ABSTRACT THE NORMAL.MAMMARY GLAND DEVELOPMENT OF RATS 10 TO 100 DAYS OF AGE by Yagya Nand Sinha Normal development of the rat mammary gland between 10 and 100 days of age was studied using mammary area and mammary deoxyribonucleic acid (DNA) content as indices of growth. Mammary area was measured from whole- mounts of the right abdominal mammary gland with a polar compensating planimeter. DNA was extracted by a modified Schmidt and Thannhauser procedure. Ribonucleic acid (RNA) and lipid content of the gland were estimated simultaneously. The mammary gland area increased from.9.26 mm2 at 10 days to h50.93 mm? at 100 days of age. This increase was allometric relative to either the body surface area (0( =3.00) or body weight (0( =1.95) of the animal. The total DNA content of the mammary fat pad, containing the six abdominal-inguinal mammary glands increased from 0.33 mg at 10 days to 3.52 mg at 100 days of age. This increase was isometric (a( =l.0h) relative to body weight but allometric (a( =l.56) relative to body surface area of the animal. No change in the slopes of log DNA plotted against either log (body weight) or log (body weight)2/3 was observed. This in- dicated a constant growth rate of the mammary fat pad between 10 and 100 days of age. The DNA/100 g body weight increased from 1.53 mg at 10 days to 1.70 mg at 100 days of age. The slope of the linear regression curve was 0.001 which suggested that DNA/100 g body weight remained constant between by Yagya Nand Sinha 10 and 100 days of age. The correlation of total mammary fat pad (TMEP)-DNA with mammary area was 0.82 (P< 0.01). The total RNA content of the TMEP increased from 0.27 mg at 10 days to 2.56 mg at 100 days of age and these increases paralleled those of total DNA. The RNA/DNA ratio tended to decline as age advanced suggesting that protein synthesis per mammary fat pad cell was retarded with advancing age. The total lipid content of TMFP increased from 21A.9 mg at 10 days to 2,163.1 mg at 100 days of age. The growth of the mammary gland appeared to be associated with a disappearance of the adipose tissue. The total DNA content of the specific mammary area (SMA), containing the left and right abdominal mammary glands increased from 0.06 mg at 10 days to 0.53 mg at 35 days of age. This increase was 1.26 times faster than body weight increases and 1.89 times faster than body surface area increases. No change in the slopes of log DNA plotted against either log (body weight) or log (body weight)2/3 was observed which suggested that mammary gland growth, based on DNA, occurred at a constant rate between 10 and 35 days of age. The DNA/100 g body weight of SMA increased from 0.28 mg at 10 days to 0.h9 mg at 35 days of age and followed a pattern similar to the total DNA. The correlation of SMA-DNA with mammary area was 0.77 (P<:0.01). The total RNA content of the SMA increased from 0.05 mg at 10 days to 0.38 mg at 35 days of age and these increases paralleled those of total DNA. by Yagya Nand Sinha The RNA/DNA ratio did not reveal any significant trend among the age groups, implying that the rate of protein synthesis remained almost constant between 10 and 35 days of age. Dedicated to the memory of my mother Late Rajeshwari Sinha THE NORMAL MAMMARY GLAND DEVELOPEMENT OF RATS 10 TO 100 DAYS OF AGE By Yagya Nand Sinha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 196M : QC? 419 J S/a‘a/Cnt BIOGRAPHICAL SKETCH Yagya Nand Sinha was born at Rohua, Muzaffarpur, Bihar, India on October 21, 1936. He received his high school education at Muzaffarpur Zilla School and passed the Matriculation Examination in February, 1952. In July, 1952, he entered Langat Singh College, Muzaffarpur and passed the Intermediate Examination in Science in February, 195A. The same year he entered Bihar Veterinary College, Patna and graduated in March, 1957. He worked for Bihar State, India, as a Veterinary Assistant Surgeon at Nathnagar from.Apri1, 1957 to March, 1958; as a Veterinary Assistant Surgeon in charge of the Artificial Insemination Center at Surajgarha from April, 1958 to September, 1959; and as a research assistant in Biological Products at the Livestock Research Station, Patna from October, 1959 to September, 1961. He joined the graduate school of Michigan State University in September, 1961. He received the Nbster of Science degree in the Department of Dairy in June, 196A. His primary area of interest is reproductive and mammary physiology. ii ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. H. Allen Tucker for his untiring interest, inspiration, and guidance during the course of this study and preparation of the manuscript. Acknowledgement is made to Dr. Charles A. Lassiter and Dr. Harold D. Hafs for their help during the course of this study. The assistance of Claude Desjardins in the preparation and study of the vaginal smears of the rats is greatly appreciated. The author is grateful for the financial support provided by his father, and by the Department of Dairy, Michigan State University. The author wishes to express his sincere thanks to his wife, Savitri, for her encouragement, tolerance, and sacrifices without which this study would not have been possible. iii TABLE OF CONTENTS BIOGRAPHICAL SKETCH . . . . . . . . . ACKNOWLEDGEMENTS . . . . . . . ... . LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . m RODUCII' ION . O O O O O O . O O O 0 REVIEW OF LITERATURE . . . . . . . . Normal Development of the Mammary Gland. Embryonic and fetal development . . From birth to puberty. . . . . . . . . . Following puberty and before pregnancy . During pregnancy . . . . . During lactation . . . . . During involution . . . . Ribonucleic Acid Content of the Mammary Gland . Lipid Content of the Mammary Gland . . . . . . . Techniques of Assessing Mammary Development. . . Whole-mount preparations . Thick histological sections . mammography . . . . . . . Total mammary gland area . . . Analysis of degree of arborescence . . . Total surface area of the secretory epithelium Volume of glandular tissue . . . Relative growth analysis . iv 0 lo 0 lo . ll Deoxyribonucleic acid content . . Alkaline phosphatase and iron content MATERIALS AND METHODS Animals Mammary gland area experiment . . Total mammary fat pad experiment Specific mammary area experiment Mammary Gland Area Measurement Nucleic Acid Analyses . . . . . Lipid Analysis . . . . . . . . Relative Growth Analyses Statistical.Analyses . . RESULTS AND DISCUSSION . Area of Mammary Glands . Nucleic Acid Content of Mammary Glands Total mammary fat pad . . Specific mammary area . . . . . . SUMMARY . . . BIBLIOGRAPHY . Page 13 13 in 11+ 15 15 l5 15 16 18 19 20 21 21 26 26 36 1+3 1+6 LIST OF TABLES Page Mammary gland area of rats 10 to 100 days of age. . . . . . .. 22 Mammary gland area of rats in various stages of the estrous cycle. . . . . . . . . . . . . . . . . . . . . . . . 22 Nucleic acid content of the total mammary fat pad of rats 10 to loo days Of age 0 O I O O O O I I O O O C O O O O O O O 28 DNA content of the total mammary fat pad of rats in various stages of the estrous cycle . . . . . . . . . . . . . 31 Lipid content of the total mammary fat pad of rats 10 to 100 days of age . . . . . . . . . . . . . . . . . . . . 31 Nucleic acid content of the specific mammary area of rats 10 to 35 days of age. . . . . . . . . . . . . . . . . . 