIALS AND GENERAL METHODS . . . . . 7 Experimental Animals and Slaughter 7 Feeding Procedure . . . . 8 IV. BODY GROWTH AND ESTROUS CYCLE OCCURRENCE 9 Review of Literature 9 Body Growth . . . . . . 9 Estrous Cycle Occurrence. . . . 12 Influence of Nutrition on Body Growth and the Occurrence of Puberty . . . . . . . . . l3 Chapter Hereditary Factors Influencing Body Growth and the Onset of Puberty Materials and Methods Body Growth. Estrous Cycles. Results Body Growth. Estrous Cycle Occurrence Discussion V. PITUITARY AND HYPOTHALAMIC FUNCTION. Review of Literature. Exterioceptive Factors and Pituitary Functions . . . . . Effect of Temperature Effect of Season . Pituitary Gonadotropins in the Pre-. and Postpuberal Animal Hypothalamic Neurohumors Influencing Puberty Materials and Methods The Pituitary The Hypothalamus Results Pituitary Gland Weights. . Levels of Pituitary FSH and LH Levels of Hypothalamic LH- RF Discussion VI. ENDOCRINE AND REPRODUCTIVE ORGAN GROWTH AND MORPHOLOGY. . . . . . Review of Literature. Growth and Morphology . . The Ovary . . . The Oviducts vi Page 1A 1A 1A l5 15 15 15 16 2O 2O 2O 22 23 2A 27 30 30 .1 32 32 39 Al A7 A7 A8 50 Chapter Page The Uterus . . . . . . 51 The Cervix and Vagina . . . . . 52 The Adrenal. . . . . . . . . 52 The Thyroid. . . . . . . . . 53 The Thymus . . . . . . . . . 53 Materials and Methods . . . . . . 5A Tissue Weights and Morphology. . . 5A Biocheical Analysis . . . . 55 Statistical Analysis. . . . . . 56 Results . . . . . . . . . . . 56 The Ovary and Oviduct . . . . . 57 The Uterus . . . . . . . . . 61 The Cervix . . . . . . . . . 65 The Vagina . . . . . . . . . 68 The Adrenal. . . . . . . . . 71 The Thyroid. . . . . . . . . 75 The Thymus . . . . . . . . . 75 Discussion . . . . . . . . . . 80 The Ovary and Oviduct . . . . . 80 The Uterus . . . . . . . . . 83 The Cervix . . . . . . . . . 85 The Vagina . . . . . . . . . 86 The Adrenal. . . . . . . . . 87 The Thyroid. . . . . . . . . 88 The Thymus . . . . . . . . . 88 VII. SUMMARY AND CONCLUSIONS. . . . . . . 00 Body Growth and Estrus . . . . . . 90 The Pituitary . . . . . . . 90 The Reproductive Organs. . . . . . 91 Adrenal, Thyroid and Thymus . . . . 93 BIBLIOGRAPHY. . . . . . . . . . . . . 9A APPENDICES . . . . . . . . . . . . . 108 vii LIST OF TABLES Table Page 1. Age at First Estrus, Stage of Estrus at Slaughter and Length of Consecutive Estrous Cycles . . . . . . . . . 18 2. Average Weight of Whole, Anterior and Posterior Pituitary and the Average Concentration of LH and FSH from Birth Through Puberty. . . . . . . . . 3A 3. Ovarian and Oviduct Development from Birth Through Puberty. O O O I O O O O 58 A. Uterine Development from Birth Through Puberty . . . . . . . . . . . 62 5. Cervical Development from Birth Through Puberty . . . . . . . . . . . 66 6. Vaginal Development from Birth Through PUber’ty c o o o o o o o o o 69 7. Adrenal Development from Birth Through Puberty . . . . . . . . . . 73 8. Thyroid Development from Birth Through Puberty . . . . . . . . . . 76 Thymus Development from Brith Through Puberty . . . . . . . . . . . 78 \0 viii LIST OF FIGURES Relationship Between Body Weight and Age Average Weights of the Posterior, Anterior and Whole Pituitary from Birth Through Puberty . . . . . . . . . . Average Concentrations (solid lines) of Pituitary LH and FSH (ug-equivalents of NIH-LH-B2 or NIH—FSH-S2 per mg of Fresh Anterior Pituitary) from Birth Through Puberty. Dotted Lines Refer to Average Concentrations of Hormones Ad- justed for Stage of Estrous Cycle. . Average Concentrations of Pituitary LH and FSH ug—equivalents of NIH-LH-BZ or NIH-FSH-S2 per mg of Fresh Anterior Pituitary) During the Estrous Cycle. Vertical Lines Refer to Standard Errors. Average LH/FSH Ratios from Birth Through Puberty Calculated from Data Adjusted for Stage of Estrous Cycle . . Average Ovarian Weights, Numbers of Small Follicles (i 5 mm diameter) and Numbers of Large follicles (> 5 mm diameter) per Heifer from Birth Through Puberty. Average Weights per Heifer, Lengths and Epithelial Cell Height (u) of the Ovi- duct from Birth Through Puberty Average Uterine Weights, Lengths, Epithelial Cell Heights, DNA and RNA/ DNA Ratios per Heifer from Birth Through Puberty. . . . . . ix Page 19 33 36 37 A0 59 60 63 Figure 9. Average Cervical Weights, Lengths, Epithelial Cell Heights, DNA, RNA/DNA Ratios per Heifer from Birth Through Puberty. 10. Average Vaginal Weights, Lengths, Epithelial Cell Heights, DNA, RNA/DNA Ratios per Heifer from Birth Through Puberty. 11. Average Adrenal Weights, Widths of Zona Glomerulosa, Combined Widths of Zona Reticularis-Fasciculata (R-F), DNA and RNA/DNA Ratios per Heifer from Birth Through Puberty. . . . . . . 12. Average Thyroid Weights, Acinor Cell Heights, DNA, RNA/DNA Ratios per Heifer from Birth Through Puberty. . . 13. Average Thymus Weights DNA, RNA and RNA/DNA Ratios per Heifer from Birth Through Puberty . . . . . . . Page 67 7A 77 79 Appendix Table 1. Figure 1. Figure 2. LIST OF APPENDICES Body Weight, Anterior Pituitary Weight and Pituitary Concentration of FSH and LH from Birth Through Puberty Photomicrographs. Photomicrographs. xi Page 109 113 115 CHAPTER I INTRODUCTION1 Puberty occupies a pivotal position among postnatal developmental events. It signifies the onset of repro- ductive activity leading to sexual and physical maturity. To the husbandman, the arrival of reproductive activity signifies an increase in an animal's economic potential based upon its ability to produce offspring. During the past decade, more intensive animal pro- duction practices have focused increased attention on aberrant reproductive functions such as infertility and sterility. Reproductive failures may be the major cause of loss of productivity among dairy cattle. Little is known of the direct causes of these reproductive failures, especially of those due to factors other than infectious diseases. However, a considerable body of evidence has accumulated to show that reproductive efficiency of the adult may be drastically affected by the prepuberal en- vironment. Therefore, accurate information on endocrine 1This thesis research was a portion of a longer study which included mammary growth parameters. The mammary parameters were measured by Y. N. Sinha and will be published under his name. and reproductive organ development before and during puberty may shed light on the normal function of these organs in the adult. Although the debut of puberty has been qualitatively recognized through certain behavioral manifestations in most laboratory and farm animals, the physiological changes occurring in the endocrine and reproductive organs during this time have not been extensively quantified; and, conse- quently, knowledge of controlling mechanisms is meager, especially in the bovine female. For example, the levels of pituitary gonadotropins during prepuberal development have never been described for the bovine. Similarly, quantitative biochemical studies on the growth and develop- ment of the other endocrines and reproductive tissues dur- ing puberty are not available for this species. The present study was initiated to quantify endocrine and reproductive organ development of normal heifers from birth through puberty. Levels of pituitary gonadotropins were measured because it was anticipated that changes in these might precipitate puberty. Reproductive organ development was quantified by measurement of DNA as an index of cell numbers, and by measurement of RNA, protein and lipid as indicies of cellular synthetic activity. These hormonal and biochemical parameters were then re- lated to each other and to morphological and micromor- phological measurements on each reproductive organ in an effort to uncover cause and effect relationships, particularly where rapid changes were observed at the time of puberty. Heifers were very good experimental animals for this research because they provided sufficient quantities of tissues to assess several different parameters on each tissue and, thereby, permitted assessment of functional activity on a within-animal basis. It was anticipated that this "uniformity" study would improve our understand- ing of the endocrine control of reproductive organ develop- ment, as well as providing bases for future research on puberty during specific stages of pre- and post—puberal development. CHAPTER II GENERAL LITERATURE REVIEW Definitions of Puberty Although there have been many attempts, an exacting definition of puberty has proven difficult. In the first place, the spontaneous onset of puberty and gradual tran— sition from puberty to sexual maturity permits no sharp demarcation of this stage of development. Marshall (1922) suggested that: Puberty, or the period at which the organism becomes sexually mature, is marked by the occurrence of those constitutional changes whereby the two sexes become fully differentiated. It is at this period that the secondary