1.. a‘ u. .1‘ n. .a. r. r. .4 r” .~ aa~ um “u. ‘ru. L. .a. .‘h :n r~ ‘4. "c ABSTRACT ENDOCRINE, REPRODUCTIVE AND MAMMARY DEVELOPMENT OF HEIFERS AND RATS DURING THE ESTROUS CYCLE by Yagya Nand Sinha The relationship between the endocrine function and reproductive and mammary development of bovine and murine female during the estrous cycle was investigated. Endocrine function was studied by measuring luteinizing hormone (LH) and prolactin in the pituitary gland, meta- bolic status of the uterus and ovarian weight changes. Reproductive and mammary develOpment was assessed by deoxyribonucleic acid (DNA), ribonucleic acid (RNA), pro- tein, lipid and collagen content of the organs as well as histological observations. The length of the estrous cycle of heifers averaged 20.6 i 0.2 days. The age of vaginal opening of rats averaged-36.7 : 0.13 days. Vaginal opening coincided with proestrus or estrous smears in 58% of the rats. The first (5.5 days) and second (5.2 days) estrous cycles were significantly longer than the third (4.8 days) and fourth (A.9 days) estrous cycles. Uterine DNA of rats did not change between pro- estrus (0.88 mg/100 g BW) and estrus (0.85 mg/lOO g BW) ‘I 4. ‘1' r.— :a A-- Yagya Nand Sinha or metestrus (0.70 mg/100 g BW) and diestrus (0.70 mg/100 g BW) but declined 18% between estrus and metestrus, sug- gesting a significant loss of uterine cells during the early luteal phase of the cycle. Cumulatively, uterine DNA increased from the first through the third estrous cycles but not thereafter. Uterine weight, RNA, RNAzDNA ratio and lipid content were maximum at proestrus, de- clined at estrus and metestrus but increased during di- estrus. Pituitary LH concentration, which averaged 0.72, 0.31, 0.27 and 0.33 ug/mg at proestrus, estrus, metestrus and diestrus, respectively, closely paralleled the changes in the RNA:DNA ratio of the uterus. Mammary DNA of heifers increased from 97.8 mg/lOO lb BW at proestrus (day 20) to 213.7 mg/lOO lb Bw at estrus, then declined during metestrus (day 2, 169.1 mg/lOO lb BW) and diestrus (days 4-18, 1A2.8 mg/lOO lb BW), suggesting proliferation of mammary cells during the estrogenic phase of the cycle and loss of cells during the progestational phase. Mammary RNA and RNAzDNA ratio followed a pattern similar to mammary DNA. Mammary protein, collagen and lipid content also increased during the estrogenic phase of the cycle but, unlike the nucleic acid content, they did not decline until after metestrus. Pituitary pro- lactin concentration increased linearly from a minimum of 0.012 IU/mg on day 2 of the estrous cycle to 0.045 IU/mg on the day of estrus. Prolactin concentration paralleled ’-—-~ ...*_. - ‘oa‘l ‘3‘! ... ._, u,- u... “6: :- ‘i-. u,” ' .- .v ’1 '1’! hut " Yagya Nand Sinha mammary development during the estrogenic phase of the estrous cycle but not during the progestational phase. Mammary DNA of rats increased 8% between proestrus and estrus but did not change thereafter. Mammary RNA and RNAzDNA ratio, however, increased between proestrus and estrus, remained constant at metestrus but declined 9% at diestrus, suggesting stimulation of mammary growth during the estrogenic phase of the cycle and involution during the luteal phase. Cumulatively, mammary DNA increased from the first through the fourth estrous cycles but not thereafter. Pituitary prolactin concentration during proestrus (0.032 IU/mg) and metestrus (0.0Al IU/mg) were significantly greater than during estrus (0.015 IU/mg) and diestrus (0.015 IU/mg). Thus, like the heifer, pituitary prolactin concentration of rats paralleled increases in mammary growth during the estrogenic phase but not during the pro- gestational phase of the cycle. It is concluded that the pubertal development of the reproductive and mammary apparatus of the heifer and the rat is completed within a few estrous cycles. And, al- though there are intrinsic differences in the mechanism of reproductive and mammary growth, both are closely re- lated to the estrous cycle. ENDOCRINE, REPRODUCTIVE AND MAMMARY DEVELOPMENT OF HEIFERS AND RATS DURING THE ESTROUS CYCLE By Yagya Nand Sinha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1967 la DEDICATED TO my father Sri Baidyanath P. Sinha 1“! dv-y; A .-‘~“ ."P-vu ~nw.. "vs A... ‘Y\ 1 BIOGRAPHICAL SKETCH I was born at Rohua, Muzaffarpur, Bihar, India, on October 21, 1936. I received my high school education at Muzaffarpur Zilla School and passed the Matriculation Examination in February, 1952. In July, 1952, I entered Langat Singh College, Muzaffarpur and passed the Inter- mediate in Science examination in February, 195A. The ~same year I entered Bihar Veterinary College, Patna and graduated in March, 1957. I worked for the government of Bihar, India, as a 'Veterinary Assistant Surgeon at Nathnagar from April, 1957, 'to March, 1958; as a Veterinary Assistant Surgeon in charge of’the Artificial Insemination Center at Suranarha from AprdJ, 1958, to September, 1959; and as a research assist- ant in Biological Products at the Livestock Research Station, Bihar, Patna, from October, 1959, to September, 19610 I Joined the graduate school at Michigan State Uni- versity in September, 1961, and received the Master of Science degree in the Department of Dairy in June, 196U, and the Doctor of Philosophy degree in September, 1967. My primary area of interest is reproductive and mammary physiology. iii ACKNOWLEDGMENTS The task of completing graduate work is an arduous one, especially when one has to begin from scratch. The one man to whom goes most of the credit for helping me out constantly is Dr. R. Allen Tucker, my major professor. He has been my teacher, my mentor, and my friend all at once for the past five years, and no amount of words can repay the debt I owe to him. Without his guidance, his interest and his inspiration this work and my mission would never have been fulfilled. My gratitude to Dr. Harold D. Hafs is equally deep. His vast knowledge of biological statistics and his pen- chant for its correct use in research is surpassed only by his gift of imagination and I have profitted immensely from my association with him. I want to express my sincere thanks to Dr. E. P. Reineke, Dr. W. C. Deal, and Dr. L. D. McGilliard for Serving on my doctoral committee, their advice and their help in preparing this manuscript. While the responsibility for the shortcomings is entirely mine, assistance in this research was rendered by many people. Prominent among them were my colleagues, DP. Claude Desjardins who helped in the performance of iv LH assays, and Drs. M. J. Paape, K. T. Kirton, Jock McMillan, A. J. Hackett and W. W. Thatcher, who helped in many other ways. The afternoon coffee-hour discussions in the laboratory was one activity of the graduate school whose memory I will always cherish. They provided not only a very enjoyable intellectual exercise but often- times important ideas for research. It is almost impossible to acknowledge individually every person who helped in this project, but the acknow- ledgment will not be complete unless I mention the assis- tance of Mrs. Helga Hulkonen, who performed many technical chores so beautifully and with such singular patience. I take this opportunity to extend my appreciation to ‘the Ayrst Laboratories, The Upjohn Company, and the Endo- f the corpus luteum is paramount. The uterus undergoes nnarked changes in preparation for the nourishment of the eInbryo. It lasts for 14-15 days in the cow (Salisbury alud VanDemark, 1961) and about 57 hours in the rat (Asdell, 196st Although the above is a convenient division of the estxrous cycle, more recently it has also been defined on true basis of the hormone effective at the time. Thus, the tean "follicular" or "estrogenic" phase is frequently used tCD describe the part of the cycle when the influence of true estrogenic hormones is predominant and "luteal" or "Drudgestational" when the corpus luteum develops and pro— Sesterone becomes the predominating hormone (Salisbury and VarflDemark, 1961). These terminologies are especially use- ful. in the study of comparative reproductive physiology. C. Vaginal Smear During the Estrous Cycle The four phases of the estrous cycle--proestrus, eStrus, metestrus and diestrus—-are associated with cyclic changes in almost all parts of the female reproductive tract including the ovaries and the mammary glands. But it is the discovery of their occurrence in the vaginal smear of the guinea pig (Stockard and Papanicolaou, 1917) that opened the way to the detailed investigation of the estrous cycle in several species. The two classical studies that quickly followed were those of Long and Evans (1922) on the rat and of Allen (1922) on the mouse. Ever since, examination of the vaginal smear has provided an easy and fairly accurate method for following the estrous cycle in living animals and has contributed much to the advancement of our knowledge of reproductive physiology. The basic constituents of the vaginal smear are nucleated epithelial cells, cornified cells and leukocytes (Allen, 1922; Long and Evans, 1922), although by the use of special staining, various subgroups of these three cell types can be recognized (Hartman, 1944). The relative proportion of each of these three cell types varies charac- teristically during different phases of the estrous cycle and forms the basis for identification of the stage of the cycle. A During proestrus, the vaginal smear consists largely of small, round, lightly staining, nucleated epithelial cells which occur singly or in small sheets. Leukocytes are totally absent (Long and Evans, 1922). At estrus the vaginal smear is "cheesy" and consists exclusively of non-nucleated cornified cells which stain a brilliant orange-red with Shorr's stain. This phase is occasionally divided into two stages (Long and Evans, 1922); during the second stage cornified cells are more abundant, although the female no longer accepts the male. In late estrus cornified cells begin to diminish and the leukocytes appear in the vaginal smear. At the same time, large basophilic epithelial cells with vesicular nuclei (so-called "Shorr" cells; Hartman, 1944) appear. During metestrus and early diestrus the smear is thick and consists mostly of leukocytes and a few corni— fied cells, Shorr cells and basophilic epithelial cells. During diestrus the vagina usually contains a little mucus in which are entangled a large number of leukocytes and a few nucleated basophilic and sometimes vacuolated epithelial cells (Mandl, 1951a). Although the vaginal smear is a reliable technique for diagnosing stage of the estrous cycle in the rat and also some other species, it cannot be used accurately in the case of the cow. Changes in the vaginal mucus occur, but they are not as clear cut in the cow as they are in many other species, probably because of the low hormone level in the cow at all times and also the complex nature Of the vaginal epithelium (Hansel, Asdell and Roberts, 1949). Hammond (1927), for