37 vi LIST OF FIGURES Mammary gland area of rats 10 to 100 days of age . . . . . Relation between mammary gland areazmmi(body weight)2/3 of rats 10 to 100 days of age . . . . . . . . . . . . . . . Relation between mammary gland area and body weight of rats 10 to 100 days of age . . . . . . . . . . . . Nucleic acid content of the total mammary fat pad of rats Relation between DNA of the total mammary fat pad and body weight of rats 10 to 100 days of age . . . . . . . total mammary fat pad and (body weight)2 3 of rats 10 to 100 days of age . . . . . Figure l 2 3 A 10 to 100 days of age . . 5 6 Relation between NA of the 7 Nucleic acid content of the rats 10 to 35 days of age 8 Relation between DNA of the body weight of rats 10 to 9 Relation betweeB/DNA of the (body weight) specific mammary area of specific mammary area and 35 days of age . . . . . specific mammary area and vii 3 of rats 10 to 35 days of age . . . . . Page .23 25 27 29 33 3h 39 A1 A2 INTRODUCTION In the past, many investigators have used rather subjective techni- ques to measure mammary gland growth. Recently, however, measurements of the mammary gland area or deoxyribonucleic acid (DNA) content have been used as quantitative indices of mammary gland growth. Mammary area measurements have been restricted primarily to studies of mammary growth before the onset of pregnancy, whereas DNA has been used to study mammary gland development under normal and experimental conditions of pregnancy, lactation, and involution. However, no systematic attempt has been made to study normal mammary gland development, using DNA as an index of growth, in young animals before the onset of pregnancy. Furthermore, mammary DNA and mammary area measurements have never been compared. Experiments, therefore, were designed to determine the DNA content and the area of the mammary glands of rats between 10 and 100 days of age. Ribonucleic acid (RNA) and lipid content were estimated during this period of mammary gland growth to determine the changes in the protein synthetic activity and the role of the fat pad, respectively. -1- REVIEW OF LITERATURE Normal Development of the Mammary Gland Turner (1939), Folley (1952), Reece (1958), Mayer and Klein (1961), and Cowie and Folley (1961) have reviewed the growth of the mammary gland during different stages of sexual development. Embryonic and fetal development. The embryonic and fetal develop- ment of the mammary gland of a large number of mammals, including man, was studied in the latter half of the nineteenth century by such workers as Huss (1873), Gegenbaur (1876), Rein (1881), and Klaatsch (188A). Later, Brouha (1905) studying bats and man, Lustig (1916) studying man, Myers (1917) studying rats, Gisler (1922) studying cats, Uehlinger (1922) studying horses, Turner (1930, 1931) studying cattle, and Turner and Gomez (1933a) studying mice, guinea pigs (1933b), and goats (1936) carried out extensive investigations of embryonic mammary development. In general, these studies indicated that the course of gland develop- ment was quite similar among species. The more important developmental stages included the formation of the mammary band or streak by a thicken- ing of the ectoderm on either side of the ventral mid-line. The mammary streak was then transformed into the mammary line. Mammary buds appeared along this line and these buds determined the number of glands that eventually developed. The mammary bud invaginates from its proximal end to form the primary sprouts. The primary sprouts then were canalized. Secondary and tertiary sprouts proliferated from the proximal end of the primary sprouts. Henneberg (1900) observed in the albino rat the appearance of the mammary streak in eleven-day embryos. In the albino mouse, Turner and Gomez (1933a) observed that the appearance of the mammary streak occurred at 10 days, the mammary line at 12 days, -2- -3- the mammary bud at 1A days, and the secondary sprouts at 20 days of age. Continued growth of these sprouts formed the duct system of the mammary gland. The extent of sprout development and the amount of canalization of the fetal mammary gland depended upon the length of the intra-uterine period of the fetus (Reece, 1958). From birth togpuberty. Puberty is defined as that age when the animal commences to release ova or becomes capable of bearing young. In primates, the accepted sign of puberty is the first menstruation, in rats, it is the vaginal introitus (Everett, 1961) and concurrent starting of the estrous cycle which occurs about the 35th to A2nd day of age. Great variations, however, in the onset of puberty are found according to breed, environment, and individuals within species. At birth, the mammary gland is rudimentary and consists of a restricted duct system. From birth to puberty, the type of mammary gland growth appears to be species-specific. In the rabbit (Ancel and Bouin, 1911), cat (Turner and DeMoss, 193A), and dog (Turner and Gomez, 193A) the mammary gland retained its infantile characteristics exhibit- ing only a small degree of ductular growth between birth and puberty. 0n the other hand, in species such as the rat (Myers, 1917), exten- sive growth of the duct system has been observed during the hth and 5th weeks after birth although this observation was interpreted to be of doubtful significance. Similar growth patterns have been observed for the guinea pig (Turner and Gomez, 1933b), mouse (Gibson, 1930; Turner and Gomez, 1933a; Cole, 1933; Weiser, 193M), calf (Hammond, 1927), and rhesus monkey (Folley, Guthkelch, and Zuckerman, 1939). Relative mammary growth analysis in monkeys (Folley at al., 1939), -h- rats (Cowie, 19A93 Silver, 1953), and mice (Flux, 195A) revealed that the mammary duct growth during early life, as measured by mammary gland area, increased at the same rate as the body surface area increased (isometry). However, the mammary gland growth shifted in advance of puberty to a rate significantly greater than body surface area increases (allometry). In rats, this allometric growth occurred about the 23rd day of age. The mammary DNA content of rats increased in a linear manner between 21 and A2 days of age (Tucker, 1962). Following_puberty and before prggnancy. During this period, mammary gland development is influenced by the type and number of estrous cycles experienced by the Species in question. Myers (1917) using rats and Gardner and Strong (1935) using mice observed large increases in duct development about the 10th week of age. Very little further development was observed after this period. Sutter (1921) observed a relationship in rats between the stage of the estrous cycle and appearance of the mammary gland. During estrus there was sprouting of the ducts as well as formation of new duct buds. Mammary regression occurred at metestrus although successive cycles exerted a cumulative effect on the gland growth. In the mouse, Bradbury (1932), Turner and Gomez (1933a), Cole (1933), and Wieser (193A) Observed similar changes with the estrous cycle stage. During estrus, there was marked elongation of the end-buds and distension of the ducts with fluids, followed by regressive changes of the gland during metestrus. In animals which have long estrous cycles (15-30 days), where one would expect some growth of the alveoli, very little, if any, alveolar proliferation was observed. Hammond (1927) did not mention finding -5- alveoli in virgin heifers. Turner and Gomez (1936) found extensive arborization of the ducts in goats, yet no true alveoli. Turner and Gomez (1933b) observed many duct buds but no formation of alveoli in nulliparous guinea pigs. Speert (1948) has described the cyclic changes in the mammary gland of rhesus monkeys with respect to the menstrual cycle, but no similar studies have been conducted in