sexual characters first become conspicuous, and the essential organs of repro— duction undergo a great increase in size. Asdel (1965) provided a more practical definition when he suggested that: "Puberty is the time at which repro- duction first becomes possible, i.e., when germ cells are released." Hammond and Marshall (1952) considered an animal to reach puberty when "the sexual organs had be- come fully developed, the sexual instincts prominent and reproduction could be completed." Most recently, Donovan and van der Werff ten Bosch (1965) have reviewed several definitions of puberty especially where they may apply to primates and man. During the past years, most authors have tacitly agreed that the appearance of the first estrus was tantamount to the attainment of puberty. The latter definition will be used throughout this thesis. Parameters Used for Assessing Puberty in the Female Since this thesis pertains entirely to females, only those parameters used to study puberty in this gender were reviewed. The time of opening of the vagina has been shown to be highly correlated with the occurrence of the first ovulation and first estrus in rodents. For example, vaginal opening has been the accepted sign of puberty in the mouse (Allen, 1922), rat (Long and Evans, 1922) and guinea-pig (Stockard and Papanicolaou, 1917). Behavioral signs appear to be the best indicators of first estrus and the onset of puberty in most domesticated animals. Several manifestations of estrus in these species include increased vascularity and turgidity of the vulva, mucous discharge from the vulva, frequent bellowing, persistent trailing and attempted mounting of other animals. However, the most conclusive behavioral criterion of estrus in heifers appears to be standing when mounted by other animals in and out of estrus (Tanabe and Almquist, 1960). Current evidence suggests that there is a specific brain center regulating estrous behavior (Gorski, 1966). The majority of evidence presented so far supports the concept that this center is under the influence of the sex steroids and becomes fully mature at the time of puberty coincidental with the first maturation of an ovarian follicle and the first ovulation (Gorski, 1966). General Endocrine and Reproductive Organ Changes Occurring During Puberty In general, puberty has been associated with two types of changes taking place in the endocrine and repro- ductive organs. These are: (a) changes occurring in the physiological activity of the endocrine glands and (b) changes occurring in the reproductive and skeletal systems which are brought about by the secretions of the endocrine organs. These changes in the endocrine organs result in a general increase in physiological activity and cause several developmental events to occur which in turn are responsible for securing the most favorable conditions for reproduction. Some of these changes are specific and pre- dictive of a particular stage of puberty (i.e., mammary gland growth in the puberal heifer). Some others, however, may be incidental to a particular period of puberal develop- ment. For example, vaginal opening is apparently com- pletely coincidental with the time of the first ovulation in the rat. However, vaginal opening also occurs in rats castrated at infancy, though it is considerably delayed. Generally, puberty accelerates and completes develop- mental processes which in the absence of a period of rapid endocrine and reproductive organ development would require additional time to mature. CHAPTER III MATERIALS AND GENERAL METHODS Experimental Methods and Slaughter Two groups of 30 female Holstein calves were ob- tained from commercial dairy herds located in Dane and Greene Counties in Wisconsin. A11 calves were sired by registered bulls and born from production tested dams, selection criteria intended to provide a more homogeneous group of experimental animals. Calves in the first group were born between the 11th and 17th of November, 1963. These animals were transported (November 18, 1963) to and reared at the University farm until the time of slaughter. Five heifers were randomly chosen and slaughtered five at a time at 1—month intervals beginning when the animals were 7—months old. Calves in the second group were born between the 5th and 11th of May, 196A. These were transported (May 12, 196A) and reared similar to the calves in the first group. Five calves from the second group were randomly chosen and slaughtered five at a time at l-month intervals beginning when the ani- mals were l-month old. An additional group of five calves (born on the 13th to 15th of September, 196A) were purchased locally and slaughtered on the 16th of September, 196A to provide the animals here-after referred to as 0 months of age. The calves were housed in individual pens until they were about 3-months old. Subsequently, calves were reared for about 2—months in pens containing about 10 animals. From 5 months of age, the calves were managed communally in a dry-lot with access to an open shed. Animals were transported from the farm to the Univer- sity Meats Laboratory on the morning of slaughter. The five heifers within an age—group were always killed on the same day and by means of a captive bolt immediately followed by exsanguination. Feeding Procedure The calves received an average of 3 kg of whole milk per day during the first 3 weeks after arriving at the University farm. From the fourth through sixth weeks, the animals received an average of 5 kg of whole milk daily and from the seventh through sixteenth weeks they received an average of A kg daily. During the latter period the calves received a 16 per cent protein calf starter and water and good quality alfalfa hay supplied free choice. During the fourth and fifth months, the calves received an average of 2 kg of ground ear corn grain mix (1A per cent crude protein) along with alfalfa hay provided free choice. Corn silage or mixed hay was provided from the fourth month of age when it was available and in varying quantities. While the total nutrient intake was not accurately measured, the total intake was intended to provide nutrients sufficient to sustain normal growth for Holstein heifers. That this was achieved was demon- strated by the measured growth characteristics presented in the next chapter. CHAPTER IV BODY GROWTH AND ESTROUS CYCLE OCCURRENCE Review of Literature Body Growth The first estrus (puberty) in cattle usually oc- curred when about 30 per cent of the mature body weight was attained (Brody, 19A5). The greatest rate of somatic cell hypertrophy and hyperplasia occurred during prenatal and not during postnatal growth (Brody, 19A5), although it has been a popular contention that maximum growth oc- curred just before or during puberty (Tanner, 1962). Postnatal growth has been divided into two segments which Brody (19A5) identified as the "self accelerating phase" and the "self inhibiting phase." The point at which acceleration ceases and a deceleration has not yet begun represents a stage of physiological age equivalence. During this time, most animals seem to pass through puberty and about 30 per cent of the mature body weight has been attained. Thus the inflection point in a graph of body size occurred in female rats at about 30 days, coinciding with the onset of vaginal opening (Kleiber, 1961). In children, the inflection occurs between 12 and 15 years 10 ll of age, corresponding to their average age of puberty. For dairy cattle, the inflection occurs at 8-10 months concurring with the time of puberty in this species (Ensminger, 1959)- Additional evidence demonstrating the relationship between body growth and the onset of puberty was provided by Boas (1932) who concluded that when the maximal post- natal growth occurred early in the life of the human fe- male, the onset of puberty was early. This early intense growth rate was of short duration. In contrast, when the period of maximal postnatal growth was delayed, its in- tensity was slight but its duration was long. Boas con- cluded that in cases of precocious puberty, skeletal maturation was accelerated, while in hypogonadism, it was delayed. In general, animals of the smaller breeds within a species attain puberty at an earlier age than the larger breeds. For example, Hammond and Marshall (1952) noted that the small Polish rabbit attained puberty about 6 weeks earlier than the larger Belgian breed. Similarly, Jersey heifers showed signs of estrus about 13 weeks earlier than did the larger Holstein breed of dairy cattle (Eckles, 1915). Termination of skeletal growth usually coincided with the loss of growth potential of the long bones and has been attributed to calcification of the epiphyseal plates. Greulich (195A) and more recently Morscher, 12 g£_§l. (1955) gave evidence that closure of the epiphyseal plate of the phalanges coincided extremely well with the occurrence of the first menses in girls. Greulich (195A) even suggested that the skeletal status of the hand per- mitted the selection, some years before puberty, of children who would mature early and those in whom matur- ation was delayed. Unfortunately, there seem to be no data on the relationship between puberty and the calcifi— cation of the epiphyseal plates in domestic animals. Estrous Cycle Occurrence The age at first estrus has received little attention in the heifer. Sorensen eg_e1. (1959) indicated that 10 Holstein heifers exhibited their first estrus at A9 : 6.3 weeks of age. These same workers indicated that the oc- currence of first estrus could be advanced or delayed depending on the level of feeding employed. The data pre- sented by Sorensen et_§1. (1959) do not indicate whether or not ovulation was concurrent with the appearance of the first estrus. However, in laboratory species like the mouse (Allen, 1922), rat (Long and Evans, 1922) and the guinea pig (Stockard and Papanicolaou, 1917), the first estrus was usually followed immediately by the first ovulation. 