the first time, studied the 10 smears taken from the vulvar region of the vagina of heifers. According to him, smears a few hours before heat are characterized by few vaginal epithelial cells present owing to their dilution with mucin. For about 6 days after the onset of heat smears from the vagina generally show abundant leukocytes and often red blood cells 2 to 4 days after the beginning of heat. During the remainder of the cycle only vaginal epithelial cells occur with now and then an occasional leukocyte. Since the part of the vagina closestto the vulva con- sists largely of mucus—secreting cells where true corni- fication does not occur, Hansel gt_§1. (1949) studied vaginal smears aspirated from the cervical end of the vagina but still did not observe any sharply distinguish- able pattern. And whatever changes they observed varied so much between individuals as to render it useless as a diagnostic tool. In contrast, naked eye observations of the vaginal discharge at different stages of the cycle are fairly characteristic in the cow (Hammond, 1927). The vagina is comparatively dry during the greater part of the cycle; a day or two before heat it becomes rather moist, at the beginning of heat it shows a flow of clear fluid mucus; and toward the middle or end of the heat the flow contains small cheesy yellowish-white lumps. About the end of heat the mucus becomes thicker and just after heat frequently (Ii 11 whitish in color and very much less in amount. In most heifers this is followed about the second or third day after the beginning of estrus by a flow of mucus stained with blood which usually lasts from 6-20 hours but may continue intermittently for 3 days. It must be noted, however, that although prevalence of a certain type of cell in the vagina is highly corre- lated with the ovarian cycle of the animal, it is by no means a completely foolproof index of the physiological state of the animal. For example, estrus in the rat may occur in the absence of a cornified smear, and vice-versa (Young, 1941). According to Blandau, Boling and Young (1941), the "copulatory response" is a better test of sexual receptivity. Cyclic vaginal changes, similar in general character and duration, though not in intensity to those present in normal animals, also occur in ovari- ectomized rats (Mandl, 1951b). D. Uterine Changes During the Estrous Cycle Despite occasional aberrations, the estrous cycle forms the nucleus of mammalian reproductive function and the uterus and other reproductive organs undergo marked morphological and biochemical changes during its various phases. Increases in water content of the uterus during the estrogenic phase of the cycle have been observed by many workers (Astwood, 1939). Uterine weight—-dry as well as 12 wet--increases dramatically during the estrogenic phase in virtually all species studied (Astwood, 1939; Dirschel, Schriefers and Bruener, 1955; Olds and VanDemark, 1957), as does volume and length of uterine horns in the rat (Cullen and Harkness, 1964). These physical changes are aaccompanied by increases in such biochemical entities as LHTA (Drasher, 1952), nitrogen (Morgan, 1963) and non— ccfillagen protein (Mortan, 1963), all signifying growth of tune organ. But these changes represent growth of all com- pcnuents of the uterus: epithelial, myometrial as well as ccnanective tissue. Since each of the above components may IKESpOfld differently under different physiological con— cdijsions, some experiments have directed attention to the Strudy of individual components. In order to study alterations in the connective tSIJssue framework, hydroxyproline, an index of collagen (bheuman and Logan, 1950) and hexosamine, an index of Brnound substance (Allison and Smith, 1965) have been mea- Sttredw Contrary to the growth parameters (weight, DNA and Ililzrogen) the collagen content of the uterus decreased to lcnn values during estrus from maximal values during di- €38tzrus (Morgan, 1963; Smith and Kaltreider, 1963). This Suggested a catabolism and/or reduction in the formation of‘ connective tissue in transition from diestrus to eStxrus, but simultaneous increases in nitrogen and non- cOlllagen protein suggested a replacement by non-connective tisSue elements. 13 On the other hand, hexosamine continued to increase during this same transition period (Morgan, 1963), indi- cating that laying down of ground substance proceeds dur- ing the growth of each type of tissue component. Other parameters of uterine metabolism also Show cyclic fluctuations. RNA and RNA:DNA ratio (indices of protein synthetic activity) in mouse uterus attained maxi- Inal levels during estrus and minimal values during di- estrus (Drasher, 1952). Oxygen consumption in the rat uterus was greatest during late diestrus and proestrus (Saldarini and Yochim, 1967). B-glucuronidase activity in the mouse uterus (Leathem, 1959), but not in the rat (Saldarini and Yochim, 1967), reached high levels during Lxroestrus and declined during diestrus, although the re- liationship between this enzyme and the growth process is not clear. Reduced diphosphopyridine nucleotide (DPNH) oxidase 118 an essential mediator of several DPN-linked dehydro- geniation reactions and, together with lactic dehydrogenase, (nontrols the reversible oxidation and reduction of lactate and.pyruvate at the end of the glycolytic sequence of re- actions. Both of these enzymes in the rat uterus dis- played maximal activity during proestrus and early estrus and minimal activity during diestrus (Bever, Velardo, Telfer, Hisaw and Goolsby, 1954). Another study (Rosa and Velardo, 1959) revealed similar changes in two of the 14 other oxidative enzyme systems of the uterus: DPN- diaphorase and succinic dehydrogenase. Lipid components of the uterus also vary during the estrous cycle. Lecithin and free cholesterol, but not cholesterol esters, increased in the pig uterus during the progestational phase of the cycle (Okey, Bloor, and Corner, 1930). In the mouse uterus, progesterone in- jection slightly enhanced lipid concentration, whereas estrogen reduced it significantly (Leathem, 1959). The function of uterine glycogen has always been more or less of a mystery. Because it appeared in large amounts in the primate endometrium in the progestational phase, a time when the uterus prepares to receive the egg, it was considered a nutritive agent (Van Dyke and Chen, 1936). But in the rat, glycogen probably plays a differ- ent role, since it was present in high concentration dur- ing the estrogenic phase and low at the time of implan- tation (Boettiger, 1946). Glycogen is also deposited in large amounts in the myometrium and is thus considered a source of energy for contraction (Telfer, 1953). The morphological changes that occur in the uterus during the estrous cycle have been reviewed extensively by Eckstein and Zuckerman (1956) and the account that follows has been adapted mainly from them. In the cow, during proestrus, the epithelium lining the uterine cavity is tall columnar and pseudostratified 15 with basal nuclei. Rarely it may be ciliated. But the glandular epithelium is not as tall as that lining the uterine cavity. The basal glands are spiral and the stroma very vascular. Although no extravasation of blood occurs, the epithelial cells lining the uterine cavity may contain blood pigment. At estrus, the glandular epithelium of the uterus is taller; the stroma more edematous. And scattered throughout the stroma are many cells including erythrocytes. Two days after estrus the basal glands are more coiled and filled with secretion, while the stroma is less edematous. Glandular hypertrophy and vascularity of the stroma are most marked 8 days after estrus and signs of regression appear about the 15th day. In the rat, during proestrus, uterine horns fill with watery fluid and the epithelium is lined with cuboidal cells. Estrus is marked with the signs of degeneration of the epithelium such as loss of the basement membrane, vacuo- lar degeneration and leukocytic invasion. But denudation of the uterine epithelium does not occur; few or no mitoses are ever seen. Uterine glands are small at this time but there are no visible degenerative changes either in the glands or in the stroma. During metestrus, degeneration and regeneration pro- ceed together and the uterus rapidly returns to the con- dition typical Of diestrus. During diestrus, the uterus 16 is small, avascular and has a slit-like lumen. It is lined by a simple columnar epithelium. Frequent mitoses occur in the endometrial glands of the mouse uterus dur- ing late diestrus (Allen, 1922). But in the rat, mitoses could not be observed in the glands and only rarely in the surface epithelium (Allen, 1931). E. Changes in the Mammary Glands During the Estrous Cycle To the best of my knowledge there have been practically no biochemical studies of the mammary glands during the estrous cycle. The only study available is by me (Sinha, 1964) in which I measured the DNA content of mammary glands of rats killed at specific ages without regard to stage of the estrous cycle. When the data were later pooled accord- ing to stage of the estrous cycle the results were not con- clusive. Yet the literature abounds with observations on the gross morphological changes in the mammary glands of numerous species during the estrous cycle (Turner, 1939). And in general, all agree that like the uterus, the mammary gland undergoes profound changes during different phases of the estrous cycle. In species with short estrous cycles, such as rats (Sutter, 1921) and mice (Bradbury, 1932; Turner and Gomez, 1933a; Cole, 1933) the duct system proliferated with the onset of the first estrous cycle while each subsequent estrus caused a further slight burst of growth resulting in a gradual cumulative growth of the duct system with the 17 succession of estrous cycles. During proestrus buds formed at the ends of the ducts while in early estrus the ducts distended with fluid and the buds elongated and sprouted. Regression and collapse of the duct system occurred during metestrus and diestrus. But alveoli and intralobular ducts did not form in the mammary glands during the estrous cycle. In animals with long estrous cycles, such as guinea pigs and cows, where some growth of the alveoli would be expected due to the long progestational phase of the cycle, very few, if any, alveoli form. Hammond (1927) has de- scribed changes in the mammary glands of virgin heifers during the estrous cycle. Like rats and mice, the lumen of the ducts were large and filled with secretion just before estrus and the epithelium was cuboidal. Eight days post-estrus, the lumina were small and the epithelial cells were almost columnar. But he did not find any alveoli at any stage of the cycle in virgin heifers. Nor was any observed in goats (Turner and Gomez, 1936) or guinea pigs (Turner and Gomez, 1933b). In the rabbit, which is in constant estrus, indi— cating an indefinite follicular phase, marked development of the duct system with terminal enlargements occurred (Ancel and Bouin, 1911), but in the ferret, which is also in constant estrus (Hammond and Marshal, 1930), no significant development of the duct system occurred. 