humans (Reece, 1958). In species such as the rabbit, which are in constant estrus, in- dicating an indefinite follicular phase, marked development of the duct system with terminal enlargements occurred (Ancel and Bouin, 1911). However, in the ferret, Hammond and.Nbrshall (1930) observed no signi- ficant development of the duct system. During pregnancy. In primiparous animals, the extent and type of growth that occurs is influenced by the number of sexual cycles prior to conception (Reece, 1958). However, the pattern of growth is strikingly similar in most species. Lane-Claypon and Starling (1906), Ancel and Bouin (1911), Schil (1912), and Hammond and Marshall (191%) observed that growth of the duct system in primiparous rabbits continued through- out early gestation and this growth was followed by a rapid proliferation of the lobule-alveolar system which was completed by mid-pregnancy. In other species such as the rat (Turner and Schultze, 1931), mouse (Turner and Gomez, 1933a), guinea pig (Turner and Gomez, 1933b), cow (Hammond, 1927); and goat (Turner and Gomez, 1936) similar observations were re- corded. on the other hand, Cole (1933) and Jeffers (1935) studying mice and rats, respectively, found mammary gland hyperplasia to occur throughout pregnancy. Similarly, Reece and Warbritton (1953), using the colchicine -6- technique, observed mitotic activity throughout gestation in the rat, the maximum rate of mitosis occurring on the 10th day. DNA analyses have further confirmed this observation. Griffith and Turner (1959; 1961a) found that 20.9% of the total mammary growth in rats occurred during the first 10 days of pregnancy and 59.3% occurred by the 20th day of pregnancy. Development was 98.3% complete by the third day of lacta- tion. Similarly, Tucker and Reece (1963a) Observed consecutive increases in mammary DNA throughout pregnancy, representing a total 261% increase over virgin controls. In mice, Brookreson and Turner (1959) found that 30.6% of the total growth occurred during the first 12 days of pregnancy, A5.7% occurred during the last one-half of pregnancy, and 21.9% additional growth occurred between parturition and day 14 of lactation. In the rabbit, Denamur (1961) observed a rapid increase of mammary DNA during pregnancy. In multiparous guinea pigs, Nelson, Heytler, and Ciaccio (1962), however, found only a slight increase in the DNA content during pregnancy. During lactation. Loeb and Hesselberg (1917) and Kuramitsu and Loeb (1921) found that mitotic activity ceased as soon as intense lactation commenced in guinea pig mammary glands. Cole (1933) detected no mitoses seven days after parturition in the mouse. Altman (19A5) using cows, and Maeder (1922) and Weatherford (1929) using rats, rarely found mitoses during lactation, whereas Jeffers (1935) occasionally observed mitoses. Reece and Warbritton (1953) observed mitoses in less than 1% of the secretory cells on the fifth day of lactation in the rat. 0n the basis of these observations, Reece (1958) concluded that the increases in milk production during lactation were largely due to an increase in the -7- secretory rate rather than further growth of the mammary gland. Recent DNA studies presented a somewhat different picture of lactational mammary growth. Greenbaum and Slater (1957) using rats found that the DNA-P content doubled between the end of pregnancy and the third day of lactation. Griffith and Turner (1959; 1961a) found that mammary hyperplasia was only 59.3% complete by the end of pregnancy, whereas mammary development by the third day of lactation was 98.3% complete. Tucker and Reece (1963b) using hooded Norway rats found that the total DNA/100 g of body weight increased 9.8% between the 20th day of pregnancy and the first day of lactation and increased 25.9% between the first and fourth day of lactation. Maximum.mammary development occurred on the 12th day of lactation and 2A.8% of the total mammary growth occurred during lactation. In other species such as the mouse (Lewin, 1957; Brookreson and Turner, 1959), rabbit (Denamur, 1961), and guinea pig (Nelson.§t_al., 1962) a similar increase in DNA content during early lactation was observed. After the peak of lactation has been reached, the amount of milk secreted gradually decreases. Histological studies suggested a decline in the secretory activity of the mammary parenchyma. Lenfers (1907), Kuramitsu and loeb (1921), Hesselberg and Loeb (1937), Cole (1933), Jeffers (1935), and Blackburn and Macadam (l95h) reported a gradual loss of secretory tissue during lactation. The recent DNA studies of Smith and Richterich (1958), Brookreson and Turner (1959), Moon (1960), Turner (1960), Denamur (1961), and Nelson _e_t_ 3;. (1962), in agreement with the histological studies, suggested a reduction in the number of cells with advancing lactation -8- but Tucker and Reece (1963b) observed no significant decrease in mammary DNA until after the 2Ath day of lactation when adequate suckling stimulus was provided. During involution. The involutionary changes that occurred in the mammary gland have been studied using histologic techniques (Myers and Myers, 1921; Kuramitsu and Loeb, 19215 Maeder, 1922; Turner and Gomez, 1933a; Cole, 1933; Fekete, 1938; Hooker and Williams, 19A0; Williams, 19A2; Hesselberg and Loeb, 1937; Turner and Reineke, 1936; Reece and Warbritton, 1953). The mode of regression of the mammary glands in most species was quite similar (Turner, 1939). Following weaning, the gland became engorged with milk causing a rise in intramammary pressure. This engorgement persisted for 2A to A8 hours, then gradually decreased upon resorption of the milk. The alveoli collapsed, the epithelial cells lining the alveoli disintegrated, and the ldbules gradually dis- appeared. The main difference among species was the length of time re- quired for complete involution of the lobule-alveolar system to a duct system (Turner, 1939). Complete involution was observed 13 days after weaning in the mouse (Cole, 1933; Williams, 19A2), 9 days in the rat (Maeder, 1922), 35 days in the guinea pig, and 75 days in the goat (Turner and Reineke, 1936). Recently, biochemical changes occurring in the mammary tissue during involution have been studied. DNA estimations have generally agreed with the histologically observed loss of cells phenomenon (Kirkham and Turner, 1953; Turner, 1960 ,- Griffith and Turner, 1961b). Slater (1962), on the other hand, observed an increase in the mammary DNA content during the first A8 hours after weaning and Tucker and -9- Reece (1963c), observed a slight but non-significant increase in DNA between the 2lst day of lactation and the first day after weaning in the mammary gland of the rat. DNA decreased significantly only three days after weaning. McNaught (19563 1957) suggested that on the basis of a decrease in oxygen uptake, respiratory quotient, glucose uptake, and an increase in lactic acid production, the involutionary changes could be seen as early as 8 to 12 hours after weaning. Ribonucleic Acid Content of the Mammary Gland The RNA content of tissues is considered to be intimately related to the biosynthesis of proteins by the tissues. However, rapid syn- thesis of proteins in the mammary gland tissue probably does not occur until the onset of pregnancy. The RNA content of the rat mammary gland before pregnancy has received little study. Kirkham and Turner (1953) reported that the total RNA and RNA/DNA ratios were low in undeveloped glands but increased gradually from pregnancy through lactation, reaching a peak at the end of lactation. Tucker (1962) observed that the RNA content of the specific area of the right abdominal gland increased from 0.1A mg at 21 days of age to 0.3A mg at A2 days of age. The RNA/DNA ratio, however, did not change signifi- cantly. Lipid Content of the Mammary Gland Studies on the lipid content of the mammary gland during various phases of sexual cycle are very scant. Harkness and Harkness (1956) determined the lipid content of the mammary glands of the albino rat during pregnancy, lactation, and involution. The lipid content diminished during lactation suggesting that the growth of the mammary -10- gland was associated with local disappearance of adipose tissue. Bitman.