13 Influence of Nutrition on Body Growth and the Occurrence of Puberty Accelerated growth due to increased energy intake was shown to advance puberty as determined by the age at first estrus (Sorensen e£_e1., 1959). Reid e£_e1. (196A) confirmed and extended the above report indicating that heifers on a low or high plane of nutrition had later or earlier puberal ages, respectively, than heifers on a normal plane of nutrition. In other studies on dairy heifers, Creichton e£_§l. (1959), Hanson (1956) and Joubert (195A) all noted that energy intakes less than that normally recommended delayed puberty in these species (McCance, 1960). Huseby e£_§1. (19A5) found that mice which had been reared from weaning on a restricted caloric intake had delayed sexual development. Kennedy and Mitra (1963) observed similar results in rats. The underlying cause for the delay in sexual development resulting from a reduced caloric intake after weaning was probably mediated through the endocrine system and has sometimes been referred to as pseudo-hypophysectomy (Mulinos and Pomerantz, 19AO). More recently, Kennedy (1966) advanced the theory that the ef- fects of low or high caloric intake on sexual maturity are under the control of hypothalamic neurohumors. He showed that in rats the maturation of the hypothalamus could be accelerated or decelerated depending on the plane of nutrition. 1A Hereditary Factors Influencing Body Growth and the Onset of Puberty Significant variation in the age at which an animal attains puberty has been attributed to hereditary factors. For example, Mirskaia and Crew (1930), studying mice, and King (1915a, 1915b) and Blunn (1939), studying rats, all found hereditary differences in the age at vaginal opening in different strains of animals in these species. Squiers e£_§1. (1952) obtained evidence that crossbred gilts reached puberty at an earlier age than inbred gilts. Foote e£_el. (1956) reported that puberal age of swine was nonadditively inherited and Warnick e£_§1. (1951) found differences in age at puberty among inbred lines of swine. These workers, as well as Robertson et_e1. (1951), all observed a negative association between age of puberty and body growth. Hawk et_e1. (195A) reported that inbreeding delayed puberty in a group of Holstein heifers. Each of these researches with mice, rats, swine and cows indicated hereditary factors can influence the growth rate of these animals and that when puberty was advanced, it was associ- ated with accelerated growth rates. Materials and Methods Body Growth Body weights were measured at l-month intervals be- ginning at 30 days of age. Slaughter weight was estimated 15 by obtaining the average of three weighings made at 2A— hour intervals just before slaughter. Estrous Cycles Animals were observed twice daily for estrus between 7:30-8:30 AM and between A:30-5:30 PM beginning when the heifers were 5 months of age. The criteria used to evaluate estrus were (1) standing when mounted by other animals, (2) repeated attempts to mount other animals (in or out of estrus), (3) swollen external genitalia, (A) copious vaginal mucous secretion, and (5) general restlessness. Results Body Growth Body growth was linearly related to age (P < 0.05) between 0 and 12 months, as evidenced by the data pre— sented in Figure and in Appendix Table 1. The data pre— sented in Figure 1 also indicated that the growth rates observed in the present experiment paralleled those re- ported by Morrison (1956) which have been generally ac- cepted as a standard describing the relationship between body weight and age. Estrous Cycle Occurrence The data concerning the age at first estrus and the length of consecutive estrous cycles are presented in Table 1. The age at first estrus for the 2A heifers 16 which had passed puberty ranged from 20.1 to AA.5 weeks with the average age at first estrus occurring at 29.7 i 1.3 weeks. The average length of 93 estrous cycles from the 22 animals which had completed at least one cycle was 20.5 t 0.6 days. In general, the data on the length of consecutive estrous cycles indicated that once estrus occurred (puberty), regular and consistent cycles were observed thereafter. Discussion The linear relationship between age and body weight demonstrated in the present experiment was in good agree— ment with the observations of previous researchers using animals of similar age and breed (Morrison, 1956; and Sorensen e£_e1., 1959). Therefore, it was assumed that the growth rate observed in the present experiment exerted the normally expected effects on the growth and development of the endocrine and reproductive systems. The data from the present experiment indicated that the age at first estrus was A to 6 weeks earlier than that recorded by other investigators (Sorensen e£_§l., 1959) for Holstein heifers. The Body weight data indicated that accelerated body growth was not the cause of the earlier age at first estrus. The discrepency between the age at first estrus reported here and that reported by Sorensen e§_el. (1959) may have been due to the differences in housing. Animals in the present study were raised 17 after 5-months in a loose housing system, whereas Sorensen's animals were stanchioned and allowed out for two 30-minute intervals each day. While objective data are not available, stanchioned heifers may be deprived of adequate opportunity to acquire the behavior pattern associated with estrus in this species and consequently may not fully exhibit the normal behavioral patterns associated with estrus. Once estrous cycles were begun, they appeared fairly regularly in all of the animals which attained puberty. However, there were a few cycle lengths of either ab— normally short or long duration. Some of the apparently long cycles may have involved a missed estrus of very short duration or abnormal estrus behavior. Although the duration of estrus was not specifically measured, inter— polation of the morning and afternoon estrous cycle ob- servations suggested an average estrus interval between 12 and 2A hours which appeared to be in agreement with previous reports reveiwed by Asdell (1965). The small standard error of estrous cycle length for the heifers in the present experiment was also in agreement with the value of 20.23 i 0.05 days presented by Asdell e£_el. (19A9) for unbred heifers. Estrous cycles for cows were of slightly longer duration but with similarly small variation (21.28 i 0.06 days). .Amozv wdppmm CH Dm>menO Lw>mzn .LopnwzmHm opoooh mappmo ummH wcHBOHHom when mo Lopezzm 18 w.ms.mm”emnmmuomnmm AH H.mm mm . . AH mm NH om ms eH m.mm mm AH.o.ms.ma.Hm.mH.mH 0H o.mm mm cm.sH.mH.eH m m.Hs Hm mNAHm.m .sm.eH. m.Hm.em HH _ 2.3m MH NH .n» ,da do .u d o «.3 «HMaWN.QOQH.F. DO O.N.m 3m CH dH co co Hm mo c 2 Or am AH.AH.Am.oa.eH.mm mm s.mm mm . .omHmHHmm mA m.mm e NH om Hm mm mm HH 0.0m w HH om.mH.CH mH m.mm mm - H 0.0: om CHAOH.CH.om.mH m :.em mm a a a a .ma H 3.3: m cm mH AH on 3H on e o.em m CH AH.mH a 0.0m am A... .1 men men om on CH 0H cm 0 mm OH . cm.mm SH A.mm NH AA AH.AH.Qm.cH.AH a m.eu m m . . wH.AH ow H.0m mm m at HA.m.Au m H.3m em mm, whflk OH .