18 IF. Changes in the Hormones of the Ovary, Anterior Pituitary and Hypothalamus During the Estrous Cycle Most of the changes described in the uterus and the rnanmmry glands are brought about by ovarian hormones, eestrogen and progesterone, secreted in varying amounts (during the estrous cycle. Although relatively little is kniown about the exact levels of secretion of these hor- nnones during the normal estrous cycle, the general pattern (3f their secretion is apparent in several species. Estrogens. Follicular fluid of the cow ovary con- izained ten times more estrogenic activity during the :follicular phase than during the luteal phase (Paredis, 31950), and estrogens in the peripheral blood plasma fWDllowed a similar pattern (Ayalon and Lewis, 1961). But Ineasurement of estrogen activity in the urine of cycling clows has not yielded consistent results (Mellin and Erb, 31965). In the sow, however, urinary estrogen excretion iJTvariably was greatest just before or at the onset of eBStrus (velle, 1958; Raeside, 1963; Liptrap and Raeside, 1966). Presumptive evidence that the level of estrogen Secretion increases with the enlargement of the follicles was also provided by the demonstration that more and more eStrogen was required to maintain perineal turgescence in baboons (Gillman and Gilbert, 1946). In animals with short estrous cycles, such as rats and mice, while no actual data are available, it has . F qsrg'fl, ' EVDDV. ac.-. . . b :«wu 2f‘ 6-...‘. v u...\ . " avg-1a .. "V~ ‘: ’l . . ~ 0 an" ‘oi-‘u V" n"? : ‘Iou _7 '3 Q 19 generally been assumed that estrogen is secreted maxi- Inally at the time of estrus, based on the observation tfluat cornified vaginal smears and estrus behavior occur sinufltaneously. But since it takes 48 to 72 hours for \Laginal epithelium to proliferate and then degenerate iJnto cornified cells, estrogen must be elaborated several knours before estrus (Young, 1961). The dilation of the titerus with fluid late in proestrus (Astwood, 1939) is zalso evidence that effective estrogen secretion had been eslaborated 24-30 hours earlier. Progesterone. The concentration of progesterone in tbovine corpora lutea showed an initial rise between day l arui 5, remained constant until day 9 or 10 and then in— <3reased to a peak on day 14 and 15 followed by a decline 130 the next estrus (Mares, Zimbleman and Casida, 1962; (30Ues, EStergreen, Frost and Erb, 1963). Concentration < macho amaoso msonpmm Ho swepm msosumm 141i .mmHoso msosumm m>Hm pmHHH 03p wcHHSU mums Ho panos OCHHOH: mwmpm> 0.05) between proestrus (0.88 mg) and estrus (0.85 mg) or between metestrus (0.70 mg) and diestrus (0.70 mg), it decreased 18% (P < 0.01) between estrus and metestrus sug- gesting a significant loss of uterine cells during the early luteal phase of the cycle. These losses in uterine DNA were, however, consistently regained between diestrus and the subsequent proestrus as reflected by a 26% increase (P < 0.01) in DNA between these periods. Similarly to uterine weight, uterine DNA increased (P < 0.01) cumulatively between the first and second (23%) and the second and third (16%) cycles but no further cumu- lative increases occurred between the third and fifth cycles. The weight of the uterus continued to increase between the third and fifth cycles (Table 2). Thus it would appear that the small increases in uterine size after the third cycle may be due to hypertrophy. One can conclude from the cumulative DNA data that pubertal growth of the uterus is largely complete after the third estrous cycle. 57 .HOHHO UHMUCMHm H :mmz** .mumh NH pmcHMHCOO macaw comm: 0a.0 00.0 00.0 00.0 mmssmsa 00.0 00.0 H 0a.0 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 0 00.0 00.0 H 0s.0 00.0 H 0s.0 00.0 H 00.0 00.0 H 00.0 0 00.0 00.0 H 0s.0 00.0 H 00.0 No.0 H 00.0 00.0 H 0H.H 0 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H aa.o m 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 H 0030 0 00H\0s manpmmHo manpmmpoz mappmm mskpmmopm .oz mwmpm>< amHoso msoppmm Ho mwmpm mmwmmmm .mmHomo msonpmm O>HH pmHHH one mchsp mums Ho pcmpcoo <20 mchmubll.m mqm< 0H.H 00.0 H 00.0 00.0 H 05.0 00.0 H mm.H 00.0 H 00.H 0 0H.H 00.0 H 00.0 00.0 H 05.0 0H.0 H 00.H 0H.0 H o5.H 0 0H.H 00.0 H 00.0 00.0 H 05.0 0H.0 H 0H.H 00.0 H o0.H 0 00.H 00.0 H 00.0 00.0 H 00.0 0H.0 H 00.H 0H.0 H 00.H m 00.0 00.0 H 00.0 00.0 H 00.0 0H.0 H 00.0 0H.0 H 0H.H H 0030 0 00H\0s manpmmHo mappmmpoz mappmm mappwmosm .oz mmmsm>< macho *OHozo msoppmm Ho OMMHm muonpmm .mmHoso msompmm 0>HH pmHHH on» wcHHSU mama Ho pcmucoO < mHoso *mHoso msompmm Ho owmpm mzopumm .mmHoso msompmm 0>HH pmHHm map wCHHSU mums Ho OHpmH 0.05). This suggested a uniform rate of metabolic activity in the uterus during all the five estrous cycles studied. Lipids. Variations in the lipid content of the uterus (Table 6) paralleled the changes in the uterine weight (Table 2). From a maximum of 37.1 mg at proestrus, the lipid content declined 13% at estrus and a further 26% at metestrus, then increased to 27.6 mg at diestrus. Cumulatively, it increased at a decreasing rate, the total increase between the first and fifth cycles being 27%. It is remarkable that analogous to uterine RNA, the decline in lipid content began initially during estrus--a time when the uterine cells (DNA) have not yet regressed. This close parallelism between lipids and RNA would imply that lipids in the uterus are quite intimately associated with uterine metabolism during the estrous cycle. Indeed, en- hancement of lipid synthesis is one of the earliest de- tectable responses of the uterus to estrogen treatment (White, Handler and Smith, 1964). But the exact role lipids play in uterine metabolism is only speculative. They may furnish the constituents of the cell membrane in the for- mation of new cells. Whether they also serve as a source of energy in the growth and metabolism of the uterus or are involved in some other ways remains to be determined. .Honpm ppwpcmum H cmmz00 .mpmH NH pmchpcoo adopw comm: 62 0.50 0.00 0.00 H.50 mwmsm>< 0.00 0.0 H H.00 0.0 H 0.00 0.0 H 0.00 0.0 H 0.00 0 5.50 5.0 H 0.00 0.H H 0.H0 0.0 H 0.00 0.H H 0.00 0 0.00 0.0 H 0.H0 0.0 H 0.00 0.0 H 0.00 H.0 H 0.50 0 0.H0 0.0 H 0.00 0.0 H 0.00 0.0 H 0.00 0.0 H 0.00 0 0.00 0.0 H 0.00 0.0 H 0.00 0.0 H 0.50 0.0 H 0.00 H 0030 0 00H\0s msmpmmHo mappmmpmz manpmm mappmmopm .oz mmmsm>< mHomo smHozo msopumm Ho mwmpm mzospmm .mmHoso mSOHHmm 0>HH pmHHH map mcHszp mpmH Ho pampcoo UHQHH mcHHmppnu.0 HHmHB 63 Morphology. Photomicrographs of the uterus during proestrus, estrus, metestrus and diestrus are shown in Figure 4(a-h). At proestrus, the uterine lumen was large due to the distention of the horns with uterine fluid and the epithelium lining the uterine cavity appeared cuboidal in shape. The endometrium contained numberous well de- veloped uterine glands. At estrus, the uterine lumen shrank and the epithelium lining it changed to tall colum- nar in shape and revealed marked signs of degeneration such as loss of the basement membrane and vacuolar disin- tegration. The uterine glands were smaller than at pro- estrus. At metestrus and diestrus, the uterus remained small and avascular and possessed a slit-like lumen. The lumen was lined with simple columnar epithelium and the endometrium contained a few small uterine glands. The structural appearance of the uterus concurred in general with the changes in the biochemical components of the uterus. The endometrium and uterine glands were con- siderably reduced during metestrus and diestrus as com- pared with proestrus and estrus. The DNA content of the uterus also decreased at this time signifying a loss of cells from the uterus. Thus, regression of the endometrium and the uterine glands may contribute partially to the loss of DNA between estrus and metestrus. However, the contri- bution of leukocytes and other uterine cellular elements may also be involved in these shifts in the uterine DNA. Figure 4a.--Photomicrograph of the rat uterus during proestrus. X87 Ilillviil all- :"I‘- «KID-Din. tilt-50:01; 5 AI...) F‘ 0 {Vaughn awn-E Figure 4b.--Photomicrograph of the rat uterus during proestrus. X435 66 Figure 4c.—-Photomicrograph of the rat uterus during estrus. X37 Figure 4d.—-Photomicrograph of the rat uterus during estrus. X435. 68 Figure 4e.-—Photomicrograph of the rat uterus X87 during metestrus. Figure 4f.--Photomicrograph of the rat uterus ' during metestrus. X435 70 Figure 4g.--Photomicrograph of the rat uterus during diestrus. X8 Figure 4h.——Photomicrograph of the rat uterus during diestrus. X435 72 C. Mammary Development The influence of the estrous cycle on mammary development was studied both in dairy heifers and rats. In the case of heifers, mammary growth was assessed with parameters such as nucleic acids, protein, collagen, lipid and histological preparations whereas in the mammary glands of the rat only nucleic acids were deter- mined. Bovine Changes in the mammary glands of heifers were studied at days 2, 4, 7, 11, 18, 20 and 0 (day of estrus) of the estrous cycle. Unlike the rat experiment, no effort was made to categorize the groups according to the number of estrous cycles experienced. That is, in a particular group, the animals may have undergone from one to several estrous cycles. Nucleic Acids. The mammary gland weights and nucleic acid data of heifers are summarized in Table 7. In Table 8 the same data have been classified according to the con- ventional stages of the estrous cycle. The differences in any of the parameters among stages of the estrous cycle could not be found significant (P > 0.05) upon evaluation of the data by analysis of variance. But from the magni- tude of the standard errors of the means (Table 7) this was not unexpected. In fact, calculations in the case of 73 mnpcoe N.0H pmwmnm>0 mHmmHmn HHm HQ 000 030 .00000 pmmccwpm H cmmz00 .00005 HH.0 Ho soHpmHsmo 00000000 0 00H; .mHmHHmn m pmszocH @5000 30000 0H.0 H 00.0 0.00 H 0.00H H.00 H 5.0H0 0.0H H 0.00 000000 00.0 H 05.0 0.HH H 5.05 0.0H H 0.50 0.0 H 0.H0 00 00.0 H 05.0 0.0H H 5.HHH 0.0H H 0.H0H 0.0 H 0.H0 0H 00.0 H 05.0 5.0H H 0.50 0.0H H 0.00H 0.H H 0.50 HH 00.0 H 00.0 0.HH H 0.00H 5.5H H 0.HOH 0.0 H 0.00 5 00.0 H 05.0 0.0H H H.00H 5.00 H 0.50H 0.0 H 0.00 0 00.0 H 00.0 0.HH H 0.00H 0.5 H H.00H H.0 H 0.00 0 0H000 30 0H 00H\0s 30 0H 00H\0s 0030 0H 00H\0 0201020 020 020 panms 00H000 0000000 Ho 00o .mHomo 0300000 0:» wcHHsp mHmHHms 0HH0© Ho pcmpcoo pHom OHmHosc mpmeemzll.5 mHmcoo map 00 wchHooom mHmHHmn spHmp no 0300300 pHdHH 330 mcHHOHQHxOszn .CHmHOHQ .meHom 0H0H033 sameemzll.m mHm00 00000000 0 0003 .000008 m.ma 000000>0 0000H00 HHO mo 0&0 009 .000000: m 0005H00H mso0w £00m: 77 o.m H 0.00 0.03 H 0.00 m.0 H 0.00 020000 m.o H 0.3 3.0 H 0.m0 0.0 H 0.0 om 0.0 H 3.0 0.00 H m.M3 0.0 H 0.00 00 0.0 H 3.0 0.0 H m.mm 0.0 H 0.00 00 3.0 H 0.0 3.0 H 3.0m Tm H 0.2 