§t_al, (1963a) determined that the lipid content of normal sheep mammary glands accounted for 27% of the total mammary gland weight in young animals, whereas in mature animals the lipid content accounted for about 12% of the gland weight. Techniques of Assessing Mammary Development Whole-mount preparation. The classical whole-mount preparation of the mammary gland was first introduced by Lane-Claypon and Starling (1906) in their study of the nature of the stimuli causing growth of the mammary gland. Later it was used extensively by Ancel and Bouin (1911) and is widely used even today. The method consists of mount- ing the entire gland between two glass slides after removing all muscular and adipose tissue and suitably staining the mammary paranchymal tissue. However, whole-mounts can be applied only to those species such as the rat, mouse, rabbit, and monkey in which the mammary gland is a relatively flat sheet of tissue. In species such as the guinea pig, cow, sheep, goat, and human, the gland develops equally in three dimensions and this technique has much less value. In such cases, thick histological sections of the gland have been used to examine the tissue. Thick histological sections. This technique consists of cutting rough, serial slices of the tissue, ranging from 0.5 - 5.0 mm in thick- ness, followed by staining the tissue. Dabelow (19A1) used thick sections to study the human mammary gland. Graumann (1952) and Pfaltz (19A9) used this technique to study neo-natal human mammary glands while Schalm and Haring (1939) have used this to study the bovine gland. -11- Richardson (19A7) suggested that the gland, while it is in a state of full distension with stored milk, should be fixed by vascular perfusion prior to obtaining the sample because handling of an unfixed gland caused considerable distortion of the cell structure. Mammography. Mammography is soft tissue roentgenography of the breast. This clinical technique evaluates quantitative changes in the mammary structure of the human breast. Warren (1930) has reported on the clinical application of mammography. Leborgne (1951) refined the technique and subsequently Egan (1960), Ingleby, Moore, and Gershon- Cohen (1957), Engleby and Gershon-Cohen (1960), and Egan (1963) have made considerable use of this technique. Total mammary gland area. The mammary area covered by the duct and lobule-alveolar systems of whole-mount preparations have been quantitatively measured. Folley at al. (1939) determined the mammary area of rhesus monkeys by tracing the periphery of the whole-mounted glands onto xylol-soaked papers and measuring the area either with a planimeter or squared paper. Cowie and Folley (19A7), Cowie (19A9), and Silver (1953) using rats and Flux (195A) using mice have obtained mammary gland areas by this method. Macdonald and Reece (1960) used a technique to measure mammary gland area which eliminated the use of the planimeter or counting the squares. These workers took negative pictures of the wholeamount preparations, cut the pictures around the periphery of the gland, weighed the resulting photograph and compared 2 standard. Using this technique, they studied the re- it to a 1 cm sponse of rat mammary glands to various mammogens (Macdonald and Reece, 1962; 1963). -12- Analysis of degree of arborescence. The area of the mammary gland alone does not indicate the morphologic differences within the area, and therefore, Cowie and Folley (19A7) devised a scoring system to quantitate the arborescence of the duct system, side-buds, endébuds, and alveoli. Silver (1953) in determining the degree of arborescence of the rat mammary gland adopted the grid technique of Short (1950) which was initially developed for measuring the internal surface area of lung alveoli. In this technique, the number of intersections made by side- buds and ducts per cm of grid were counted. Flux (195A) counted the number of junctions between the ducts in estimating the arborescence of the mouse mammary gland, whereas Benson EE.§A° (1957) devised a scoring system for the duct and alveolar development of guinea pigs. Total surface area of the secretgpy epithelium. Attempts were made by Cowie _a_t_ a_1_._. (1952) to estimate the total internal surface area of the mammary alveoli and to correlate this with milk yield. Richardson (1953) applied the method of Short (1950) and Chalkley (19A3) to determine the surface area of the secretory epithelium of the goat mammary gland. Volume of glandular tissue. Benson ap.a1. (1957) used a procedure in guinea pigs to determine the volume of the glandular tissue from area measurements of serial sections of the gland. This procedure, though useful for three-dimensional glands, is extremely time-consuming (Cowie and Folley, 1961). Relativepgrowth analysis. The concept of relative growth analysis, as developed by Huxley (192A) whereby the rate of growth of a particular organ is related to the growth of the animal as a whole was first applied to the mammary gland by Folley pp a1. (1939). The body surface area is -13_ approximately proportional to the function (body weight)2/3, and this function was used as a standard of reference to which the changes in mammary area were related. Cowie (19A9) and Silver (1953) using rats and Flux (195A) using mice applied this technique to the study of normal and experimental mammary growth. Aberle (193A) compared the rate of growth of the mammary gland in rhesus monkeys with the rate of weight increase of the kidney, the kidney serving as a reference standard. Deoxyribonucleic acid content. Davidson and Leslie (l950a,b) suggested that the DNA content may be used as a measure of cell numbers in a tissue. Kirkham and Turner (1953) were the first workers to use the DNA content as a measure of mammary gland growth. Tucker and Reece (1962) observed that the DNA content per nucleus was constant in mammary tissue of pregnant and lactating rats, indicating that DNA analysis may be used directly as an index of gland growth. Numerous publications have appeared using DNA as a measure of normal (Griffith and Turner, 1957; Brookreson and Turner, 1959; Griffith and Turner, 1961a; and Tucker, 1962) as well as experimentally induced gland growth (Kirkham and Turner, 195A; Anderson, Brookreson, and Turner, 1961; Moon, Griffith, and Turner, 1959; Yamamoto and Turner, 1956; and wada and Turner, l959a,b). Alkalinepphosphatase and iron content. Huggins and Mainzer (1957; 1958) determined the alkaline phosphatase content of the mammary gland and suggested its use as a method of assessing mammary development. Rawlinson and Pierce (1950) have suggested that the iron content of the .gland measured the gland development. However, neither of these techniques has received widespread application. MATERIALS AND METHODS Animals Female rats of the Sprague-Dawley strain were used throughout this study. The animals were housed in a room maintained at a temperature of 2A0 :.10 C and illuminated with fluorescent lights from 8:00.AM to 8:00 PM. The animals were given ad libitum water and a ration consisting of: Ground shell corn A60 1b Soybean oil meal, AA% protein 200 lb Fishmeal, 65% protein 100 lb Alfalfa meal dehydrated, 17% protein 50 lb Dried skim.milk powder 100 lb Sugar (as beet sugar-sucrose) 50 lb Corn oil 30 lb Trace mineral salt 5 lb Vitamin B complex 1 1b 1. Riboflavin, 2,000 mg/lb 2. Pantothenic acid, A,000 mg/lb 3. Niacin, 9,000 mg/lb A. Choline chloride, 90,000 mg/lb Vitamin A and D mixture 0.5 lb 1. 