-. men m wcr on : m HA m.mm mm A m 0.0m mH Am.cu A e.mm m mum on mH mo: sue: HH 5 moHomo mzoemmm whopnmsmHm um oHomo wsepmm pmnHm Lopezz HoucwsmHm pm e0 Am>mpv cpmcoq msoepmm no mmmpm pm ow< no mxooz LomHo: mm< mo mnucoz noHomo msopumo o>Husoowcoo mo CumcmH pcm nopnmsmHm Hm manpwo go owmom .mzeumo meHu um om 0.25). Levels of Pituitary FSH and LH The average concentrations of pituitary FSH and LH from birth through puberty were summarized in Table 2 and 33 .zphooso nmsoesp nuHHh.EmHm.szbH5pHQ oaonz pew Hoahopcm .Hoaemumoo map Ho menmams owmho>¢tv m .wam m0< mo mrbzoz N. = O. m D h w m ¢ m N _ >¢<.CBE ¢O_CNP§N oOoooooooooocooooco.o .00 >¢¢HBUo w: mm pommmndxo mucouomx 00.H H 00.0 00.0 H 0e.0 00.0 H 03.0 00.0 H 03.H 00.0 H 00.H 0H 00.H H 00.0 50.0 H H0.0 00.0 H 00.0 HH.0 H 03.H 0H.0 H 00.H HH 00.H H 00.0 0H.0 H 30.0 00.0 H H0.0 00.0 H 03.H 00.0 H He.H 0H 03.0 H H0.0 00.0 H 00.0 00.0 H 30.0 00.0 H 03.H 00.0 H 0e.H 0 00.H H 00.e 00.0 H H0.H 00.0 H 00.0 0H.0 H H0.H HH.0 H e0.H 0 00.0 H 0H.0H 0H.0 H 00.0 00.0 H 0H.0 00.0 H 00.H 00.0 H 33.H e H0.H H 03.3 AH.0 H 00.0 00.0 H 00.0 30.0 H 00.H 00.0 H 33.H 0 H3.H H 00.0 00.0 H 30.0 00.0 H 00.0 00.0 H 00.H HH.0 H 00.H 0 00.H H 03.3 00.0 H 00.0 00.0 H 0H.0 HH.0 H 00.0 0H.0 H.00.H 3 00.0 H_00.0 0H.0 H 00.0 00.0 H 0H.0 30.0 H 00.0 00.0 H 00.H 0 05.0 H 00.0 00.0 H 00.H 00.0 H 0H.0 00.0 H 00.0 30.0 H 00.0 0 00.0 H e0.0 03.0 H 00.0 30.0 H 00.0 30.0 H 03.0 00.0 H H0.0 H 3H.0 H 33.0 00.0 H 00.H 00.0 H 00.0 00.0 H 33.0 00.0 H 00.0 0 In: uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu Amm H :mmevuul lllll I uuuuuuuuuuuuuuuuuuuu 0H mma 00Hh00000 soHpmpce mHogz Hmepcozv *CHQOHuopmcoo mw< HHMHHSHHm mo COHpmHucmocoo va mpanoz HHMHHsuHm emcee .mueohsd nwsoesp spHHh Eogm mmm new $4 mo :oHpmepcoozoo mwmeo>m on» new mHmHHSHHo HOHHoumoo use HOHHopcm AoHogz Ho pthos mwmho> 0.15). As discussed in the review of literature, the concen- tration Of pituitary LH is known to fluctuate with the stage of estrous cycle in some species. Heifers in the present experiment began regular estrous cycles at about 7 months of age and the stage of estrous cycle at the time of slaughter undoubtedly influenced the post-puberal values. Consequently, the pituitary FSH and LH concen- trations obtained from heifers which had been cycling previous to slaughter were studied in a separate analysis to determine pituitary levels of gonadotropin at various stages of the estrous cycle. The cylic pattern of pituitary gonadotropin was illustrated in Figure A. The smallest concentration of LH was observed immediately 36 toggling-gang...- 5 O .0... OOOOOOI LH CONCENTRAHON mubumqmm 5 I I n A l 4 5 s 7 3 9 IO II l2 MONTHSOFAGE D F O N 0‘ Fig. 3.—-Average concentrations (solid lines) of pituitary LH and FSH (ugwequivalents of NIH—LH—B2 or NIH—FSH—S2 per mg of fresh anterior pituitary) from birth through puberty. Dotted lines refer to average concentrations of hormones adjusted for stage of estrous cycle. 37 1 LOS p 8 FSH CONCENTRATION § (ITS' E LH CONCENTRATDN 5 hi (I «O 0' GD 3‘ (I 19 O-I 2--5 6-9 IO- - z-J DAYS FOLLOWING esmus Fig. A.-—Average concentrations of pituitary LH and FSH ug equivalents of NIH—LH-B2 or NIH—FSH-S2 per mg of fresh anterior pituitary) during the estrous cycle. Vertical lines refer to standard errors. 38 following ovulation. This was followed by a gradual accumulation of pituitary LH and finally a dramatic ele- vation in the final days before the time of the next ovulation. These cyclic changes represented an eight- fold increase in pituitary LH concentration from the beginning to the end of the estrous cycle. Apparently, about 80 or 90 per cent of pituitary LH was released from the pituitary during the day just before ovulation. No appreciable variation in pituitary FSH concentration was observable between day 0 and day 13 of the cycle (Figure A). However, the data suggested that pituitary levels of FSH may have declined during the last A or 5 days before the next ovulation. Because of these variations in pituitary FSH and LH concentrations due to stage of estrous cycle at the time of slaughter, the average concentrations of pituitary FSH and LH by age (Figure 3) were probably biased, at least after puberty. In an effort to correct this, the pituitary concentrations of FSH and LH of the cycling animals were adjusted to the average observed on the 11th day of the estrous cycle. Endocrinologically, prepuberal animals probably most closely resemble postpuberal ani- mals at this stage (diestrus) of the cycle. The graph of the adjusted pituitary FSH and LH concentrations (Figure 3--dotted line) indicated that the precipitous decrease in pituitary LH concentration beginning at puberty was not due to variations caused by the stage of 39 the estrous cycle. Rather, the data suggested that, while the prepuberal pituitary synthesizes LH, it does not re- lease appreciable quantities into the blood until puberty. The ratio of LH:FSH was determined for each animal using the data corrected for stage of the estrous cycle and these observations are plotted in Figure 5. The ratio LH:FSH increased rapidly until 3 months of age, was charac- terized by considerable variation from 3 to 7 months of age, and then decreased rather regularly to the lowest level at 12 months of age. The overall change in the ratio of LH:FSH from 3 to 8 months represented an eight-fold de- crease in this ratio. Due to the relative constancy of pituitary FSH during the cycle, the LH:FSH ratio paralleled cyclic vari- ations in the concentration of pituitary LH observed in the postpuberal heifers (Figure 3). The most striking feature of LH:FSH ratios during the estrous cycle was the sudden decline in the ratio just before the time of the next ovulation. Levels of Hypothalamic LH—RF Injection of extracts derived from the equivalent of 0.8 calf hypothalami failed to induce ovulation in the test rats. Larger quantities of the extract resulted in severe toxicity and death of the rats. Consequently, it was not possible to determine whether or not changes in the hypothalamic LH-RF content occurred during puberty. H.3— A0 N. .oaomo msonpmo mo ommpm How poemsnom memo EOHH oopmHSOHmo muhohso Lmzopnp LHHHQ Eomm mOHHmH mmm\mq owmgo> 5mm) follicles were noticed at 1 month of age. A marked increase in the number of both small and large follicles occurred between 1 and A months, and this was followed by a decrease between A and 8 months of age. Numbers of small and large follicles on the ovary were relatively constant from 8 to 12 months of age, the ages during which estrous cycles were observed in the present eXperiment. The average changes with age in oviduct weight, length and epithelial cell height are illustrated in Figure 7 and listed in Table 3. Average oviduct weight increased about 3g between birth and 12 months of age. Although the average oviduct weights for the 13 age groups .mm H 2002* 58 +I 0.0 3.00 0.H H 0.H0 0.0 H 0.0 3.0 0.3 0.0 H 3.0H 0H 0.0 H 0.00 3.0 H 0.H0 H.0 H 0.0 0.H 0.0 0.0 H 0.0H HH 3.0 H H.00 3.0 H 0.0H 0.0 H 0.0 3.0 0.0 0.0 H 3.0 0H 3.0 H H.H0 0.H H 0.00 e.0 H 0.0 0.0 0.0 0.H H 0.0 0 0.0 H 0.00 0.0 H 0.00 0.0 H e.0 0.H 0.0 0.H H H.e 0 0.0 H 0.00 0.0 H 0.0H 0.0 H 0.H 3.H 0.3H 0.0 H 0.0 N 0.0 H 0.00 0.0 H H.0H 0.0 H H.0 3.0 0.0 0.H H 0.0 0 0.0 H 0.00 0.0 H 0.0H 0.0 H 0.H 0.0 0.0H 0.H H H.e 0 0.0 H 3.H0 0.0 H 0.0H 0.0 H 0.H 3.0 3.00 0.0 H 0.0 3 0.0 H 0.00 0.0 H H.0H 0.0 H 0.H 0.H 3.HH 0.0 H 0.3 0 0.0 H 0.00 3.0 H H.3H H.0 H 0.H 0 0.0 3.0 H 0.0 0 0.0 H 0.0H 0.0 H 0.HH H.0 H 0.0 0.H 0.H H.0 H 0.H H 0.0 H 0.00 3.0 H N.HH H.0 H 0.0 0 0 *H.0 H 0.0 0 panmWAWHmo H500 A00 es 0 A as 0 w va HmHHmanam npwcmq DemHmz HgmHmz Hmepcoev poseH>o HospH>o poseH>o mmHOHHHom mo .02 new: 0Hm>o UOHHmm mm¢ .HHHODBQ hmsoehp cpHHh EOHH pcmquHo>oe HospH>o pcm :mHHm>OII.m mHm 0.25). Average oviduct length increased about 11 cm be- tween birth and 12 months, but the differences did not approach significance (P > 0.25). Despite this lack of significance, the data approximated linear growth of oviduct length at least to 8 months of age. Epithelial cell height measurements from the upper third of the oviduct revealed a marked decrease in epithelial cell height between 0 and 1 month, followed by rapid in- l’?-' crease between 1 and 2 months of age. No appreciable changes in this criterion were apparent after 2 months of age. The epithelium in this region of the oviduct was always ciliated even in the very young prepuberal animals. Although the number of cilia were not counted, inspection suggested that fewer cilia were observed in prepuberal animals than in puberal and postpuberal animals. Similarly, more secretion blebs were observed at the luminal extremities of these cells in postpuberal animals than in prepuberal animals. At about the average age of first estrus and thereafter morphological examination suggested the presence of ex- truded nuclei, which were evident in the photomicrographs, Appendix Figure l. The Uterus The data in Figure 8 and Table A indicated that the average uterine weight increased linearly between birth Lipid (g) in Total Prote V'A/ r110? Cell eight‘ (u) H Length (cm) 7.7 0.H 5.7 i 05* 5.u + Table U.-Uterine development from birth through puberty. C" Lf‘. C7 (‘u (7\ (D (I) Lfl r-‘I C) Ln r—I L) H r-1 0 (\J W KB LO \0 co (\1 +I +I +I +1 +I +I +I +I +I +I +I F~ 3 C“ C‘ O‘ (I) m 0 Ln m (I) :7 C‘ r—4 (\. [\ 0\ if OJ CI,“ (*3 O‘. (“J r—1 H :1 M Li" :1 0.: (" m {I (‘0 0‘ (”J :3 N O\ {7‘ C5 C“ C C‘ O C- r-4 H C) O C“) +I +I +I +l +I +I +| +l +I +I +I \ F C F‘ KC — T Q ‘0 K ‘ H m " ‘ ‘ ‘ “'V‘ 5 , L. K: t - L-‘ r“ \o r-‘I H 9—4 ( r' J . r a m ~ C“ r r—I M (j . . ‘ (~ \ r: ("T '0": \f? r—I C) .. C I‘ k.