0 m.0 H 3.00 0.0 H 0.03 0.0 H 0.00 3 0.0 H m.00 3.0 H 0.00 0.m H 0.00 m 30 pH ooa\0 30 00 ooa\0e **30 00 ooa\0 cHQHq 0CHH00meo0uzm 0H00o0m *0Hozo 0:0000m 00 >09 .0Homo 0500000 000 wcfi030 0000000 m0H00 no 0000000 0HQHH 000 0CHHo0amxo0000 .0H00o0a >00EE0zll.m mqm< 00.0 00.0 H 30.0 00.0 H mm.0 00.0 H 00.0 00.0 H 00.0 m 00.0 30.0 H 00.0 00.0 H 00.0 30.0 H 00.0 30.0 H mm.0 3 30.0 00.0 H 00.0 m0.0 H 00.0 00.0 H 00.0 m0.0 H 33.0 m M3.0 30.0 H 03.0 00.0 H 00.0 00.0 H 0m.0 m0.0 H 00.0 m 0m.0 00.0 H 0m.0 00.0 H 0m.0 m0.0 H 00.0 00.0 H 00.0 0 .020 0 000\00 03000000 030000002 0300mm 030000000 .02 0w000>< 00000 300000 030000m 00 00000 030000m .000000 0300000 0>00 00000 000 000030 0000 00 0000000 <20 000EE02:|.00 m0m 0.05). However, changes in mammary DNA content among the stages were not consistent from cycle to cycle. For example, in cycles 1 and 2, the increases in mammary DNA initiated during proestrus or estrus continued through metestrus, whereas in cycles 3, A, and 5, the peak DNA content attained at estrus declined somewhat during met- estrus. In this respect, there seemed to be a clear dis- tinction between cycles 1 and 2 and cycles 3, U and 5. Mammary DNA also increased cumulatively; 10% (P < 0.01) between first and second, 8% (P < 0.01) between second and third, 3% between third and fourth and less than 1% between fourth and fifth estrous cycles. From this it appeared that most of the pubertal mammary growth in the rat was completed by the fourth cycle. If ammmary DNA does not increase cumulatively after the fourth cycle, there are two possibilities with regard to the phasic changes in mammary DNA in the subsequent cycles: (1) the over-all increase in mammary DNA observed between proestrus and estrus does not occur, or (2) if it does occur, it must be offset by a proportional decrease in DNA during the luteal phase. The data of the present experiments do not provide conclusive evidence either way but if the rat mammary glands grow similarly to the heifer mammary glands during the estrous cycle, the latter alter- native is more probable. Indeed, an average of cycles 3, 4 and 5 (Table 11), which are more representative of a 87 mature cycle, does show such a trend: after an initial rise during proestrus, mammary DNA decreased during met— estrus with no appreciable change during diestrus. Such an hypothesis would also explain the morphological re- gression observed in the mammary glands during the luteal phase of the cycle by earlier workers (Turner, 1939). Although mammary DNA did not increase cumulatively after the fourth cycle, trimmed and defatted weight of the mammary glands (Appendix VIII) increased during this per- iod. This suggested that growth of the connective tissue elements of the mammary glands may continue even after the bulk of the parenchymal growth had been achieved. The over-all changes in mammary RNA content (Table 12) during proestrus (1.86 mg) and estrus (2.10 mg) were similar to the changes in mammary DNA-—an initial rise (P < 0.01) of 13% during these two stages. At metestrus, the RNA content (2.10 mg) remained constant. But between metestrus and diestrus (1.92 mg), unlike mammary DNA, the RNA content decreased 9% (P < 0.01). An analysis in terms of the first two and the last three cycles (Table 11) re- vealed that although in the first two cycles RNA increased 5% between estrus and metestrus, it decreased 5% in the last three cycles'during the same period. This decline of RNA during metestrus and the over-all decline during dies- trus (Table 12) together with the decline in DNA during :metestrus of the third, fourth and fifth cycles (Table 11) 88 .00000 00000000 M 000200 .0000 00 000000000 03000 0000* 00.0 00.0 00.0 00.0 00000>0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0,0m.m 00.0 0 00.0 m 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 m 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 m 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 0 **3m 0 oo0\00 03000000 03000000: 030000 030000000 .02 0m000>< 00000 000000 0300000 00 00000 0300000 .000000 0300000 0>00 00000 000 000030 0000 00 0000000 <20 000EE02II.N0 0000B 89 suggested that the mammary glands of the rat involute dur- ing the luteal phase of the cycle as observed histologically by earlier workers (Turner, 1939). This involution may con- sist-of lowered cellular activity (loss of RNA) as well as probably some atrophy of the mammary cells (loss of DNA). Unlike uterine RNA:DNA ratio, mammary RNA:DNA ratio (Table 13) was neither less variable nor very consistent from cycle to cycle. However, on an over-all basis, it followed a pattern similar to the mammary RNA. The RNA:DNA ratio increased 8% (P < 0.05) between proestrus (1.30) and estrus (1.40), remained constant between estrus and metestrus (l.fl0) but decreased 9% (P < 0.05) between met- estrus and diestrus (1.28). Significantly, the mammary RNA:DNA ratio was considerably higher during the first (1.u6) and also somewhat higher during the second (1.36) cycles than during the third (1.32), fourth (1.28) and fifth (1.32) cycles. This fact provided another indication that the first and second estrous cycles may be different than the third, fourth and fifth cycles in yet another aspect: rate of metabolism. Having experienced the pinnacle of activity during the first and second cycles, the mammary gland, as it were, settles down to a more subdued rate of metabolism in the later cycles. The im- plication is that changes in the mammary glands during the first few cycles may not be quite representative of the changes that occur after puberty has been fully attained. 90 .00000 00000000 H 0002** .0000 00 000000000 03000 0000* 00.0 00.0 00.0 om.0 00000>< 0m.0 No.0 0 00.0 20.0 0 0m.0 No.0 0 mm.0 00.0 0 om.0 m 00.0 00.0 0 00.0 00.0 0 0m.0 00.0 0 00.0 00.0 0 00.0 : mm.0 00.0 0 00.0 00.0 0 :m.0 00.0 0 :m.0 00.0 0 00.0 m 0m.0 00.0 0 00.0 00.0 0 0m.0 00.0 0 00.0 no.0 0 om.0 m 00.0 00.0 0 02.0 00.0 0 00.0 00.0 0 00.0 00.0 0 :m.0 0 *000000 03000000 030000002 030000 030000000 .02 0m000>< 00000 000000 0300000 00 00000 0300000 .000000 0300000 0>00 00000 000 000030 0000 00 00000 0200020 000EE02II.m0 000 0.05). There was some indication, however, that the anterior pituitary weights may be low during diestrus (day 4 to 11) and high from shortly before to shortly after estrus (day 18 to day 2). TABLE lu.--Anterior pituitary weight of heifers during the estrous cycle. Day of No. of Anterior Pituitary* Cycle Animals (g) 2 5 1.59 i 0.18 0 5 1.33 i 0.00 7 5 1.35 i 0.07 11 5 1.36 i 0.08 18 5 1.57 i 0.08 20 5 1.u1 i 0.06 Estrus 5 l.fl3 : 0.04 *Mean 1 standard error. The anterior pituitary weights of rats during the five estrous cycles are given in Table 15. The anterior pituitaries gained weight progressively from the first through the fourth cycle, increasing from 5.2 mg during .00000 00000000 H 0002* .0000 00 000000000 03000 0000* 92 0.0 m.» m.» 0.0 00000>< 0.0 00.0 0 0.0 00.0 0 0.0 00.0.0 0.0 00.0 0 0.0 m 0.0 00.0 0 0.0 00.0 0 0.0 00.0.0 0.0 00.0 0 0.0 0 0.0 00.0 0 0.0 00.0 0 0.0 00.0 0 0.0 00.0 0 0.0 m 0.0 00.0 0 0.0 00.0 0 0.0 00.0 0 0.0 00.0 0 0.0 m 0.0 0m.0 0 0.0 00.0 0 0.0 00.0 0 0.0 00.0 0 0.0 0 0008 03000000 030000002 030000 030000000 .02 00000>< 00000 000000 0300000 00 00000 0300000 .000000 0300000 0>00 00000 000 000030 0000 00 000003 000003000 00000000ll.m0 0000B 93 the first cycle to 8.1 mg during the fourth cycle. Among stages of the cycle, they appeared to gain weight through all the stages in the first and second estrous cycles. But in cycles 3, A and 5, they gained weight only during proestrus and estrus whereas they lost weight during met- estrus and diestrus. The latter three cycles would indi- cate that there may be a cyclic fluctuation in pituitary weight according to the hormonal state of the cycle: high during estrogenic phase (proestrus and estrus) and low during the luteal phase (metestrus and diestrus). The ovarian weights during the estrous cycle of the rat are listed in Table 16. The ovarian weight increased progressively throughout the five estrous cycles but most of the increase (7%) occurred between proestrus and estrus. The formation of new corpora lutea after ovulation on the morning of proestrus may have contributed to the pro- nounced ovarian weight increase at this time. At no stage of the cycle was the ovarian weight significantly reduced, suggesting that the reduction in ovarian weight due to regression of old corpora lutea is compensated for by the concurrent growth of new follicles. E. Pituitary Luteinizing Hormone The LH concentration of the rat pituitaries during the various stages of the first five estrous cycles is given in Table 17. Due to insufficient amount of tissue available from single pituitary, several pituitaries .00000 00000000 H 0002*: .0000 00 000000000 03000 0000* 9U 0.0m m.0m m.mm 0.00 00000>< 0.00 0.0 0 0.00 0.0 0 0.m0 0.0 0 0.00 0.0 0 0.00 m 0.00 0.0 0 0.00 0.0 0 0.00 0.0 0 0.00 0.0 0 0.00 0 0.00 0.0 0 0.00 0.0 0 0.00 0.0.0 0.00 0.0 H 0.00 m 0.00 0.0 0 0.00 m.0 0 m.mm 0.0 0 0.00 0.0 0 0.00 m 0.0m 0.0 0 0.00 0.0 0 0.0m 0.0 0 0.00 0.0 0 0.0m 0 *000 03000000 030000002 030000 030000000 .02 00000>< 00000 *00000 0300000 00 00000 0000000 .000000 0000000 0>00 00000 000 000030 00000>o 000 000 00 000003|I.00 0000B 955 .0000000 00000600 000 00 00>00000 0000000000 00000500 “mm... .000000 00000 00 003000000 000 00 00000500 00000>0 00000003 000 00300>uc .00000000000 00 000000000 03000 0000 00 000000 00 000530 00 00000>0 000 00 0300> 0000. 00.0 u 00.0 00.0 n 00.0 00.0 u 00.0 00.0 n m0.0 0.0.0.0 000 00.0 00.0 0m.0 00.0 ..0>< neoo 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00000>< 00.0 n 00.0 00.0 00.0 0 00.0 .00000.0 00000.0 00000.0 00000.0 0 00.0 n 00.0 00.0 00.0 0 00.0 00000.0 00000.0 00000.0 00000.0 0 00.0.