80% A, 10,000 IU/g 2. 20% D, 9,000 IU/g Vitamin 312, 6 mg/lb 3.75 lb Rats for these experiments were obtained from litters adjusted within 2 days of birth to six pups per mother. The rat pups were weaned at 20 days of age and were then housed 20 rats (maximum) per cage (cage area = 22 x 20 in.) until 50 days of age. At this time rats _1h- -15- were transferred to another cage (cage area = 25.5 x 9.5 in.) and were housed 10 rats per cage. IMammary gland area experiment. Rats were sacrificed at 10, 20, 23, 25, 30, 33, 35, A0, 60, 80, and 100 days of age and the skin containing the right abdominal mammary gland was removed for mammary area measure- ments. Vaginal smears from rats 60 days of age and older were prepared prior to sacrifice for ascertaining the stage of the estrous cycle. Total mammapy fat pad (TMFP) experiment. Rats were sacrificed at 10, 20, 30, A0, 50, 60, 70, 80, 90, and 100 days of age and the entire fat pad containing the two abdominal and four inguinal mammary glands was removed for nucleic acid analyses. Vaginal smears of the rats were pre- pared at the time of sacrifice from rats AO days of age and older with the exception of the 50-day group. Spacific mammary area (SMA) expariment. Rats were sacrificed at 10, 15, 20, 21, 22, 23, 2A, 25, 30, 31, 32, 33, 3A, and 35 days of age. The two areas containing the right and left abdominal mammary glands were removed for nucleic acid analyses. Preliminary studies of whole mounts revealed the location of this specific area. The lymph nodes, which served as a landmark for this specific area, were removed prior to analysis. Mammapy Gland Area Measurement The skin-mammary gland complex was removed from the rat, pinned on a cork board, and fixed in Bouin's fluid for at least 2A hours. The tela subcutanea, containing the mammary gland, was separated from the skin, and with the aid of a binocular microscope, the major lymph nodes, blood vessels, and nerves were dissected out. The Bouin's fluid was -16- removed from the gland by washing several times with 2% NHAOH in 70% ethanol. The fat was removed by placing the gland in 95% ethanol for several days. The gland was subsequently stained with Meyer's hemalum for 12-2A hours, washed several times with 1% potassium alum and differentiated in acid-alcohol (2.0% H01 in 70% ethanol) until the ducts and alveoli of the gland presented a sharp contrast to the matrix. The gland was further dehydrated in 80%, 95%, and absolute alcohol for five, five, and eight minutes, respectively; cleared in xylene-ethanol (1:1) followed by 100% xylene for three and five minutes, respectively; and then mounted in permount (Fisher Scientific Co.). The wholeamount was placed in the negative chamber of a photographic enlarger and the periphery of the enlarged image was traced onto paper by two persons. The indentations of the gland were followed only where the indentation was twice as wide as the length of the adjacent duct. The outline of the gland thus obtained was then traced with a planimeter (Keuffel and Esser Co. Compensating Polar Planimeter Model No. 620015) by two persons. The area obtained was corrected for magnification (the magnification was about 8 for glands under 30 days of age and about 3.2 for glands of 30 days and above) and the average area in mm? for each gland was calculated. Nucleic Acid Analyses The animals were sacrificed by cervical dislocation and the entire fat pad (TMFP) containing the six abdominal-inguinal glands, or the specific area (SMA) containing the left and right abdominal glands were removed and placed immediately in ice-cold 0.25 M.sucrose. The tissue was stored at -200 C until assayed for nucleic acid content. -17- The nucleic acid extraction was performed by the Schmidt and Thannhauser (19A5) procedure as modified by Tucker (196A). The tissue was thawed, blotted on paper towels, and weighed on an analytical balance. The entire TMFP or SMA was cut into small pieces with scissors and sus- pended in ice-cold glass distilled water in the ratio of 1 g per 20 ml of TMFP and l g per A0 m1 of SMA. The tissue was homogenized for 2 minutes in either the 1000 ml Pyrex glass container or the 25 to 250 m1 semi-micro metal (Cenco 172A6-2) container of a Cenco PB-5 waring Blender. Fat pads from animals 70 to 100 days of age were, however, homogenized for 3 to A minutes to obtain a satisfactory homogenate. Duplicate 2 ml or A ml samples of the homogenate, depending on the dilution, were removed into polypropylene tubes and 8 ml or 6 ml, respectively, of 95% alcohol was added. The samples were incubated at least 12 hours at room temperature with continuous shaking and were then centrifuged in a Servall RC-2 centrifuge at 1-30 C. .All centri- fugations were performed at 32,000 x.g for 20 minutes. The supernatant fluid was poured offl and nine m1 of methanol-chloroform (2:1) Was added to the residue. .After incubation at room temperature for at least 2A hours, the samples were centrifuged, supernatant fluids poured offl, and nine m1 of ether was added to the residue. The samples were then incubated for at least 12 hours at room temperature, centrifuged, and the ether fraction removedl. The residue was extracted twice with 5 m1 of 10% ice-cold trich- loroacetic acid which was removed by washing with 5 m1 of ice-cold 95% ethanol saturated with sodium acetate. lSee lipid analysis, page 18. -18- The samples were digested with 2 ml of 1N KOH for 15 hours at 370 C. The digest was acidified with 0.3 m1 of ice-cold 6N HCl and 2.5 ml of 10% perchloric acid (PCA). The samples were centrifuged and washed twice with 2 ml each of ice-cold 5% PCA. These combined acid super- natant fluids, containing the RNA, were adjusted to 10 m1 and were analyzed for RNA-ribose by the colorimetric orcinol procedure of Mejbaum (1939) as described by LePage (1957). This procedure consisted of mixing a 3 ml aliquot of the above supernatant fluids with 3 m1 of a 1.0% solution of orcinol in 0.1% FeCl3. 