“ K ‘ (_‘ Ca \A 7.? \A C' F O L +I +I +1 +1 +l +I +4 -+I +1 +l +I «.1 g E I I I r“. . “ o ‘ h) (‘x l}. -, r» ("l (n C"- "W D , » .‘- ‘i (V “- r—4 F". (u. 0" CD -7 (V) I \7 ‘ T T C C‘ r-4 ‘: C: T , o , I L 7, fl rx :0 C‘ L N a“ I“; u o I o o o o a o o o |. " "I ‘I t' f - {V '\ " ; I" :3 {‘x T: r 4 .- IAJ L; H +I +l +l +l +I +l +I +I +l +| +I T T ' ‘ F 1 (T f‘» C; I O I ‘ ‘ r‘ W» 1: w w 0 C‘: (T m ‘ C t ' {T (T ‘7‘ 0 H I\ (T) H (‘1 r—I r—I ,: ‘ . m r“ L." 0: " (7“ (D \O O I O D O O . I O . I I C‘I q " 7’21 (‘3 C; CI} r—‘I H C‘.‘ m :1 r' 4 (“\J r-~I H (“I") u“. L."\ (X; (1) :3 +l +1 +l +I +I +l +4 -+I +l +I +l Q‘ “.1 #44 0" C\ L\— m \0 \C m KO v. - "‘ (N- \: ’\. r—I {N p. n :7 N .71,» H4 -. “ (fl [\ (I " r—1 N C P" Lfl r 4 r—4 H H (‘J (“I (V) :3 W‘- ‘. ’VW L. \D (‘J CD :I \3 L1” -3 \o C“ CW (W L- "W C“ C“ O C? O O +l +I +I +I +I +I +I +I +l +I +I o. 34 ‘ “ a”. H CO m C‘ m O \Q [\ r4 XLT P: C) ~17 :7 N CT\ m H r 4 r1 H H (‘J r4 (\1 (\J (\J m \0 up LL \ N r-4 CO H) (\J O\ 1‘ O\ C) O 3 O r—i C‘ (:7 H O O N +I +I +| +I +I +| +I +I +I +I 4" KO N (\J' (I) if (\I [\ m :7 m r—I r4 r4 H H H r4 H (‘0 (\J OJ +Endometrial epithelium cells. oo r4 r4 ON 0 m ox ow [- b- \o m “-4 on m H O H :: t\ \o oo '—4 H r—1 . +| +l +l +l +I +l +I +l +| +I +I L1] U] ‘~O =.7\ C“ (\J O \D O 3 r-I H (I) O I O O O I O O I I O + | no om m H Ln 3 oo oo co N O\ 04 c (\J :r (\1 Ln Ln {\ m m :r C H H H (U (D E u: 12 RNA/DNA 3 2| cainemwtu) UTERINE CHARACTERISTICS I» .a LENGflIkmfl -N no I20 mom I.) 8 IS 8 63 l’.--‘"—'--'/ H,’~~—--->¥ MONTHS OW'AGE Fig. 8.--Average uterine weights, lengths, epithelial cell heights, DNA (mg), and RNA/DNA ratios per heifer from birth through puberty. FFT‘TWT-‘infm. ... 'u. 6A and 6 months of age (P < 0.01), but uterine weight increased Inore rapidly after puberty and consequentry uterine growth from 0 to 12 months was best described by a quadratic curve (P < 0.01). Thus, increases in uterine weight were great- est between 6 and 12 months of age. Uterine length in- creased linearly from birth to 12 months of age (P < 0.01), ‘but the rate of increase was greatest after puberty. A significant linear increase in total uterine DNA, 3 IRNA and protein occurred during the first 6 months of life i (P < 0.01), but subsequent increases in these biochemical components were more rapid with the result that these para— xneters were best described by a quadratic response (P < 0.01). The ratio of uterine RNA/DNA did not change significantly from birth to 12 months of age (P > 0.25). Total uterine RNA and paralleled total uterine DNA during all phases of uterine growth. Total uterine lipids varied considerably within the various ages and consequently, the averages for the age groups were not significantly different (P > 0.25). .Micromorphological investigation of the endometrial cell heights indicated a significant quadratic growth curve (P < 0.10). Epithelial cell heights decreased between 0 Eind 6 months of age, then generally increased to a maximum fleight of 33v at 12 months of age. Both the superficial zand basal uterine glands were absent until A or 5 months (bf age but became well developed and lined with tall czolumnar epithelial cells by 6 or 7 months of age. These Changes were evident in the photomicrographs (Appendix Figure 2). 65 The Cervix The averages of some of the criteria used to deter- mine cervical growth were graphed (Figure 9) and the average results of all cervical measurements were sum— marized (Table 5). Cervical weight increased linearly between birth and 6 months of age (P < 0.01), but increases in weight were more rapid after puberty and consequently increases in weight between 0 and 12 months of age were quadratic (P < 0.01). Consequently, the greatest changes in cervical weight took place during puberal and postpuberal develop— ment. Cervical length also increased with increasing age suggesting a linear trend; however, these data were vari- able and the age groups did not differ significantly (P > 0.25). Additional data concerning cervical growth between birth and 12 months of age were the total DNA, RNA and protein contents of the cervix. Analysis of these three biochemical criteria revealed that these criteria of cervical growth were similar to cervical weight--linear between birth and 6 months of age but quadratic from 0 to 12 months of age. Analysis of the RNA/DNA ratio data presented in Figure 9 suggested that this ratio increased at about 8 months of age. This increase in the RNA/DNA ratio at this time paralleled similar increases in cervical weight, total DNA, total RNA and total protein as well as TABLE 5.-—Cervica1 development from birth through puberty. r—I RKA/ +- {le *4 J I") V 2.U 3.5 i 0.3* O + I (1‘) ‘) #4 i‘\ i) r) . a i. .- 9»: + i) 87 + 1.0 . I J +I \0 PI 12.1 1 2.3 0.7 1v; . f) 6.1 + 1.2 3 0.3 10.2 + 3.14 i 0.2 11.3 i 0.11 (‘J N +l (V1 +I (\J (\I +I (13 \i‘, ;T _ 0.3 0.5 12.0 + “.2 18.5 + 2.3 22.2 + 1.8 8 :I (W O +I (n \O 31.14 i 6.7 9 10 .1 A 0.8M + 0.07 (‘J + I 214.8 + 0.6 214.1 + 0.7 + 0.3 3.9 0.70 95.3 i 12.8 81. 23.3 + 0.6 11.9 i 0.3 39.0 + 2.5 113.6 + 5.8 11 2.5 17.9 + ’1 0.7 0.75 t 0.01: 5.5 5.7 .6+ 60 8.9 + I 11.9 i 0.2 _ l2 *Mean + SE. +Epithelium. CERVICAL CHARACTERISTICS LENGTH (cm) 033 \ / \ / \ \ 1* RNA I DNA 23° '2“ / \ \ \ l ( \ ( a: a» 5 00 DNA (mo) 0 .5 O N O UHJ.HE£HTHO 33 5 ’5 \“ {335 ”9'0! ob: VI .5 (I J 0.. o. . N N N“ I I I i I WEIGHT (9) m on A o o o O A 1 l L l n I 1 5 6 7 MONTHS OF AGE Fig. 9.--Average cervical weights, lengths, epithelial cell heights, DNA, RNA/DNA ratios per heifer from birth through puberty. 68 epithelial cell height, but differences in RNA/DNA among the age groups were not significant (P > 0.25). Results of the lipid analyses of cervical tissue from the 13 different age groups were extremely variable (P > 0.25); and, consequently, this was not a useful parameter for evaluating cervical growth. Cervical epithelial cell heights decreased immedi- ately after birth, suggesting some hormonal stimulation of these cells during gestation. Between birth and 6 months of age, the height of the epithelial cells changed only slightly. After 6 months of age, the height of these cells increased at least 50 per cent to reach a peak at 10 months of age. Cervical epithelial cells were pseudostratified throughout this study. Secretion blebs derived from the cervical epithelium were especially conspicuous during puberal and postpuberal development indicating an increased activity of these cells at this time. In contrast, almost no secretory blebs were observed during prepuberal develop— ment. The Vagina The averages of all parameters used to determine vaginal growth from birth through puberty were summarized in Table 6. Some of these results were graphed in Figure 10 to facilitate interpretation of the data. The relation- ships between age and vaginal weight, total DNA, total RNA and total protein were all linear (P < 0.01) up to — an... a IuA. -A(\“'d§' .- . It“. E 6.-Vaginal development from birth through puberty. TABL L“ 3 w k . .ot rv‘ nr-I Q) A J») M O\ A f) y: deight (s) v 0 Age (months _ 0.7 11.9 + 1.0 11.3 + rm J 3.0 + I \0 (VW + I ON C) (1) ur.7 : 7.5 ML) ('1 + I (1. Ln _ 0.7 10.2 + 8 7".) <1. _ 6.1 78.A + 7 16.2 11.6 + 0.7 _ 9.5 84.3 + (I) (\J + I 3.2 122.3 + 11.5 o“ E. J I 17.8 + 0.5 10.2 t 0.9 11.A + 0.“ 7.8 102.5 A 3. 1" 151.2 + 2U.2 280.1 + 83.0 + 300.2 20.0 + 0.5 25.7 + 0.9 190.3 1 72.6 10 0.06 + 0.95 .2 + 10.2 _ 158.3 + 19.3 10.6 i 0.6 1U.1 112.5 + 11 t 13.2 U6.0 0.99 3 0.05 10.7 i 0.9 , 0 lb 15.3 + 1u9.5 25.A + 1.1 10.2 i 1.0 _ 8.0 115.3 + 12 *Mean : SE. r...- ' M‘IJ “In-11.4 Int .. \1~ VAGINAL CHARACTERISTICS CELL HEIGHT (HI RNA/DNA LENGniIum WEIGHT (9) ao—”---"’ ,— ,— ISO ~ 60 20E L 1 1‘. O l 2 3 4 5 6 7 8 9 K) H MONTHS OF AGE h Fig. 10.-—Average vaginal weights, lengths, epithelial cell heights, DNA, RNA/DNA ratios per heifer from birth through puberty. I 1"} 70?? |mn lax-fl.)- m'nn :1 I...