- 00.0 00.0 00.0 0 00.0 00000.0 00000.0 00000.0 00000.0 0 00.0 n 00.0 00.0 00.0 0 00.0 00000.0 00000.0 00000.0 00000.0 0 00.0 n 00.0 00.0 00.0 0 00.0 .00000.0 00000.0 0000m.0 00000.0 0 05\0n 0.0.0.0 000>< 00000>< 03000000 030000002 030000 030000000 0wwm0 umm 0500 .000000 0000000 00 00000 0300000 .000000 0300000 0>00 00000 000 000030 0000 00 0000000000000 0005000 00000000030 000003000I|.00 000<9 96 had to be pooled for the assay. The values in parentheses indicate the number of such pools assayed in each group. Since the LH content per pituitary (Appendix VI) dis- played almost identical trends, it is not included in the text. A preliminary statistical analysis of the data by the hierarchical method showed no significant change (P > 0.05) in the average LH concentration from the first through fifth estrous cycle. Therefore, to test for differences among the stages of the estrous cycle, the data from all cycles were pooled and analyzed with a one- way analysis of variance. This procedure resulted in a considerable increase in the degrees of freedom for error mean square and thus an increase in the sensitivity of the test. The analysis disclosed highly significant (P < 0.01) differences in LH concentration among stages of the estrous cycle. From a maximum of 0.72 ug/mg at proestrus, the LH concentration declined 57% to 0.31 ug/mg at estrus, re- mained relatively constant at metestrus (0.27 ug/mg) but increased 22% at diestrus (0.33 ug/mg). The correlation coefficients between average pitui- tary LH content and average uterine weight, uterine DNA and uterine RNA:DNA ratio were 0.59 (df = 18, P < 0.01), 0.uu (df = 18, P < 0.05) and 0.58 (df = 18, P < 0.01), respectively. The interpretation of the pituitary content data of any hormone is very hazardous. High as well as low levels of hormone in the pituitary gland have been associated 97 with both high and low secretion rates of the hormone. And in many instances both interpretations have been found to be correct. But one basic feature seems to hold true in most of the cases: under very abrupt and acute conditions of physiological stimulation, release of the hormone in blood is marked with a depletion of the hor- mone in the pituitary whereas under conditions of gradual and chronic stimulation, enhanced secretion of the hor- mone in the blood is accompanied by increases in the pituitary content of the hormone as well. Sometimes, the response of a target organ, whose specific relationship with the particular hormone is known, also helps inter- pretation of pituitary content data. The high concentration of LH in the pituitary ob- served on the day of proestrus in the present experiments was interpreted as indicating high levels of secretion of the hormone at proestrus. In fact, changes in the LH con- centration or content of the pituitary gland paralleled closely (r = 0.58) the changes in the uterine RNA:DNA ratio (Fig. 6). In this connection, it is known that LH is required together with FSH to stimulate estrogen secretion from the growing follicles (Everett, 1961), and estrogen promotes RNA synthesis in the uterus (Telfer, 1953; Gorski and Nelson, 1965). Thus, I interpret these data as indicating that LH influenced estrogen secretion which in turn stimulated the uterine RNA:DNA ratio. Uterine RNA:DNA ratio Pituitary LH (no/mg) I '60 I40 l-ZO l-OO 0’80 0'60 0°40 0°20 98 l L I j P E M 0 STAGE or ESTROUS CYCLE Figure 6.-—Eelationship between pituitary LH and uterine RNA:DNA ratio of rats during the estrous cycle. 99 During estrus, the LH concentration in the pituitary and the uterine RNA:DNA ratio, both declined. The drop in pituitary LH may have been caused by the feed-back action of estrogen or progesterone or both on LH, since estrogen in high amounts (McCann and Ramirez, l96u) and progesterone (McCann, 1962) both suppress LH secretion. On the other hand, the fall in the uterine RNA:DNA ratio may have occurred due to (l) a reduction in the blood titers of estrogen following ovulation on the morning of estrus or (2) inhibition of estrogen effect by progesterone (Velardo, 1959) which is secreted from the preovulatory follicles (Astwood, 1939) or (3) a combination of both factors. Metestrus was the period of lowest uterine protein synthetic activity (RNA:DNA ratio) as well as lowest LH concentration in the pituitary. But during diestrus the uterine RNA:DNA ratio increased significantly. This en- hancement of uterine metabolic activity may indicate stimu— lation of the uterus by estrogen at this time. Since it is known that there is a 6- to Zu—hour lag period between initial estrogen treatment and rise in RNA content of the uterus (Telfer, 1953), it would appear that significant estrogen secretion by the growing follicles is probably initiated early in diestrus. This hypothesis is supported by the observed increase (22%) in the pituitary LH con- centration between metestrus and diestrus. Although this 100 increase was not significant in the present study, Schwartz and Bartosik (1962) observed a similar increase in pituitary LH content at this time. The LRF content of the stalk median eminence (Ramirez and Sawyer, 1965; Chowers and McCann, 1965) is also elevated during diestrus which further supports the idea of increased LH and thus estrogen secretion during diestrus. From the preceding discussion the relationship be- tween LH and uterine metabolism during the rat estrous cycle may be summed up as follows: during metestrus, the LH and estrogen secretions are at basal levels and thus the uterus is quiescent. But low levels of estrogen dur- ing metestrus release the feedback inhibition of LH and therefore LH secretion increases during early diestrus. The LH, in synergism with FSH, stimulates estrogen secretion from the growing follicles. Since low levels of estrogen can stimulate LH release (Callantine, Humphrey and Nesset, 1966), the estrogen in turn stimulates more LH secretion culminating in the "ovulatory surge" of LH during pro- estrus. The estrogen is secreted maximally during pro- estrus which results in maximal uterine metabolic activity at this time and gradual suppression of LH during estrus. Then the estrogen secretion is either reduced after ovu- lation or its action on uterus is counteracted by pro- gesterone which is also secreted at a high level during proestrus (Porter, Siiteri and Yates, 1967; Hashimoto and 101 Melampy, 1967) resulting in lowered uterine metabolic activity during estrus and metestrus. And this cycle of events is repeated over and over again. F. Pituitary Prolactin Bovine The prolactin content of the heifer pituitaries dur- ing the various stages of the estrous cycle is given in Table 18 and Appendix VII. Pituitary gland from each heifer was assayed individually and the values in the table are the average of the five heifers in each group. TABLE 18.--Pituitary prolactin of heifers during the estrous cycle. ' **Mean 1 standard error. Day of Prolactin** Estrous Cycle* Concentration , Content IU/mg IU/pituitary 2 0.012 t 0.004 19.39 1 7.8 4 0.013 t 0.003 17.53 i 3.9 7 0.030 t 0.018 39.51 i 24.0 11 0.021 1 0.013 28.9u i 17.7 18 0.031 1 0.012 51.01 i 21.4 20 0.035 1 0.007 50°02'I 10.5 Estrus 0.095 f 0.027 67.16 t “2.2 *Each group included 5 heifers. 102 The prolactin content per pituitary paralleled the pro- lactin concentration data (Table 18). As usual with the heifer parameters, the within animal variation in pro- lactin concentrations was very large. Therefore, the changes in prolactin concentration among stages of the estrous cycle were statistically not significant (P > 0.05) when analyzed with a one-way analysis of variance. How- ever, evaluation of the data by regression analysis dis— closed a significant slope at a = 0.08 level of signifi- cance. Or, use of a t-test revealed significant differences between various comparisons such as day 2 vs day 20 of the cycle. Thus, even though the changes in prolactin concen- tration were of borderline statistical significance, some valid trends were still apparent. From a minimum of 0.012 IU/mg on day 2 of the estrous cycle, the prolactin concen- tration increased to a maximal value of 0.0H5 IU/mg on the day of estrus-—a sizeable increase of 275%. These results agreed with the findings of Day gg_a1. (1959) who, in the sheep, observed a significant linear increase in pituitary prolactin potency from day 2 to day 18-19 of the estrous cycle. The relationship between pituitary prolactin concen- tration and mammary RNA:DNA ratio is shown in Figure 7. Between day 18 of the cycle and the day of estrus, mammary RNA:DNA ratio and pituitary prolactin concentration in- creased 37% and “5%, respectively. During this interval Mammary RNA:DNA ratio Pituitary prolactin (lU/mg) 0'96 0°88 080 0-72 0045 0035 0025 005 0005 103 j J l l l 1 l 2 4 7 ll l8 20 E DAY OF ESTROUS CYCLE Figure 7.—-Re1ationship between pituitary prolactin and mammary RNA:DNA ratio of heifers during the estrous cycle. 104 estrogen is also secreted at an enhanced level. It appears, therefore, that high level of prolactin in the pituitary gland during this period reflected elevated levels of secretion of the hormone which synergized with estrogen to stimulate mammary metabolism. The drop in prolactin level of the pituitary be- tween estrus and day 2 of the cycle probably indicated a very high secretion rate of the hormone at this time, so much so that synthesis could not keep up with the release of the hormone. And this high level of prolactin secretion probably maintained the mammary RNA:DNA ratio at a rela- tively high level at day 2 of the cycle (0.8“ vs 0.96 at estrus) even though estrogen secretion is diminished by this time. After day 2, synthesis may have caught up with release of prolactin once again because the pituitary concentration began to rise steadily. There was no appreciable change in either pituitary prolactin or mammary RNA:DNA ratio on day A of the cycle. The slight increase in prolactin content on day 7 of the cycle may be wholly due to chance but the parallel rise in mammary RNA:DNA ratio at the same time may not be so: it may actually reflect the intimate re- lationship between prolactin and mammary growth during the estrous cycle. Between days 7 and 18 of the cycle, despite the gradual rise in pituitary prolactin concentration, mammary metabolic activity declined and reached its lowest 105 level. During this period estrogen secretion is also at its lowest ebb. Therefore, it appears that the level of prolactin secreted during this interval could not by it- self maintain mammary metabolism. Indeed, it is known that it takes very large amounts of prolactin to stimu- late mammary growth in the absence of estrogen (Talwalker and Meites, 1961). But stimulation of mammary growth is not the only function that prolactin performs: it is also said to be luteotrophic in rats and mice (Meites and Nicoll, 1966). Whether or not it has a similar role in the cow is contro- versial at the moment. Some researchers (Hansel, 1966) have produced evidence that LH is the luteotrophic hormone in the cow. But another view is that there may not be any one luteotrophic hormone but instead a "luteotrophic com- plex" in most species (Rothchild, 1966). In mice (Brown- ing, Larke and White, 1962) LR and prolactin seem to be the members of that complex; in hamsters (Greenwald, 1967) prolactin and FSH. Whether prolactin together with LH constitutes a part of that complex in the bovine remains to be shown but the indications of the present data are suggestive. A comparison of prolactin content with luteal pro- gesterone and pituitary LH content (data from Hackett, Hafs and Armstrong, 1967) of the same heifers used in this experiment is shown in Figure 8. Changes in pituitary Luteal Progesterone (uq/q) Pituitary LH (ug/mq ) Pituitary prolactin (Miami 106 60*- 4o- 20 P 350 - 2°50 - l-50 - 0°50 *- 0-040 - O°03O '- 0-020 '- b I- I 1 J 1 I 2 4 7 ll IS 20 E DAY OF ESTROUS CYCLE O-OIO Figure 8.——Re1ationship between pituitary prolactin and LH and luteal pro— gesterone of heifers during the estrous cycle. 