6 H20 dissolved in concentrated HCl. The reaction mixture was boiled in a water bath for 30 minutes, and the resulting color development was determined in a Beckman D B Spectrophotometer at 670 M In, . The RNA content was calculated from a standard curve obtained using highly purified yeast RNA (Worthington Biochemical Corp.). The DNA was extracted from the residue, remaining after cold PCA treatment, with 5 m1 of 5% PCA heated to 700 C for 15 minutes. After centrifuging, the residue was washed twice with 2 m1 of ice-cold 5% PCA. These DNA-containing supernatant fluids were combined and ad- justed to 10 ml with 5% PCA. The absorbancy of the DNA-containing solution was read at 268 M7“; in a Beckman DB Spectrophotometer. Highly polymerized DNA (worthington Biochemicals Corp.) was used as the standard. Lipid.Analysis The alcohol, methanol-chloroform, and ether extracts of the tissue, obtained in the process of extracting the nucleic acids were used to estimate the lipid content of the total mammary fat pad. The three lipid-containing extracts were pooled into tared beakers and allowed to -19- evaporate for 2A hours at room temperature. Such treatment removed most of the ether. The remaining lipid solvents and moisture were evaporated at 650 C on a hot plate. The beakers containing the lipid fractions were placed into a dessicator for at least 2A hours and then weighed on an analytical balance. Relative Growth.Analyses Several studies (Cowie, 19A9; Silver, 1953; Flux, l95A; Folley, 1956) have related mammary gland development with the growth of the body or the increase in the body surface area. It was of interest, therefore, to present an analysis of mammary development relative to increases in body weight and/or body surface area. The simple allometric growth law (Huxley, 192A) is usually expressed as y=bx‘ , where y = the quantity undergoing allometric growth, x = the reference quantity, and b and 0‘ are constants, the more biologically significant of which, termed the "equilibrium constant" is c( . This relation may also be expressed in the form log y = b + 0( log x. When data relating to growth rates conform to this law, a straight line is obtained by plotting log y against log x. The lepe of such a straight line is equal to a( . In the case of_isometric growth, which may be considered a special case of allometric growth, the second expression becomes log y = log b + log x and the straight line obtained by logarithmic plotting of the data will have a slope equal to unity. The function (body weight)2/3, which is approximately proportional to body surface area, was the reference standard of choice to which mammary area was related. However, mammary area was also related to -20- the body weight increases of the animal for comparing with mammary DNA data. On the other hand, total body DNA, a measure of body cell numbers, was probably the most reasonable reference standard with which changes in mammary DNA could be related. However, since body DNA measurements would be difficult to obtain, the body weight of the animal was used as the reference standard. It was assumed that the rate of increase in the body weight was approximately proportional to the rate of increase in body DNA. The mammary DNA was also related to (body weight)2/3, a function of body surface area, for comparing with mammary area data. Statistical.Analyses Standard errors of means, coefficients of correlation, linear regression analysis, and analysis of variance were computed (Snedecor, 1956; Dixon and Massey, 1957). RESULTS AND DISCUSSION Area of Mammary Glands 2 The area in mm of the right abdominal mammary gland of rats 10 to 100 days of age is presented in Table 1. The abdominal mammary gland area of rats increased from 9.26 mm2 at 10 days of age to A9A.57 mm2 at 80 days of age, representing a 5,2Al% increase. The mammary gland area increased 65% between 10 and 20 days, l,857% between 20 and A0 days, and 65% between A0 and 80 days. The area then declined 8.8% between 80 and 100 days of age. Inspection of the growth curve (Fig. 1) indicated that the acceleration in the growth rate of the mammary gland area was established in the Sprague-Dawley rat as early as 20 days of age, whereas Cowie (19A9), using hooded Norway rats, observed this rapid increase in mammary growth after 30 days of age. This divergence of results may represent strain differences. Moreover, Cowie (19A9) measured the area of all 12 mammary glands, whereas in the present experiment only the area of the right abdominal mammary gland was measured. The average mammary area of rats exhibiting proestrus or estrus and metestrus or diestrus is presented in Table 2. Average mammary area of lOO-day old rats exhibiting proestrus or estrus was A58.83 mm2 compared with 3A5.1A mm2 in rats of the same age group exhibiting metestrus or diestrus. But the mammary area of 60- and 80-day old rats exhibiting proestrus or estrus was less than the mammary area of rats of the corresponding age groups exhibiting either metestrus or diestrus. These observations failed to support the phenomenon of increased duct pro- liferation during estrus reported by several workers using histological techniques. This contradictory finding was attributed to the presence of -2]_- -22- Table l. Mammary gland area of rats 10 to 100 days of age. App No. of rats Avg. body wt. (g) Area (mme) 10 12 22.0 9.26 : 0.9A 20 12 A2.0 15.29 i 1.A2 23 12 57.0 35.5A : 3.27 25 12 62.0 A8.75 :_ 3.99 30 12 68.0 67.06 : A.32 33 12 92.0 138.02 i 6.65 35 12 99.0 178.31.:. 8.72 A0 12 122.0 299.22 i 1A.82 60 12 159.0 A15.90 1 12.90 80 12 188.0 A9A.57 :_27.39 100 12 217.0 A50.93 i 2A.07 Table 2. Mammarypgland area of rats in various stages of the estrous cycle. No. of *Rats in ,Av . area *Rats in Avg. area Age rats P or E imme) ,M or D A(mm2) 60 12 5 A09.96 7 A20.15 80 12 9 A52.50 3 620.75 100 12 8 A58.83 A A35.1A * P = proestrus, E = estrus, M = metestrus, D = diestrus. several uncontrolled variables in the present experiment, such as the body size of the animal, the number of estrous cycles experienced by each individual animal, and the individual rate of mammary growth of the animal. The agreement of the data with the law of simple allometry was .wwd no w>m<22<2 4.0..”- 3.2-Emma oo. om om ox. cm on 2. on om o. d d I 11 1 1 1 dc! d4q -23- OO- CON con 00¢ oon VBUV 3mm) -2A- tested by plotting log mammary area against log (body weight)2/3, a function of body surface area (Fig. 2). Since Cowie (19A9) fitted similar data by two straight lines, intersecting arbitrarily in the neighborhood of log (body weight)2/3 = 1.1, it was of interest, to fit the data with three straight lines, as follows: (a) including all the points; (b) including points from log (bodyweight)2/3 = 0 to log (body weight)2/3 = 1.1, which represented rats 10 to 20 days of age; and (c) including points above log (body weight)2/3 = 1.1, which represented rats 23 to 100 days of age. The respective coefficients of equilibrium (fi‘) for the three linear regression lines, calculated by the method of least squares, were 3.00 (P< 0.01), 1.13 (P< 0.01) and 3.00 (P<0.01), respectively. The latter two values agreed very well with the corres- ponding values of 1.10 and 3.03, respectively, reported by Cowie (19A9). Thus, the mammary gland area increased three times faster than the body surface area between 10 and 100 days of age inclusive but the mammary gland area increased only 1.13 times faster than body surface area between 10 and 20 days of age. This observation agreed very well with the data of Cowie (19A9) which suggested that allometry commenced at day 23 of age. Inspection of the plot (Fig. 2) showed that most of the data could be fitted to a single straight line. On the other hand, the data would probably have been best fitted by a cubic curve. The lower portion of this curve is explained by the c( value of 1.13 as dis- cussed above. On the other hand, it would appear that at some point approximating l.A to 1.5 log (bodyweight)2/3 the mammary gland area did not increase further despite increases in surface area. In addition, the growth rate of the mammary gland area was related LOG MAMMARY GLAND AREA 30 2-6 2-2 l-8 M 0-6 -25- I 08 IO l-2 I-4 l-6 LDG ( BODY WEIGHT )2’3 FIG. 2. RELATION BETWEEN MAMMARY GLAND AREA AND (BODY WEIGHT )2’35 OF RATS l0 T0 IOO DAYS OF AGE. _26_ to the body weight of the animal. The plot of log mammary gland area against log body weight appeared to fit a straight line (Fig. 3) although a cubic curve would probably more closely fit the data. The equilibrium constant¢£., calculated