‘ . .l 71 6 months of age but quadratic between 0 and 12 months of age, indicating that vaginal growth was greatest after 6 months of age. Vaginal lipid content, as measured here, varied considerably between age groups (P > 0.25) and consequently, was not a satisfactory parameter for evalu- ating vaginal growth. The average height of the vaginal epithelial cells decreased between birth and 1 month of age, remained relatively low until about 3 months of age, and then began gradually to increase to a maximum at 12 months of age. If; in“ unflnmzxrnnzlia 5' An unusually high peak in vaginal weight and DNA occurred at 10 months of age. The observations in this age group were obtained from two animals in mid-cycle and three animals that were close to estrus (: 2.5 days). The latter three may have influenced the average in- ordinately if these criteria varied with stage of estrous cycle. In general, measures of vaginal growth were more variable than similar estimates made on other reproductive tissues. One reason for this was undoubtedly the diffi- culty encountered in repeatably defining the limits of vaginal tissue at the time of autopsy. This difficulty may have inflated the variability in measuring the growth of the cervix. The Adrenal The averages for the various parameters used to evaluate adrenal development from birth through puberty 72 were summarized in Table 7 and some of these growth measure- ments were graphed in Figure 11. Total adrenal weight increased linearly (P < 0.01) between birth and 12 months of age. As expected, the in- creases in total adrenal DNA and total RNA paralleled the increase in adrenal weight and resulted in linear growth through 12 months of age (P < 0.01). Therefore, the adrenal did not grow in the same way as the reproductive tissues. The constancy in the RNA/DNA ratio between birth and 12 months suggested that the cells of the prepuberal adrenal were equally capable of synthesizing protein as those cells in the postpuberal animal. The width of the zona glomerulosa remained rela- tively constant from birth through 12 months of age (P > 0.25). In contrast, the combined width of the zona reticularis-fasciculata decreased between birth and 1 month of age, increased markedly at 2 months of age, re- mained relatively constant between 2 and 9 months of age, and appeared to increase gradually after 9 months of age. The changes in total adrenal lipid between the different age groups were not significantly different (P > 0.25). Consequently, this biochemical component was not useful in evaluating changes in adrenal growth. Al- though the changes in total adrenal protein due to age were significant (P < 0.01), the changes with advancing age did not fit any of the patterns established for the reproductive tissues or other endocrines. .mm H c002. '73 H 0.0 H 0.0 30.0 H 03.0 0.0 H 0.00 0.0 H 0.00 0.H H 0.HOH 0.0 H 0.00 0.H H 0.3H 0H H m.0 H m.3 00.0 H 03.0 0.H H 0.00 0.0 H 3.00 H.0 H 0.0HH 0.0 H 0.0H 3.0 H 0.0H HH H 0.0 H 0.3 30.0 H 03.0 0.m H m.00 0.3 H 0.H0 m.0 H 0.3HH 0.0 H 0.0H 0.0 H 0.0H 0H H 0.0 H 0.m 00.0 H 30.0 0.0 H 0.00 0.0 H 0.0 0.0 H 0.30H 3.0 H 0.0H 0.H H 0.0 0 H .0 H H.3 H0.0 H 03.0 0.0 H 0.30 3.3 H 3.H0 H.0 H 0.30H 0.0 H H.0H 0.0 H 3.0 0 H .0 H 3.0 00.0 H 03.0 0.H H H.H0 0.0 H 0.03 A.H H 0.00 0.0 H 0.0H 0.0 H H.0 0 H .0 H 0.0 30.0 H H3.H 0.H H 0.0H 0.H H 0.00 0.H H 0.m0H 0.0 H 0.3H 0.0 H 0.0 0 0 H .0 H 0.m 00.3 H 03.0 m.m H 0.0H 0.0 H 3.00 0.H H m.00H 0.0 H 0.3H 3.0 H 0.0 0 H 0 .0 H 0.0 00.0 H 00.0 0.H H 0.0H 3.0 H 0.03 0.H H 0.HOH 0.0 H 3.0H 0.0 H 0.0 3 H H .0 H 3.0 00.0 H 03.3 0.H H 0.0H 3.H H H.00 0.0 H 3.00 m.0 H H.0H. 0.0 H 0.3 m H H .0 H 0.H 30.. H 03.0 0.0 H 0.0H H.0 H 3.00 m.H H 0.00H 0.0 H H.0H 0.0 H 0.3 0 .H .0 H 0.0 03.1 H 03.0 H.H H 0.0 0.H H 0.00 H.0 H 3.00 m.0 H 0.0H 0.0 H 0.m H H 0.0 H H.0 30.0 H 03.0 3.H H 0.0H H.0 H 0.00 m.m H 0.00 3.0 H 0.00 00.0 H 0.0 0 100 100 A000 1000 H30 130 100 10000000 vHuHH :Hmpcxd «:3 arm «in mpmHSOHommm amonpmsoHo H00 0 H0000 \000 H0000 H0000 -memHsoHpmm 0:00 chN pcmHmz 000 0o 000H3 nmcHnsoo 0o :00H3 .mppmnsa nwzopcu :HLHQ Eopm acmEQon>mo Hmcmpp 0.25). Thyroid weight, total thyroid DNA and total thyroid RNA varied considerably from birth to 6 months of age, but only slightly from 6 to 12 months of age. The differences in the RNA/DNA ratio due to age were not significant (P > 0.20). Total thyroid RNA paralleled total thyroid DNA from birth to 12 months of age. The changes in total protein due to age were significant (P < 0.01). Thyroid acinar cell heights decreased at least 50 per cent between birth and 1 month of age. The height of these epithelial cells remained relatively constant be— tween 1 and 5 months of age. Between 5 and 12 months of age, the height of the cells gradually increased, reaching maximal heights at 11 and 12 months of age. The Thymus The average weight, total DNA, total RNA, RNA/DNA ratio, total protein and total lipid content of the thymus gland from birth through 1 year of age was summarized in Table 9, and some of these parameters were illustrated in 'Figure 13. 4 o v I .4 \ 0. N - \r'AJ‘ ‘1'2'v' :‘v‘; 1:15 -fl"l"313 rn. 11‘. _-.—. r"I T1 (TI ~c-1' r‘\ +4 [.2 hi] (Jar-1 \--’ Pfl Ll F. ('1 ~ 1 :0 '11"\ I‘ 1‘ HQ) (7) On— >—« i. "\ b-‘c I I (. I .‘1 >1 \ | I w- (G (77‘ L. ..\ w ".2 ‘11“ U : I I“. r”I r‘I Q) 7,) I) J»: :4 11‘; \ O «I .1 i} 1) ‘v ’(‘I 7:: F.) - C 10‘ .17 > u ‘0’.) 12 d: O 0. if '0 + 0.7 O (I; (‘-| L\‘ L;’"\ m H r' DNAIII a. o o c Q ~ I f .0 To \' \ \ i5 0 6 CELL HEIGHT (III 0 O I 2 13 4» 5 6 '7 8 ED IO U MONTHS OF AGE Fig. l2.--Average thyroid weights, acinar cell heights, DNA and RNA/DNA ratio per heifer from birth through puberty. I.-. E ca L—l ASL 'I" A. 78 (“'W 00 ('7. L(\ ‘~C‘ (*3 r1 ‘3 (\J m (\J “I . I _ I (“‘7 i‘ — Ii" '1‘ LIW C (D U") (‘1 r I r‘I LT (\ ("FI +I +I +I ‘-I +I +I +I +| +I +I +I +I .3 a. I ~. (“-1 v--I t~~~ C‘.‘ C“ r’I m if) I 1 '.' I'. !‘. 1'. ”‘7 C". \{I r-I (*1 ON (7“. 1 J .1 I o 1 a. f- 1,”. 0+ .- I [~— I“: on 1,, (VI ,... r-J. r~-I I‘I H r ,3 I, , :‘1 In If» g: r: "r :7 H 0;) KO I - ‘ u. ‘- I :7 .3 (V1 0"» :r m O r' I r-". r-I +I +I +I +I +l +I +I +I +I +I +I +I 'TVI (' ., f» (7‘. (7) 1'“ LA; 0 1 \C H ;f\ H rI . : - [I ‘ b- ( . I." :3 (1') Ch r, . I ' ' -I " f“ :3 ‘1) L: Q I ' I") f 04 (" :T (J r4 ‘ S‘ D f _‘ C“ F“ (D (’3 I: O I' I‘“ I 3 I I 3 \I‘ (I) I— \L.‘ (‘\- I“ L\- '...W 1‘» I _ -f (‘fi (7‘. o u . n o . o o a a o o (7“ H > - - : * r -7 ' .;\ r I {1‘ :3 F0 ' 1‘ 7 ~. .2 (‘1 -..' 5‘ \u ’30 F" \F“. v“ I r1 I" I r I +I 4| +I +| +I +I +I +I +I +I +I "I'I . I VI “x .V“ I‘ (‘4' ‘43 CD . , . -~ ~ , I "I e r-I \3 f: (\1 1 .1 -7 CL‘ ‘ r I \O C‘\ Li“ r4 O . ‘ ‘ ~ -* .. , f 3 no Q. a: r: c:- 0‘ A a r-I r—I r-«I I E I‘ I 3 \ K ' . \ (\I \C O‘ O I C‘ r-I r *1 r-‘I r~1 r—I H (D "(I (‘3 +I +I +I +I +I +I +I +I +I +I +I + <7 . r I H1 0. I \I (\1 r -I {‘0 704 (I) ‘O 03 (‘1‘ C-I I 2 CT CU (\J P". .’\J I-I r'I C.“ m H r-I r-I r‘I r—‘I r—I r'"I * :3 \\ -»\ r4 :3- f\ z N -:T 3 CO 'C . O 0 C O O . I l . . D m 1 . HT 47 4 K.) H r—I H m H C'\ v‘ i (‘0‘ \3 CV 0’1 L.’\ (1'1 (‘1 O N H +I +I +I +I +I +I +I +I +I +I +I +I II \ I‘ - ‘ {‘0 .0 03 O 0.] Ln CI) m ox u ‘ D I”) v ’ (\J C) (\— Q LI'\ KO I“ (\J ’ ) ~ ) ’\ r‘\ m «T :\, L.‘\ (\I \0 LI\ 0\ 7“ 7-4 m m cm (*1 m (*1 Ln (*1 C) H (0 PM :1 Ln KO N d) O'\ C) H I—I r—I ' "-“"""‘__H'I u 7.!"2‘1 -0! 123?} m 0 -er 590.6 1 91.3 *Mean + SE. 12 THYMUS CHARACTERISTICS DNA (a) RNA IONA :3 9 8 8 § RNA(.¢ o ca 79 _ / r- I- ’I ,. ” o“‘~ ’ b ”’ ‘~~ / I ‘ I b I ‘V’ ’---’ I I- ” I I L I --~I’ q I- '0'... .J O . . 0 o 0 o 0 .° 0 o r- : “.‘o. o..oo°oo........ .0 .0 '00000000 P .0 O ..... O Fig. RNA/DNA ratios per heifer from birth through puberty. I l l L A MONTH 8 OF AGE l3.--Average thymus weights DNA, RNA and ' r t - . TIT? n... u-l‘fi_w I 80 A five—fold increase in thymus weight was observed between birth and 12 months of age. The increases in thymus weight, total DNA and total RNA also lipid and protein due toage were significant (P < 0.01); however, no attempt was made to try to describe the growth response curve because it was too complex. The changes in the RNA/ 3*: DNA ratio due to age were not significant (P > 0.35). A close parallel existed between total thymus DNA and total thymus RNA during the entire experiment. "\ r R. .23! 0' Villh.‘ .‘v‘ A ‘_’J Discussion —w The Ovary and Oviduct Ovarian weight increased from birth to 12 months of age, and the data appeared to suggest a linear increase in weight with respect to age, but the data were too variable to confirm this trend statistically. Although absolute ovarian weight increased with age, ovarian weight per kilogram of body weight increased from 13.6 mg per kg to 37.6 mg per kg from O to 12 months of age, respectively. No drastic changes in ovarian weight were observed at the time of first estrus. This observation was not in accord with that reported by Sorensen g£_a1. (1959) who observed a marked change in ovarian weight at the time of estrus. However, these workers used groups of animals that differed in age at the time of puberty by 112 days, whereas the animals used in the present experiment differed only by 30 days, and this difference may account for the failure 81 to observe marked increases in ovarian weight at the time of puberty in the present data. The increases in ovarian weight that occurred dur- ing the first 7 months were chiefly influenced by the number and size of follicles. In contrast, corpora lutea largely contributed to increases in ovarian weight after 7 months of age. Although no histological observations were performed 3 on the ovarian tissues, direct gross morphological follicle counts indicated a peak in the number of small follicles occurred at 4 months of age, and this peak was followed by a gradual decline to normal numbers of small follicles at about 8 months of age. Although Sorensen gt_al. (1959) did not report direct follicle counts, these researchers noted a very large number of atretic follicles in the ovaries of prepuberal heifers. Consequently, their data suggested that