107 prolactin content were very similar to the changes in the pituitary LH content except on the day of estrus when LH content decreased while prolactin content was still high. But both of these hormones paralleled the alterations in the progesterone content of the corpus luteum until day 18 of the cycle. After day 18, in contrast to prolactin and LH, the progesterone content of the corpus luteum declined abruptly. Just as the similarity between LH and progesterone secretion does not prove LH's role in corpus luteum function, neither does the parallelism between prolactin and progesterone. But a probable case for the involvement of prolactin in bovine luteal function is indi- cated at any rate. Indeed, the results of Snook, Brunner and Soatman (1967), in which they found that injections of antibovine LH serum reduced corpus luteum weight and total progesterone but did not alter progesterone concentration, may suggest that factors in addition to LH are involved. And one of those factors may very well be prolactin. Additional evidence in the favor of prolactin partici- pating in the function of the corpus luteum has also ap- peared recently. Progesterone secretion during the estrous cycle in stalk-sectioned cows (thereby presumably depriving the animal of LH but not prolactin) followed a curve identical to intact controls although at a reduced level (Henricks, Oxenreider, Anderson and Guthrie, 1967). In the sheep (Thibault, 1966), progesterone levels in the 108 stalk-sectioned animals were quite comparable to those observed in intact animals during the estrous cycle. But a final picture in this regard will have to await more investigation. Murine The prolactin concentration of the rat pituitaries during various stages of the first five estrous cycles is summarized in Table 19. The values in parentheses indi- cate the number of pituitary pools assayed in each group. Statistical analyses for the test of significance were performed identically to the LH data. Prolactin concen- tration among the five estrous cycles did not differ significantly from each other. But among stages of the cycle, pituitary prolactin concentration during proestrus (0.032 IU/mg) and metestrus (0.041 IU/mg) were significantly greater (P < 0.025) than during estrus (0.015 IU/mg) and diestrus (0.015 IU/mg). Changes in the prolactin content per pituitary (Appendix VIII) were similar to the changes in prolactin concentration. The correlation coefficients of average pituitary prolactin content with average mammary DNA, RNA and RNA: DNA ratio were 0.09 (df 18; P > 0.05), 0.01 (df = 18; P > 0.05) and -0.05 (df 19; P > 0.05), respectively. The maximal pituitary prolactin concentration ob— served during proestrus in the present study is in agree- ment with the reports of Reece and Turner (1937) who .zozoooo pocHoEoo 059 no Hm>pmch mocmofiucoo nocfioeoo mmmeax .Acmmav mmfiam mo oszvmoonq on» an pmcfiosoo ommnm>m pmucwfivz mum modam>ms ..mmmocpcopmo CH noumOfincfi qsopw comm :H mammwm ho Lopez: mwmpm>m map ma 03Hm> comm: 000.0uHH0.0 000.0-000.0 000.0-000.0 050.0:mH0.0 ..*.H.0 “m0 0H0.0 H00.0 0H0.0 000.0 ..w>0 0500 m00.0 H 0H0.0 000.0 H 000.0 000.0 M mm0.0 0H0.0 fl 000.0 mwmpm>< 000.0 n 000.0 0H0.0 HH0.0 H Hm0.0 AmUOH0.0 Amvmmo.0 Am0000.0 A00000.0 m 0m0.0 s ~H0.0 0m0.0 HH0.0 “ H00.0 Am00m0.0 Am0m00.0 Am0000.0 Amvamo.0 a 0m0.0 . 000.0 NH0.0 mH0.0 H mm0.0 Amva00.0 Amvma0.0 A00~H0.0 A000m0.0 m m00.0 u 0H0.0 sm0.0 ~00.0 H 0m0.0 A00~H0.0 A000NH.0 A000H0.0 A00000.0 0 000.0 . 0fl0.0 000.0 0H0.0 H 000.0 A000H0.0 A000~0.0 Amvm00.0 A00000.0 H we\0H . . mapummfia mahpmmpmz mzhuwm mahpmmopm .oz *mmxH o :xww¢ mwmpo>< macho uuo neoc :oHomo msoppmm mo owmum mzopuwm .mmaozo mzoppmo o>Hu umpfim on» wcfiosp mama mo :ofipMLucmocoo cfiuomaopa homuHSuHmtl.mH mqm<9 110 noted an increase in prolactin concentration between di- estrus and proestrus. However, they did not observe a decrease at estrus or an increase at metestrus as found in the present study. The findings of Everett (1945), who observed transient depletion of cholesterol in corpora lutea of rats during proestrus, also suggested an increase in prolactin secretion during proestrus. No direct evi- dence in the literature in support of elevated prolactin secretion during metestrus in rats is available to the best of my knowledge. But White and Browning (1962), based on observations of hyperemia of corpora lutea formed in intraocular ovarian grafts, suggested that prolactin, in mice, was released during metestrus. If the patterns of prolactin secretion during the estrous cycle in mice and rats are similar, the increased levels of pituitary pro- lactin observed during metestrus in the present study sup- ported that contention. Presumptive evidence that prolactin may be secreted at high levels during proestrus as well as metestrus is provided by the recent findings of Hashimoto and Melampy (1967). They observed two peaks of progesterone levels in the ovarian venous blood of the rat--one during pro— esterus, the other during early diestrus (comparable with metestrus of the present study)--suggesting prolactin stimulation of the corpora lutea during both of those periods. In fact, the prolactin data of the present 111 experiments combined with the progesterone data of Hashi— moto and Melampy (1967) would suggest, as proposed by Everett (1961), that the corpora lutea of rats may be functional during the estrous cycle. And the periods of enhanced activity may include two phases of the cycle: proestrus and metestrus. The relationship between pituitary prolactin concen- tration and mammary growth (DNA) is shown in Figure 9. During proestrus, pituitary prolactin concentration was high. At this time, estrogen secretion is also maximum (Astwood, 1939) as indicated by the RNA:DNA ratio of the uterus. These hormones probably contributed to the surge of mammary growth between proestrus and estrus. The lag between peak hormone secretion at proestrus and peak mammary cell proliferation at estrus was probably due to latent periods of these hormones with regard to their action on mammary tissue. This latent period may be also responsible for the low correlation between prolactin and mammary DNA (r = 0.09) and mammary RNA:DNA ratio (r = 0.05). The lack of increase in mammary cell numbers at metestrus may be the result of reduction in prolactin activity at the preceding estrus. 0n the other hand, the absence of significant increases in mammary cell numbers during diestrus following the peak of prolactin activity during metestrus may be due to the low levels of estrogen secreted at this time. It is known that it takes very 112 E (D é; '452: . <>\\“\---1> Q ‘~ p E v l-48 - ‘z‘ o P t‘ l-44 - a E E P O 2 I40 - 13, 0-040 - E . 2 .5 0.030 b 3 g i- 9. Q 0020 _ t‘ 9- , E’ h: O'OIO 1 1 n n P E M D STAGE OF ESTROUS CYCLE Figure 9.—-Relationship between pituitary prolactin and mammary DNA of rats during the estrous cycle. 113 large amounts of prolactin (probably more than is secreted during the rat estrous cycle) to stimulate mammary growth in the absence of estrogen (Talwalker and Meites, 1961). G. General Discussion The results of this research provided a comparison of the physiology of mammary growth and prolactin secretion during the estrous cycle of two species--the heifer and the rat. Although there were some subtle differences, the similarities between the two were marked. In both of these species, the estrous cycle had a profound influence on mammary growth and metabolism. In both of these species, protein synthetic activity in the mammary tissue was signifi- cantly enhanced only during the estrogenic phase of the estrous cycle—-that is, between proestrus and estrus. And in both species, mammary metabolic activity was considerably reduced during the progestational phase of the cycle as indi- cated by lowered RNA:DNA ratio during metestrus and diestrus. The lack of mammary growth during the luteal phase of the estrous cycle in the rat was not surprising since the rat possesses such a short luteal period. But in the heifer it indicated that the progesterone secreted during the .estrous cycle was probably not enough to influence mammary growth to any appreciable extent. The conclusion is that the influence of progesterone in bovine mammary growth does not become consequential until the onset of pregnancy. 114 Before pregnancy, it is the estrogen in concert with pro- lactin that plays the main role. The changes in the pituitary prolactin concentrations of the heifer were linear; in the rat there were two dis- tinct peaks. But in both species prolactin concentration, in general, appeared to be elevated during the estrogenic phase of the cycle. Since estrogen, in moderate doses, can stimulate prolactin secretion from the anterior pitui- tary (Meites and Nicoll, 1965), the low levels of estrogen secreted during late diestrus may have stimulated pro— lactin secretion in both species during the early estro- genic phase of the cycle. But what caused a second peak of prolactin concentration in the rat during metestrus remains unexplained. Although enhancement of prolactin secretion during the early estrogenic phase seems almost certain in both species, the role prolactin plays at this time besides stimulating mammary growth and corpus luteum function is not so clear. Desjardins, Kirton and Hafs (1967) observed rapid depletion of pituitary prolactin after copulation in the rabbit and suggested that prolactin may be involved in ovulation. This hypothesis together with the possibility that prolactin may be associated with the process of im- plantation needs to be investigated. Mammary as well as uterine development has long been known to be influenced by the stages of the estrous cycle. But a study of their metabolism in the same animal revealed 115 some striking differences. Greatest metabolic activity (RNA:DNA ratio) of the mammary tissue was observed generally at estrus, a day later than that observed in the uterus. This delay of one day in the appearance of maximal protein synthetic activity between mammary and uterine tissue may reflect differences in the latent periods and/or optimal titers of the hormones necessary for mammary and uterine stimulation. In addition, mammary RNA:DNA ratio during the first and second estrous cycles was substantially higher than during the third, fourth and fifth cycles, whereas in the uterus the ratios during all cycles were virtually the same. Also mammary metabolism in the latter three cycles was at about the same level as the uterine metabolism in all cycles. There are two implications of this. Firstly, that mammary tissue is very sensitive to the hormones acting during the first and second cycles to which it becomes partially refractory during the later cycles. And secondly, that either this higher sensitivity to the hormones of the estrous cycle in the uterine tissue has occurred before the onset of the first estrus or that the refractoriness to these hormones never develops in the uterine tissue at all. Whatever may be the case, the above facts point to some important differences in the mechanism of mammary and uterine growth at the time of the initiation of estrous cycles. Similarities in the mammary and uterine growth