by the method of least squares, was 1.95 which was 35% less than the corresponding¢x value obtained in the relative growth analysis of mammary area with respect to body surface area. Thus, the mammary gland area between 10 and 100 days of age in- creased about two times faster than the body weight. This data also showed that the two reference standards, body surface area and body weight of the animal, did not increase with advancing age at the same rate; body weight tended to increase more rapidly than body surface area. This observation was in agreement with the geometric relationships between weight and surface area as discussed by Brody (19A5). Nucleic Acid Content of Mammary Glands Total mammary fat_pad (TMFP). The nucleic acid content of the entire mammary fat pad containing the six abdominal-inguinal mammary glands of rats 10 to 100 days of age is presented in Table 3. DNA is expressed as total mg of DNA and mg of DNA per 100 g of body weight. RNA is expressed as total mg of RNA. The total DNA content of TMFP increased from 0.33 mg at 10 days of age to 3.57 mg at 90 days of age. The mammary fat pad increased in a linear manner to 50 days of age (Fig. A), con- firming the observations of Tucker (1962), but declined 10%, 21%, and 17% at 60, 70, and 80 days of age, respectively, relative to 50 days. The differences among means of 50-, 60-, 70-, and 80-day groups, however, were not statistically significant (P) 0.05). LOG MAMMARY GLAND AREA 32 2-8 24 20 I2 08 0-4 -27- L °( = l-95 L L A A 1 J A l L L A 4 l-2 l-4 I°6 I-B 2'0 2'2 2'4 LOG BODY WEIGHT FIG. 3. RELATION BETWEEN MAMMARY GLAND AREA AND BODY WEIGHT OF RATS IO TO I00 DAYS OF AGE . -28- m0.0 + 1:30 3.0 H 003 00.0 H 3.3 3.0 H 30 333.0 0.03 3 03 30.0 H 00.0 3.0 H 11.0.3 3.0 H 00.3 3.0 H 3:0. 53.: 0.03 3 0a m0.0 H 30.0 10.0 H 3.3 8.0 H 3.3 00.0 H 3.3 E30 0.03 3 00 10.0 H 3.0 3.0 H m03 3.0 H 3.3 3.0 H 33 33.: 0.03 3 0a m0.0 H 3.0 m0.0 H 10.3 10.0 H 3.3 5.0 H $3 $011.: 003 3 00 00.0 H 3.0 3.0 H 3.3 3.0 H 3.3 3.0 H 30.3 330 0.03 3 00 3.0.0 H 3.0 3.0 H 3.3 3.0 H 00.4.. 3.0 H 3.3 330.3 0.03 3 0a m0.0 H 5.0 00.0 H 3.3 10.0 H m03 00.0 H 3.3 300.3 0E. 3 0m .00 H 30.0 10.0 H 3.0 3.0 H 03.3 00.0 H 3.0 0100.3 0.3. 3 3 30.0 H 30.0 30.0 H 3.0 00.0 H mmé 30.0 H 3.0 0100.0 0.3 3 3 Amav H03 Amsv Amv Amv mama oma. 333,3 3 3.309 m 0338 SS 30009 :E 0930 5.: Atom .8 .oz .omm mo wand 00H O0. 0H 00.03 mo 0.0m Rom Nam-saws Han-sop map mo HQOHSOO 0.3.0 3332 .IM 2509 .uo< “.0 m>m<22<2 1.3.0... MI... “.0 hzwhzoo . in: 004 00. 00 00 2. 00 00 0.. wk0.05). When 10g DNA was plotted against log (body weight)2/3, the data again appeared to fit a single straight line (Fig. 6) and the slope of that line was found to be 1.56 which was significantly greater than unity (P< 0.01). These data suggested that the increase in the DNA of the mammary fat pad between 10 and 100 days of age was isometric with respect to body weight and allometric with respect to the body surface area of the animal. This also implied, in agreement with the observation of the relative growth analysis of mammary area, that body surface area and body weight of the animal in- creased at different rates, body weight increasing faster than body sur- face area. There was no change with age in the slopes of the plot of log DNA against 10g body weight or log (body weight)2/3. These ob- servations suggested that the abrupt increase in mammary area, occur- ring around day 23, did not occur between 10 and 100 days of age based on the DNA of the total mammary fat pad. The correlation coefficient of the total DNA content of the TMFP LOG MAMMARY DNA -33- l-2 l-4 |°6 l-B 2'0 22 2'4 LOG BODY WEIGHT FIG. 5. RELATION BETWEEN DNA OF TOTAL MAMMARY FAT PAD AND BODY WEIGHT OF RATS l0 TO IOO DAYS OF AGE. DNA LOG MAMMARY (16 0-4 (12 412 4y4 03 IO l-2 l-4 l-6 LOG (BODY WEIGHT )2” FIG. 6. RELATION BETWEEN DNA OF TOTAL MAMMARY FAT PAD AND (BODY WEIGHT)” 0F RATS l0 T0 IOO DAYS OF AGE. 3 -35- with mammary gland area was 0.82 (P<0.01). The total DNA content of the TMFP increased from 0.27 mg at 10 days of age to 2.56 mg at 100 days of age. The increase in total RNA followed a pattern similar to the total DNA (Fig. A). The ratio of RNA to DNA tended to decline as age advanced implying that protein synthesis per mammary fat pad cell was retarded with advancing age. The lipid content of the TMFP is presented in Table 5 and is expressed in total mg of lipid and lipid as percent of fresh gland weight. The total lipid content increased from 2lh.9 mg at 10 days of age to 3,h13.1 mg at 80 days of age. The lipid content represented 30.5% of the mammary gland weight at 10 days of age and 58.9% at 60 days of age. The lipid fraction of the gland declined from 56.0% to 13.3% of the gland weight between 80 and 90 days of age during which period there was a h8% increase in the DNA content of the TMFP (Table 3). This may be interpreted as suggesting that the growth of the mammary gland was associated with a disappearance of the adipose tissue as re- ported by Harkness and Harkness (1956) in mammary glands of albino rats. The role of the fat pad in the growth of the mammary gland has not been studied extensively. However, some unpublished observations in our laboratory indicated that when the surrounding fat pad was removed surgically from rats at an early age, the mammary gland failed to grow under the stimulus of pregnancy as compared with the contralateral, intact glands. These observations suggested that the presence of the fat pad was necessary for the mammary gland to grow and that the amount of development of the fat pad may limit the extent of growth of the mammary gland. -36- Specific mammary area (SMA). The preceding data were observations on the growth of the entire mammary fat pad containing the mammary glands as well as considerable amounts of extraneous tissue including adipose tissue, other connective tissue, and lymph nodes. It was of interest, therefore, to determine the growth of the mammary tissue without extraneous fat pad tissue. For this purpose, the nucleic acid content was determined in the two specific mammary areas containing the right and left abdominal mammary glands. Wholeamount preparations of glands showed that it was not possible to remove such specific mammary areas after 35 days of age because of the overlapping of the abdominal and the first inguinal mammary glands. The nucleic acid content of rats 10 to 35 days of age is presented in Table 6. Total DNA increased from 0.06 mg at 10 days of age to 0.3A mg at 30 days of age, representing a h67% increase. The DNA declined, however, between 31 and 33 days of age to 0.27 mg and increased again between 34 and 35 days of age to 0.53 mg. The differences between the total DNA content of 30-, 31-, 32-, and 33-day groups was not statist- ically significant (P:»0.05). The failure to observe an increase in the DNA content between 31 and 33 days of age could be due to error in re- moving the SMA from the fat pad. Another more likely possibility was that rats at this age were influenced by approaching puberty. The external signs of estrus were not apparent until after the 3hth or 35th day but a 62% increase in mammary DNA between 25 and 30 days of age suggested that the mammary gland was already under the influence of the mammogenic hormone(s). The regression of mammary DNA between the 31st and 33rd days was probably caused by a lack of the ovarian hormones, -37- 8.0 H $20 N0.0 H 0.0.0 0.0.0 H 0H.0 m0.0 + 0.0.0 0000.0 0.00H NH mm N0.0 H med 8.0 H 0N.0 No.0 H 8.0 No.0 H mmd m0mm.0 0.m0 NH Hm m0.0 H 8.0 8.0 H 0N.0 No.0 H 0m.0 8.0 H 5.0 