the eventual fate of the small prepuberal follicle is atresia. Additional support for this hypothesis was provided by Rajakoski (1960) who noted that the fate of the majority of small follicles in normal cycling adult cows was also atresia. Unfortunately, at present no data are available to indicate whether or not the small and large prepuberal follicles are capable of producing steroids. The evidence provided by Roberts and Warren (1964) suggested that the fetal bovine ovary was capable of certain steroidal transformations. If ovarian steroids 82 are produced, it would be attractive to advance the theory that steroids from the ovary influenced the prepuberal growth and maturation of the endocrine and reproductive systems. The results reported on oviduct length in the pre- sent study were in agreement with those reported by Sorensen gt_gl. (1959). The majority of the increase in oviduct weight and length occurred after 6 months of age, illus— trating that the rapid phase of growth of this organ oc— curred during puberal and postpuberal development. Gross inspection of the oviduct suggested that the majority of the increase in the weight of the oviduct was due to in- creased thickness of the muscle and connective tissue of the lamina propria. Epithelial cell heights of the oviduct varied only slightly during the various stages of the estrous cycle of the postpuberal heifers, but a precipitous decrease in epithelial cell height occurred between 0 and 1 month of age. These observations suggested that the epithelium of the oviduct of the neonatal calf may be under the influence of maternal and/or placental hormones at birth but came under full influence of endogenous hormones within 2 months. The presence of extruded nuclei in puberal and postpuberal animals made in this study confirmed the report of Sorensen et al. (1959). These workers 83 suggested that these nuclei were derived from the luminal epithelial cells of the oviduct because their size, shape and tinctorial properties were similar to those observed in normal positions within the cell. Recently, Asdell (1965) suggested that "extruded nuclei: were globules of cytoplasm containing centers which stained intensely with hematoxylin. These bodies appeared to be secreted protein which were inbibing fluid at the periphery and, thus, stained less intensely in that region than at the center. The exact nature of these bodies remains an enigma. The Uterus The several parameters used to characterize develop- ment of the uterus between birth and 12 months of age paralleled each other. In general, uterine length and weight, as well as total DNA, total lipid, and total protein contents indicated that uterine growth was relatively slow and linear during the first 6 months of life. However, these same measures indicated that uterine growth was more rapid during the puberal and postpuberal phases. During this time uterine growth was best described by a quadratic curve. The marked acceleration in uterine growth, which began at puberty, was probably due to higher levels of pituitary and gonadal hormones associated with the onset of puberty. The greatest changes in uterine weight, DNA and protein occurred between 6 and 7 months of age, indicating that the stimulus for uterine growth was greatest just before the onset of first estrus. 84 An indication of the total protein synthetic activity of the uterus was provided by total uterine RNA. Since total uterine RNA paralleled total uterine DNA very closely, it was concluded that uterine protein synthetic activity was considerably elevated just prior to the on- set of first estrus. That the RNA/DNA ratio remained relatively constant throughout the different phases of uterine growth suggested that the cells of the prepuberal uterus were just as capable of synthesizing protein as those cells present in the postpuberal uterus. Thus, these data were interpreted to mean that the protein synthetic activity per cell remained constant in the pre- and postpuberal animal. It is proposed that the relation- ship between total uterine DNA and age represents the normal growth curve of the bovine uterus between birth and 12 months of age. Uterine weight, total uterine protein, and total uterine RNA described similar curves, and these criteria of response were no more variable than total uterine DNA. Although a linear increase in total uterine DNA and uterine protein was observed between birth and 6 months of age, the height of the uterine endometrial epithelium declined during this same period. In con- trast, uterine epithelial cell height paralleled uterine growth between 7 and 12 months of age. Because of the known influence of ovarian steroid hormones on uterine tissues, these data suggested that the steroidal output 85 of the prepuberal bovine ovary was small relative to its output during postpuberal uterine development. This con- clusion, based upon the data on epithelial cell heights, was supported by the absence of superficial and basal endometrial glands in prepuberal heifers. The invagi- nations into the endometrium which were lined by epithelial ipn”' cells and comprised the endometrial glands became fully , apparent after the onset of puberty. The Cervix t The estimates of cervical growth provided by measur- ing changes in cervical weight, total DNA, total RNA and total protein revealed increases which paralleled each other between birth and 12 months of age.. In addition, graphs of these different measurements with age indicated that growth was linear between 0 and 6 months of age and that a significant increase in growth occurred after this time resulting in the quadratic response between 0 and 12 months of age. That the RNA/DNA ratio did not differ be— tween age groups suggested that the protein synthetic activity per cell was similar in all age groups. Since it is known that the epithelial cell height was responsive to estrogen, the findings on the heights of the cervical epithelial cells suggested that steroidal stimulation provided by the ovary during postpuberal development was greater than that provided during pre- puberal development. These results agree well with those 86 provided by Roark and Herman (1950) for mature cows in various physiological states. Thus, it is proposed that the growth estimates reported here present the normal growth curve of the bovine cervix from birth through puberty. In addition, these data on cervical development appeared to parallel g”, closely similar data presented above for uterine growth from birth through puberty. The Vagina The measures used to evaluate vaginal growth between .; birth and 12 months were in general agreement with each other. Estimates of total DNA, total RNA and total protein all suggested that vaginal growth was linear between birth and 6 months of age and quadratic between 0 and 12 months of age. Although the vaginal RNA/DNA ratio did not differ (P > 0.25) between age groups, trends in these data were evident which suggested two growth waves. An almost identical response in epithelial cell heights coincided with the changes in the RNA/DNA ratio. Unfortunately, any explanation of these growth waves does not conform to the previous patterns described for the development of other reproductive tissues. Vaginal epithelial cell height, like that of the other reproductive tissues previously described, decreased between birth and 1 month of age lending further support 87 to the hypothesis that the reproductive tissues of the new-born calf may be under the influence of maternal or placental hormones. Once removed from this hormonal stimulus, the epithelial cell height decreased to levels which were presumably more indicative of the endocrine capacity of the neonatal calf. } f The Adrenal Adrenal growth between birth and 12 months of age was linear (P < 0.10), as measured by adrenal weight, 8 total adrenal DNA and total adrenal RNA and was in marked ‘3, contrast to the quadratic response reported above for the reproductive tissues. These data suggested that the con- trol of adrenal gland growth during the first year of life may be quite different from that of reproductive tissues during this same time. The width of the zona glomerulosa did not appear to be influenced by the onset of puberty. In contrast, the combined width of the zona reticularis—fasciculata apparently increased markedly after puberty. This ob— servation suggested an increased function in either the zona reticularis or the zona fasciculata after the time of puberty. Because the zona fasciculata has been shown to be associated with sex steroid production (Turner, 1966), it would be attractive to suggest that postpuberal growth of this tissue represented increases in function after the time of puberty. However, the staining procedures 88 used did not allow differentiation of the zona reticularis from the zona fasciculata and consequently the increase in the width of the