during the estrous cycle were also quite salient. Measured in 116 terms of DNA, the pubertal growth of both the mammary gland and uterus was largely completed by the fourth cycle. The mammary gland of the rat begins to grow at a notably accel- erated rate (Sinha and Tucker, 1966; Cowie, 1949) at approxi- mately 21 days of age. Similar information in the uterus is not available, but it may be assumed to commence growing at a faster rate at about the same age. Then, puberty in the rat, according to the definition of Donovan and van der Werff ten Bosch (1965; see review) and based on mammary and uterine development, would appear to begin at three weeks of age and be completed after the animal has undergone four estrous cycles. The fact that uterine and mammary growth in the rat is completed by the fourth cycle has yet another more signifi- cant and practical implication. Does it take so few cycles to complete the pubertal mammary and reproductive growth in the bovine female as well? The data of the present experi— ments can notadequately answer this question. But Sinha and Tucker (1965) reported that mammary DNA in Holstein heifers plateaued at 9-10 months of age and the heifers in this study were approximately 16 months of age and con- tained no more DNA than the 9-10 month old heifers. The data of Desjardins (1966) indicated lack of significant increase in uterine DNA after 10 months of age. The heifers in Desjardins' (1966) study exhibited their first estrus at an average age of seven months. Thus it would appear that in the bovine too, relatively few cycles may be required 117 to complete the major portion of the pubertal mammary and reproductive growth. If that is the case, there may not be much advantage in terms of optimal mammary and uterine development to delay breeding of dairy heifers any longer than 10 months. CHAPTER V SUMMARY AND CONCLUSIONS The relationship between the reproductive and mammary development and endocrine function of bovine and murine female during the estrous cycle was investigated in these experiments. A. Estrous Cycle Parameters l. The length of the estrous cycle of heifers averaged 20.6 i 0.2 days. The age of vaginal opening of rats averaged 36.7 i 0.13 days. Vaginal opening coincided with proestrus or estrus smear in 58% of the rats. The mean length of the first (5.5 days) and second (5.2 days) cycles was significantly greater than the third (4.8 days) and fourth (4.9 days) cycles. B. Uterine Development 3. Uterine weight of rats declined from a maximum of 211 mg/100 g BW at proestrus to 125 mg/100 g BW at metestrus but increased to 134 mg/100 g BW at diestrus. Cumulatively, the uterine weight in- creased progressively throughout the five estrous cycle. 118 119 Uterine DNA of rats did not change between pro- estrus (0.88 mg/100 g BW) and estrus (0.85 mg/ 100 g BW) or metestrus (0.70 mg/100 g BW) and diestrus (0.70 mg/100 g BW) but declined 18% be— tween estrus and metestrus suggesting a signifi- cant 1oss of uterine cells during the early luteal phase of the cycle. Cumulatively, uterine DNA increased progressively from the first through the third estrous cycles but not thereafter. Uterine RNA content of rats was maximum at pro- estrus (1.50 mg/100 g BW), minimum at metestrus (0.68 mg/100 g BW) but increased during diestrus (0.86 mg/100 g BW). Cumulatively, it increased progressively from the first through the third estrous cycles but not thereafter. Uterine RNA:DNA ratio of rats decreased from a maximum of 1.70 at proestrous to a minimum of 1.00 at metestrus, then increased to 1.22 at di- estrus. Cumulatively, it did not change from cycle to cycle. Uterine lipid content decreased from a maximum of 37.1 mg/100 g BW at proestrus to 23.8 mg/100 g BW at metestrus, then increased to 27.6 mg/ 100 g BW at diestrus. Cumulatively, it increased at a decreasing rate throughout the five estrous cycles. 8. 120 The histological appearance of the uterus re- flected in general the biochemical changes in the uterus during the estrous cycle. C. Mammary Development 90 10. ll. 12. Mammary DNA of heifers increased from 97.8 mg/lOO lb BW at proestrus to 213.7 mg/100 1b BW at estrus then declined to 169.1 and 142.8 mg/100 1b BW during metestrus and diestrus, re- spectively, suggesting proliferation of mammary cells during estrogenic phase and loss of cells during the progestational phase of the estrous cycle. Mammary RNA of heifers increased from 70.7 mg/100 1b BW at proestrus to 198.2 mg/100 lb BW at estrus, then declined to 142.6 and 109.4 mg/ 100 lb BW at metestrus and diestrus, respectively. Mammary RNA:DNA ratio of heifers increased from 0.72 at proestrus to 0.96 at estrus, then de- clined to 0.84 and 0.78 at metestrus and diestrus, respectively. Mammary protein content of heifers increased from a low of 9.6 g /100 lb BW at proestrus to 22.6 g/100 lb BW at estrus, remained at about the same level during metestrus (21.8 g/lOO 1b BW), then declined to 14.5 g/100 lb BW during di- estrus. l3. l4. 15. 16. 121 Mammary lipid content of heifers increased from a low of 4.2 g/lOO lb BW at proestrus to 10.0 g/100 lb BW at estrus, remained at about the same level during metestrus (11.5 g/lOO lb BW), then declined to 8.3 g/100 lb BW during di- estrus. Mammary collagen content of heifers increased from a low of 15.6 mg/lOO lb BW at proestrus to 62.7 mg/100 lb BW at estrus, increased slightly during metestrus (71.0 mg/lOO lb BW), then de- clined to 38.7 mg/100 lb BW during diestrus. The histological appearance of the mammary gland of heifers reflected in general the biochemical changes in the uterus during the estrous cycle. Mammary DNA content of rats increased from 1.23 mg/100 g BW at proestrus to 1.32 and 1.47 mg/ 100 g BW at estrus and metestrus, respectively, and remained constant at diestrus (1.42 mg/100 g BW) in the first and second estrous cycles. In cycles 3, 4 and 5, however, mammary DNA in- creased between proestrus (1.53 mg/100 g BW) and estrus (1.65 mg/100 g BW) but declined at metestrus (1.54 mg/100 g BW) and remained con- stant at diestrus (1.56 mg/100 g BW). 0n the over-all basis, most of the increase in mammary DNA (8%) of rats occurred between proestrus 17. 18. 122 (1.41 mg/100 g BW) and estrus (1.52 mg/100 g BW), while the changes between estrus, metestrus (1.52 mg/100 g BW) and diestrus (1.50 mg/100 g BW) were not significant. Cumulatively, mammary DNA of rats increased between the first and the fourth estrous cycle but not thereafter. Mammary RNA content of rats, on the over—all basis, increased 13% between proestrus (1.86 mg/100 g BW) and estrus (2.10 mg/100 g BW), remained constant at metestrus (2.10 mg/100 g BW) but declined 9% at diestrus (1.92-mg/100 g BW), suggesting stimulation of mammary growth during the estrogenic phase of the estrous cycle and involution during the progestational phase. Mammary RNA:DNA ratio of rats on an over-all basis increased 8% between proestrus (1.30) and estrus (1.40), remained constant between estrus and metestrus (1.40) but decreased 9% between metestrus and diestrus (1.28). Mammary RNA:DNA ratio of rats was considerably higher during the first (1.46) and second (1.36) cycles than during the third (1.32), fourth (1.28), and fifth (1.32) cycles, suggesting enhanced mammary stimulation immediately following vaginal opening. D. 123 Endocrine Function 19. 20. 21. 22. 23. Weight of the anterior pituitary gland of heifers appeared to be high during the follicular phase and low during the luteal phase of the cycle. The weight of the anterior pituitary gland of rats, although increased through all stages of the first and second cycles, was high during the follicular phase and low during the luteal phase of cycles 3, 4 and 5. The weight of the ovaries of rats increased progressively throughout the five estrous cycles but most of the increase (7%) occurred between proestrus and estrus. The luteinizing hormone concentration of rat pituitaries was maximal at proestrus (0.72 ug/mg) which declined to 0.31 ug/mg at estrus, remained relatively constant at metestrus (0.27 ug/mg) but increased 22% at diestrus (0.33 ug/mg). There appeared to be a close parallelism between uterine development and LH secretion during all phases of the estrous cycle of the rat. The prolactin concentration of the anterior pituitary of heifers increased linearly from a minimum of 0.012 IU/mg on day 2 of the estrous cycle to 0.045 IU/mg on the day of estrus. There appeared to be a close parallelism between 24. 25. 124 mammary development and pituitary prolactin concentration during the estrogenic but not progestational phase of the estrous cycle of the heifer. The prolactin concentration of the anterior pituitary gland of rats during proestrus (0.032 IU/mg) and metestrus (0.041 IU/mg) were signifi- cantly greater than during estrus (0.015 IU/mg) or diestrus (0.015 IU/mg), There appeared to be a close parallelism between mammary develop- ment and prolactin concentration during the estrogenic but not progestational phase of the estrous cycle of the rat. It is concluded that the pubertal development of the reproductive and mammary apparatus of the heifer and the rat is completed within a few estrous cycles. In addition, although there are intrinsic differences in the mechanism of reproductive and mammary growth, both are heavily dependent upon the estrous cycle whose integrity is essential for the normal reproductive function of the female. BIBLIOGRAPHY BIBLIOGRAPHY Allen, B. 1922. The oestrous cycle in the mouse. Am. J. Anat., 30:297. Allen, W. M. 1931. Cyclical alterations of the endo- metrium of the rat during the normal cycle, pseudo- pregnancy, and pregnancy. Anat. Rec., 48:65. Allison, D. J., and Smith, Q. T. 1965. Application of a rapid and reliable method for determination of hexosamine in the skin of estrogen-treated rats. Anal. Biochem., 13:510. Ancel, P., and Bouin, P. 1911. Recherches sur 1es fonctions du corps Jaune gestatif. II. Sur 1e determinisme du developpement de la gland mammaire au cours de la gestation. J. de Physiol. et de Path. Gen., 13:31. Anderson, L. L. 1966. Pituitary-ovarian-uterine relation- ships in pigs. J. Reprod. Fert., Suppl. 1, p. 21. Anderson, R. R., and McShan, W. H., 1966. Luteinizing hormone levels in pig, cow and rat blood plasma during the estrous cycle. Endocrinology, 78:976. Amstrong, D. T., Black, D. L., and Cone, C. E.- 1964. Metabolic studies with active and involuting bovine corpora lutea (Abstract), Fed. Proc., 23:462. Asdell, S. A.‘ 1946. Patterns of Mammalian Reproduction, Comstock Publishing Co., Inc., Ithaca, New York. Asdell, S. A. 1965. Patterns of Mammalian Reproduction, 2nd Ed., Cornell Univ. Press, Ithaca, New York. Asdell, S. A., deAlba, J., and Roberts, S. J. 1949. Studies on the estrous cycle in dairy cattle: cycle length, size of corpus luteum, and endometrial changes. Cornell Vet., 39:389. Astwood, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Am. J. Physiol., 126:162. 