082.0 0.00 NH mm 00.0 H 8.0 No.0 H eN.0 8.0 H mm.0 No.0 H 0N.0 03.0.0 0.5 NH Nm 8.0 H 8.0 8.0 H 0N.0 No.0 H 00:0 No.0 H 0m.0 0000.0 0.8 NH Hm No.0 H 0N.0 No.0 H 0N.0 m0.0 H 8.0 N0.0 H 8.0 080.0 0.00 NH 0m 00.0 H 00.0 8.0 H 0H.0 m0.0 H 8.0 8.0 H HN.0 080.0 0.00 NH 0N m0.0 H N08 N00.0 H 0H.0 8.0 H 8.0 N00.0 H 0H.0 008.0 0.Nm NH 8 m0.0 H Q20 8.0 H NH.0 N0.0 H Nm.0 8.0 H 0H.0 008.0 0.0: NH mN No.0 H 8.0 000.0 H NH.0 No.0 H mm.0 000.0 H NH.0 88.0 0.? NH NN No.0 H N08 000.0 H 0H.0 No.0 H 0m.0 8.0 H NH.0 0NHN.0 0N: NH HN No.0 H 8.0 80.0 H 0H.0 8.0 H 0N.0 N00.0 H mH.0 005.0 0.8 NH 0N 00.0 H $8 80.0 H 8.0 No.0 H 0N.0 80.0 H 00.0 38.0 0.Nm NH 0H m0.0 H 5.0 m00.0 H 00.0 8.0 H 0N.0 80.0 H 00.0 38.0 0.HN NH 0H EQNE Namflwwoa N wwwazm Hzflwfia 5.: WW8 .Psamwom prwmz 00.0 .mwd mo awed mm op OH meH %o Nona Nndaama oHMHommmimgp mo pampsoo wHow OHoHodz .w magma -38- whereas the rapid increase between 3% and 35 days of age could be attributed to the presence of hormones involved in controlling the estrous cycle. This cyclic effect on SMA-DNA before puberty agrees very well with the histological observations relating estrous cycle to mammary development (Turner and Gomez, 1936; Cole, 1933; Sutter,‘l92l). The mammary area data of the present experiment and that of Cowie (l9h9) suggested an allometric mammary growth rate at least two weeks before puberty whereas the SMA-DNA data indicated that this prepubertal growth of the gland is cyclic as well as cumulative. The DNA/100 g body weight increased from 0.28 mg at 10 days of age to 0.h9 mg at 35 days of age, representing a 75% increase, and followed almost the same pattern of increase with age as did total DNA (Fig. 7). The agreement of the increases in the DNA content of SMA with the law of simple allometry was tested by plotting log DNA against log body weight of the animal (Fig. 8). From inspection of the plot, the data appeared to fit a straight line. The slope (oC) of the best fit linear regression was found to be 1.26 which was significantly greater than unity (P<:0.0l). When DNA was related to the body surface area, by plotting log DNA against log (body weight)2/3, the data again appeared to fit a straight line (Fig. 9). The slope of this line was found to be 1.89 which was significantly greater than unity (P< 0.01). These 9( values suggested that the increase in the DNA content of SMA between 10 and 35 days of age was only slightly allometric with reSpect to the body weight increases but considerably allometric with respect to the body surface area changes of the animal. The difference in the two o( values again suggested that the body weight increased faster than the body .mod “.0 m>m<22<2 oifimmm NIP v.0 PZMFZOQ 0.04 Emu—0:2 .h 0E 3:5. 004 ON 0. 0. 505; >000 000.220 1 42m .53 TI. <20 n_<._.0._. I L l 0.0 _.0 N0 n0 v.0 0.0 0.0 lNBLNOO OIOV OIB'IOON bun -ho- surface area. Inspection of the plot of 10g DNA against log body weight (Fig. 8) and log DNA against log (body weight)2/ 3 (Fig. 9) did not reveal any change in the slopes of the curves at any point between 10 and 35 days of age which again suggested that mammary gland growth, as measured by the DNA content of the gland, continued at a constant rate between 10 and 35 days of age. The correlation coefficient of the total DNA content of the SMA with mammary gland area was 0.77 (P< 0.01). Total RNA of SMA increased from 0.05 mg at 10 days of age to 0.38 mg at 35 days of age. The increases in total RNA were similar to the in- creases in total DNA (Fig. 7). The ratio of RNA to DNA did not reveal any significant trend among the age groups which suggested that the rate of protein synthesis remained constant between 10 and 35 days of age. LOG MAMMARY DNA -O-B 40 42 4.4 -l-6 -ul- l 1 l 1 l 1 l 1 j 1 l 1 l2 l-4 |°6 |°B 2-0 22 l-O LOG BODY WEIGHT FIG. 8. RELATION BETWEEN DNA OF THE SPECIFIC MAMMARY AREA AND BODY WEIGHT OF RATS IO TO 35 DAYS OF AGE. LOG MAMMARY DNA -0-2 -04 -O-6 -O-B 4‘0 -I°6 I 0-6 0-8 I'O I-2 I-4 2/3 LOG (BODY WEIGHT) FIG. 9. RELATION BETWEEN DNA OF THE SPECIFIC MAMMARY AREA AND (BODY- WEIGHTIUSOF RATS l0 T0 36 DAYS OF AGE. SUMMARY 1. The mammary gland area of rats increased from 9.26 mm2 at 10 days to h50.93 mm2 at 100 days of age. This increase was allometric relative to either the body surface area (4: 3.00) or body weight (a(= 1.95) of the animal. Arbitrarily fitting a regression curve to include animals between 10 and 20 days and between 23 and 100 days of age suggested that mammary area increased only 1.13 times as fast as surface area before 20 days of age and 3.00 times faster after 23 days of age. The mammary area of the older age groups did not increase further with in- creasing surface area. 2. The total DNA content of the mammary fat pad, containing the six abdominal-inguinal mammary glands, increased from 0.33 mg at 10 days to 3.52 mg at 100 days of age. This increase was isometric (d = 1.0LL) relative to body weight but allometric (c( = 1.56) relative to body surface area of the animal. No change in the slopes of log DNA plotted against either log body weight or log (body weight)2/3 was observed. The mammary gland complex thus appeared to grow at a constant rate based on DNA, between 10 and 100 days of age. 3. The DNA/100 g body weight increased from 1.53 mg at 10 days to 1.70 mg at 100 days of age. The slope of the linear regression curve was 0.001 which suggested that DNA/100 g body weight remained constant between 10 and 100 days of age. 1+. The correlation of TMFP-DNA with mammary area was 0.82 (P<0.01). 5. The total RNA content of the TMFP increased from 0.27 mg at 10 days to 2.56 mg at 100 days of age and these increases paralleled those of total DNA. -h3- -hh- 6. The RNA/DNA ratio tended to decline as age advanced implying that protein synthesis per mammary fat pad cell was retarded with ad- vancing age. 7. The total lipid content increased from 2lh.9 mg at 10 days to 2,163.1 mg at 100 days of age. The growth of the mammary gland appeared to be associated with a disappearance of the adipose tissue. 8. The total DNA content of the specific mammary area containing the left and right abdominal mammary glands increased from 0.06 mg at 10 days to 0.53 mg at 35 days of age. This increase was 1.26 times faster than body weight and 1.89 times faster than body surface area of the animal. No change in the slopes of log DNA plotted against either log body weight or log (bodyweight)2/3 was observed, which again suggested that relative mammary gland growth, based on DNA, occurred at a constant rate between 10 and 35 days of age. A cyclic mammary growth period was noted between 30 and 35 days which suggested that the mammogenic hormones were cycling and effecting mammary development before puberty. 9. The DNA/100 g body weight of the SMA increased from 0.28 mg at 10 days to 0.h9 mg at 35 days of age and followed a pattern similar to the total DNA. 10. The correlation of SMA-DNA with mammary area was 0.77 (P<:0.01). 11. The total RNA content of the SMA increased from 0.05 mg at 10 days to 0.38 mg at 35 days of age and these increases paralleled those of total DNA. 12. The RNA/DNA ratio did not reveal any significant trend among the age groups implying that the rate of protein synthesis remained -45- almost constant between 10 and 35 days of age. BIBLIOGRAPHY Aberle, S. B. D. 193A. Growth of the mammary gland in the rhesus monkey. Proc. Soc. Exp. Biol. and Med., 32:2u9. Altman, A. D. 19h5. Izmeneniya v vimeni korov v protsesse razdoya. Vestn. Zhivotnov., No. 1, 85. .As abstracted in Dairy Sci. 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