combined zones could also be attributed only to the zona reticularis which is thought to be re- sponsible for glucocorticoid production. The Thyroid Few changes were observed in thyroid growth between 7 and 12 months of age when growth was assessed by thyroid weight or total thyroidal DNA and RNA. In contrast, marked F variance in these growth parameters were observed between v 0 and 6 months of age. The large variation associated with thyroid development during the first 6 months after birth may have in part been caused by an iodine deficient diet for these heifers. This suggestion was provoked by the fact that thyroid acinar cell heights were uniformly lower during the first 6 months of life than during the last 6 months of life. Unfortunately, there exist many exceptions to this histologic finding and this evidence alone was not sufficient to establish the functional state of the gland between birth and 6 months of age. The iodine content of the ration was not measured. The Thymus According to observations made on rats and humans (Turner, 1966), the thymus gland attained its greatest size at the time of puberty and regressed thereafter. In contrast to humans and rats, the bovine thymus gland 89 weight, total DNA and total RNA each continued to increase after puberty to 12 months of age. These data indicated that the bovine thymus gland does not involute at the appearance of the first estrus, an age which is the ac— cepted sign of puberty in this species. Whether or not the thymus involutes in the adult bovine could not be determined from these data because the present study continued only about 5 months beyond puberty. CHAPTER VII SUMMARY AND CONCLUSIONS The normal growth and development of the endocrine and reproductive systems of Holstein heifers was studied in 13 groups of five animals which were slaughtered at monthly intervals between 0 and 12 months of age in an effort to detect and quantify some of the changes occur- ring in these systems at the time of puberty. Body Growth and Estrus Body growth, as determined by body weight, con- formed to that listed by Morrison (1956). Age at first estrus was 29.7 f 1.3 weeks, and the average length of 93 estrous cycles was 20.5 i 0.6 days. These estrous cycle data agreed with those provided by Asdell (1965), and suggested that the animals used in this study were overtly normal. The Pituitary The most important observation on the pituitary relative to puberty was a progressive decline in LH concentration beginning at the time of puberty. Differ- ences in pituitary FSH concentration due to age were 90 ,-. 91 only significant (P < 0.05) during the first 3 months of age. Thereafter, pituitary FSH remained relatively con- stant although a small decrease was noted at puberty. These changes in pituitary gonadotropin suggested that puberty was initiated by a sudden release of LH and possibly a smaller release of FSH into the blood. Assays of peripheral plasma for LH and FSH are needed for con- firmation of this hypothesis. Also, experiments designed to discern the location and function of hypothalamic cyclic and tonic LH release centers appear warranted to determine the relative function of these centers in prepuberal and postpuberal animals. In addition, experiments should be designed to determine whether or not some ovarian steroidal substance may be responsible for blocking the release of pituitary gonadotropins in the prepuberal heifer. The Reproductive Organs The growth of both large and small ovarian follicles appeared to lag about 1 month behind the marked decreases in pituitary FSH which occurred at 2 and 3 months of age. After A months of age, the number of large and small follicles decreased and subsequently were relatively constant. This observation appeared to coincide with the relatively constant level of pituitary FSH during this period of growth. These observations on the prepuberal ovary, particularly at 3 and A months of age and there- after, suggest that it may possess some steroidogenic 92 capacity and that experimental comparison of steroido- genesis in prepuberal ovaries with that in postpuberal ovaries may contribute to our knowledge of any role of prepuberal ovaries in inhibiting puberty. Growth (as measured by weight and DNA) and function (as measured by protein and RNA) of the uterus, cervix and vagina slowly increased in a linear manner until E A puberty. However, each of these criteria was accelerated E beginning at puberty, presumably because of the influence of elevated levels of ovarian steroids. Assays of peripheral blood plasma for steroids could confirm this hypothesis. RNA/DNA ratios for each of the reproductive tissues remained relatively constant throughout the period of growth studied in this thesis. Consequently, the accelerated puberal growth (weight and DNA) and total function (RNA and protein) was largely attributable to hyperplasia rather than to hypertrophy. In general, the height of the oviduct, uterine, cervical and vaginal epithelia appeared to parallel each other from birth to 12 months of age. Epithelial cell heights of each of these tissues decreased between 0 and 1 month of age, suggesting that the maternal environ— ment may have exerted a stimulating effect. The height of the endometrial epithelial cells of the uterus de- creased to 50 per cent of their size at birth by 6 months of age, but the birth height was restored by 12 months of age. These data were interpreted to mean that the 93 stimulatory steroid environment of the reproductive organs was minimal Just before puberty. Adrenal, Thyroid, and Thymus In contrast, to the quadratic growth observed for reproductive tissues, the adrenal grew linearly (P < 0.01) as determined by increases in its weight, total DNA, total F““‘ RNA and total protein. Changes in thyroid growth from birth to 7 months of age were difficult to evaluate be- cause of large variations in thyroid weights observed O -‘.(.l!.‘ .J . J.) . between these ages. However, after 7 months of age thyroid weight, acinar cell height, total DNA, total RNA all in- creased, and these increases appeared to parallel each other. Thymus growth was not retarded by the onset of puberty in the present experiment. Rather a five-fold increase was observed in thymus weight, total thymus DNA and total thymus RNA between 6 and 12 months of age. The present results must be extended beyond 12 months of age to determine whether or not the bovine thymus regresses with advancing age beyond 12 months of age. The present data suggested that the thymus appeared to have no primary relationship with the onset of puberty. . _ _ fn— . . v. . I“; e‘ LMH‘ .Hlnhvl“ i.§)a1ww BIBLIOGRAPHY 91$ BIBLIOGRAPHY Allen, E. 1922. The oestrous cycle in the mouse. Am. J. Anat., 39:297. Altman, P. L. and Dittmer, D. S. 1962. Growth., Federation of Am. Soc. for Exp. Biol., Washington, D. C., p. 1A5. _ n I t5 ‘IF’ r .1 Anderson, R. R. and McShan, w. H. 1966. 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Record, 61:213. 107 Zuckerman, S. 1962. The Ovary. Press, New York, N. Y. Vols. 1 and 2. Academic tun—- APPENDICES p anterior eight, 1.-—Body w TABLE APPENDI‘ v(‘ I L ¢ i\ >1 {1 Y W4 Li w P LU S. 1;. 12 ‘l'. C) £3 (1 O L.‘ C ) 7 IT: +l . 1 n‘.‘ (\ (I) {1 KW C1. v'i y . {I ' ('1 ' ‘ ., 4 0—3 ‘ m ; i. x" 1,: T. (a L.) I i‘ ‘ .‘TZ + I (‘1 (I. “‘3 f) 1) 1:) 1;. .0. 7‘) L L 0 ~15 P 'V“ J—‘ ,L: i. ~r4 {)0 (1x :5 ~74 #3 $3 1‘ K. *4 7'?- -1: IL Cd 1.) >> 1‘: v m 0 W1 .11 .1» ’3 Animal mber \ X‘IU ()4 z‘\ U) LL< U i Gr Age (mont , 6 1 x 0.60 Q (*1 ("\J by 73 7a I“ 1_ 37. 39.3 LIN 109 r—{ (‘3 Ki‘v ‘3 ’J 36.7 + 1. 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D o W. .n.’ A“: r x I I v .C I ( _ I - \I a I J o L! _ ' o I o .. o ..- ~10\.v1 rd H a I a .1 H n «on NC I a x o «In- I .\\l m I an 5 2 _ + .. . 3o r 3 My. Hw l. I II I\ o 1 o A ufi ucI .~.JI\/V I‘ ”I r n.” C I + T WI r p... -«xr wxr (H 113 APPENDIX FIGURE 1 Photomicrographs (X330) of oviducts from 0 (upper left), 1 (upper right), 5 (lower left) and 12 (lower right) month old heifers showing changes in the luminal epithelial cell develop- ment from birth through 12 months of age. After puberty, the mucosa was thrown into a greater number of secondary and tertiary folds. Ciliated epithelia were present in all age groups. Large numbers of extruded nuclei were observed after the onset of puberty (see lower right). 115 APPENDIX FIGURE 2 Photomicrographs (x330) of uteri from 0 (upper left), 1 (upper right), 5 (lower left), and 12 (lower right) month old heifers showing endometrial changes from birth through 12 months of age. Uterine glands were absent in prepuberal ani- mals; a few developing uterine glands could be observed at 5 months of age and well developed uterine glands were noted by 12 months of age. A large uterine gland was sectioned (lower right) through the wall of the gland neck and through the lumen of the basal portion of the gland. "311113111311“