126 Be 127 Ayalon, N., and Lewis, I. 1961. Oestrogen levels in the blood and ovarian cyst fluid of dairy cattle. Proc. IVth Intern. Congr. Animal Reprod., 2:332. Bearn, J. G., Gould, R. P., and Jones, H. E. H. 1960. The response of the adrenal gland of the rat to pregnancy and lactation. Acta Anat., 41:273. Bever, A. T., Velardo, J. T., Telfer, M. A., Hisaw, Jr., F. L., and Goolsby, C. M. 1954. Changes in enzyme activity in uterus of rats during estrous cycle. Fed. Proc., 13:13. Blandau, R. J., and Money, W. L. 1943. The attainment of sexual maturity in the female albino rat as determined by the copulatory response. Anat. Rec., 86:197. Bliss, C. I. 1952. The Statistics of Bioassay. Academic Press, Inc., New York, N. Y. Bliss, C. I. 1956. Analysis of the biological assays in U. S. P. XV. Drug Standards, 24:33. Blunn, C. T. 1939. The age of rats at sexual maturity as determined by their genetic constitution. Anat. Rec., 74:199. Boccabella, A. V., and Alger, E. A. 1961. Thyroid: serum radio-iodine concentration ratio during the estrous cycle of the mouse. Proc. Soc. Exp. Biol. Med., 108:484. Boettiger, E. G. 1946. Changes in the glycogen and water content of the rat uterus. J. Cell. Comp. Physiol., 27:9. Bradbury, J. T. 1932. Study of the factors influencing mammary development and secretion in the mouse. Proc. Soc. EXp. Biol. Med., 30:212. Brown—Grant, K. 1962a. Changes in thyroid gland activity during the oestrous cycle in rats. J. Physiol., 161:557. Brown-Grant, K. 1962b. Variations in thyroid gland activity during the oestrous cycle in guinea pigs. J. Endo- crinol., 25:405. Browning, H. C., Larke, G. A., and White, W. D. 1962. Action of purified gonadotrOpins on corpora lutea in the cyclic mouse. Proc. Soc. Exp. Biol. Med., 111:686. 128 Callantine, M. R., Humphrey, R. R., and Nesset, B. L. 1966. LH release by 17 B-estradiol in the rat. Endocrinology, 79:455. Chowers, 1., and McCann, S. M. 1965. Content of luteiniz- ing hormone-releasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers. Endocrinology, 76:700. Clark, R. H., and Baker, B. L. 1964. Circadian periodi- city in the concentration of prolactin in the rat hypophysis. Science, 143:375. Cole, H. A. 1933. The mammary gland of the mouse during the oestrous cycle, pregnancy and lactation. Proc. Royal Soc., B., 114:136. Cowie, A. T. 1949. The relative growth of the mammary gland in normal, gonadectomized, and adrenal- ectomized rats. J. Endocrinol., 6:145. Cullen, B. M., and Harkness, R. D. 1964. Effects of ovariectomy and of hormones on collagenous framework of the uterus. Am. J. Physiol., 206:621. David, M. A., Fraschini, F., and Martini, L. 1966. Con- trol of LH secretion: role of a "short" feedback mechanism. Endocrinology, 78:55. Day, B. N., Anderson, L. L., Hazel, L. N., and Melampy, R. M. 1959. GonadotrOphic and lactogenic hormone potencies of gilt pituitaries during the oestrous cycle and pregnancy. J. Ani. Sci., 18:675. Desjardins, C. 1966. Endocrine and reproductive develop- ment in the bovine female from birth through puberty. Ph.D. Thesis. Michigan State University, East Lansing, Michigan. Desjardins, C., Kirton, K. T., and Hafs, H. D. 1967. Pituitary levels of LH, FSH and prolactin after coitus in rabbits (Abstract). Red. Proc., 26:534. Dirschel, W., Schriefers, H., and Breuer, H. 1955. Action of steroid hormones on glycolysis and aldolase activity of the uterus of normal and castrate rats. Acta Endocrinol., 20:181. Donovan, B. T., and van der Werff ten Bosch, J. J. 1965. Physiology of Puberty, Edward Arnold (Publishers) Ltd., London. 129 Drasher, M. L. 1952. Morphological and chemical obser- vations on the mouse uterus during the estrous cycle and under hormonal treatment. J. Exptl. Zool., 119:333- Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics, 11:1. Duncan, G. W., Bowerman, A. N., Anderson, L. L., Hearn, W. R., and Melampy, R. M. 1961. Factors influenc— ing in vitro synthesis of progesterone. Endocrinology, 68:199. Eckstein, P., and Zuckerman, S. 1956. Changes in the accessory reproductive organs of the non-pregnant female. In Marshall's Physiology of Reproduction, 3rd ed., A. S. Parkes, Ed., Vol. 1, Part 1, Ch. 6, Longmans, Green and Co., London. Edgar, D. G., and Ronaldson, J. W. 1958. Blood levels of progesterone in the ewe. J. Endocrinol., 16:378. Everett, J. W. 1945. The microscopically demonstrable lipids of cyclic corpora lutea in the rat. Am. J. Anat., 77:293. Everett, J. W. 1961. The mammalian female reproductive cycle and its controlling mechanisms. In Sex and InternaliSecretions, 3rd ed., William C. Young, Ed., Vol. I, Ch. 8. The Williams and Wilkins Co., Baltimore. Everett, J. W. 1964. Central neural control of repro- ductive functions of the adenohypophysis. Physiol. Rev., 44:373, 1964. Freudenberger, C. B. 1932. A comparison of the Wistar albino and Long-Evans hybrid strain of Norway rats. Am. J. Anat., 50:293. Gans, E., deJongh, S. E., van Rees, G. P., van der Werff ten Bosch, J. J., and Wolthuis, 0. L. 1964. Cyclic variations in the hypophyseal FSH-content in the female rat. Acta Endocrinol., 45:335. Gillman, J., and Gilbert, C. 1946. The reproductive cycle of the chacma baboon (Papio ursimus) with special reference to the problems of menstrual irregularities as assessed by the behavior of the sex skin. South African J. M. Sc., LL:Biol. Suppl. 130 Gomes, W. R., Estergreen, V. L., Jr., Frost, 0. L., and Erb, R. E. 1963. Progestin levels in jugular and ovarian venous blood, corpora lutea, and igaries of the non-pregnant bovine. J. Dairy Sci., =553. Gornall, A. G., Bardawill, C. J., and David, M. M. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem., 177:751, 1949. Gorski, J., and Nelson, N. J. 1965. Ribonucleic acid synthesis in the rat uterus and its early response to estrogen. Arch. Biochem. Biophys., 110:284. Greenwald, G. S. 1967. Luteotropic complex of the ham- ster. Endocrinology, 80:118. Hackett, A. J., Hafs, H. D., and Armstrong, D. T._ 1967. Relationships of pituitary LH and FSH to luteal and follicular activities (Abstract). J. Dairy Sci., 50:969. Hammond, J. 1927. The Physiologyof Reproduction in the Cow. Cambridge University Press, London. Hammond, J., and Marshall, F. H. A. 1930. Oestrus and pseudopregnancy in the ferret. Proc. Royal Soc., Bo, 105‘607e Hansel, W. 1966. Luteotrophic and luteolytic mechanisms in bovine corpora lutea. J. Reprod. Fert., Suppl. 1, p. 33. Hansel, W., Asdell, S. A., and Roberts, S. J. 1949. The vaginal smear of the cow and causes of its vari- ation. Am. J. Vet. Res., 10:221. Harris, G. W. 1948. Neural control of the pituitary gland. Physiol. Rev., 28:139. Hartman, C. G. 1944. Some new observations on the vaginal smear of the rat. Yale J. Biol. Med., 17:99. Hashimoto, I., and Melampy, R. M. 1967. Ovarian progestin secretion in various reproductive states and experi- mental conditions in the rat (Abstract). Fed. Proc., 26:485. Heald, P. J., Furnival, B. E., and Rookledge, K. A. 1967. Changes in the levels of luteinizing hormone in the pituitary of the domestic fowl during an ovulatory cycle. J. Endocrinol., 37:73. 131 Heap, R. B., and Linzell, J. L. 1966. Arterial concen- tration, ovarian secretion and mammary uptake of progesterone in goats during the reproductive cycle. J. Endocrinol., 36:389. Heape, W. 1900. The "sexual season" of mammals, and the relation of the "pro-oestrum" to menstruation. Quart. J. Micr. Sci., 44:1. Henricks, D. M., Oxenreider, S. L., Anderson, L. L., and Guthrie, H. D. 1967. Progesterone in systemic and ovarian venous blood and corpora lutea of cycling, hypophyseal stalk-sectioned, stalk-sectioned hyster- ectomized and stalk-sectioned oxytocin treated cows (Abstract). Fed. Proc., 26:366. Krulich, L., and McCann, S. M. 1966. Influence of growth hormone (GH) on content of GH in the pituitaries of normal rats. Proc. Soc. Exp. Biol. Med., 121:1114. Kwa, H. G., and Verhofstad, F. 1967. Prolactin levels in the plasma of female (C57BLxCBA) Fl mice. J. Endo- crinol., 38:81. Leathem, J. H. 1959. Some biochemical aSpects of the uterus. Ann. N. Y. Acad. Sci., 75:463. LePage, G. A. 1957. Methods for the analysis of phos— phorylated intermediates. In Manometric Techniques, 3rd ed., W. W. Umbreit, R. H. Burris, and J. F. Stauffer, Eds., Ch. 16. Burgess Publishing Co., Minneapolis, Minnesota. Li, J. C. R. 1964. Statistical Inference. Vol. 1,, Edward Brothers, Inc., Ann Arbor, Michigan. Liptrap, R. M., and Raeside, J. I. 1966. Luteinizing hormone activity in blood and urinary oestrogen excretion by the sow at oestrus and ovulation. J. Reprod. Fert., 11:439. Long, J. A., and Evans, H. M. 1922. The oestrous cycle in the rat and its associated phenomena. Mem. Univ. Calif., 6. Mandl, A. M. 1951a. The phases of the oestrous cycle in the adult white rat. J. Exp. Biol., 28:576. Mandl, A. M. 1961b. Cyclical changes in the vaginal smear of adult ovariectomized rats. J. Exp. Biol., 28:585. 132 Mares, S. E., Zimbleman, R. G., and Casida, L. E. 1962. Variations in progesterone content of the bovine corpus luteum of the estrual cycle. J. Animal Sci., 21:266. Masuda, H., Anderson, L. L., Henricks, D. M., and Melampy, R. M. 1967. Progesterone in ovarian venous plasma and corpora lutea of the pig. Endocrinology, 80:240. McCann, S. M. 1962. Effects of progesterone on plasma luteinizing hormone activity. Am. J. Physiol., 202:601. McCann, S. M., and Ramirez, V. D. 1964. The neuroendo— crine regulation of hypophyseal luteinizing hormone secretion. Recent Progr. Hormone Res., 20:131. McClintock, J. A., and Schwartz, N. B. 1967. Pituitary FSH content in cycling female rats (Abstract). Fed. Proc., 26:366. Meites, J. 1966. Control of mammary growth and lactation. In Neuroendocrinology, L. Martini and W. F. Ganong, Eds., Vol , Ch 1 Academic Press, New York, N. Y. Meites, J., and Nicoll, C. S. 1965. In vivo and in vitro effects of steroids on pituitary prolactin secretion. Proc. First Internat. Cong. Hormonal Steroids, 2:307. Meites, J., and Nicoll, C. S. 1966. Adenohypophysis: Prolactin. Ann. Rev. Physiol., 28:57. Mejbaum, W. 1939. Uber die Bestimmung kleiner Pento- semengen, insbesondere in Derivaten der Adenylsaure. Z. Physiol. Chem., Hoppe- Seyler' s, 258: 117. Mellin, T. N., and Erb., R. E. 1965. Estrogens in the bovine--a review. J. Dairy Sic., 48:687. Mills, J. M., and Schwartz, N. B. 1961. Ovarian ascorbic acid as an endogenous and exogenous assay for cyclic proestrus LH release. Endocrinology, 69:844. Morgan, C. F. 1963. Temporal variations in the collagen, non-collagen protein and hexosamine of the uterus and vagina. Proc. Soc. Exp. Biol. Med., 112:690. Motta, M., Mangili, G., and Martini, L. 1965. A "short" feedback loop in the control of ACTH secretion. Endocrinology, 77:392. 133 Nelson, D. M., Norton, H. W., and Nalbandov, A. V. 1965. Changes in hypophyseal and plasma LH levels during the laying cycle of the hen. Endocrinology, 77:889. Neuman, R. E., and Logan, M. A. 1950. The determination of hydroxyproline. J. Biol. Chem., 184:299. Okey, R., Bloor, W. R., and Corner, G. W. 1930. The vari- ation in the lipids of the uterine mucosa in the pig. J. Biol. Chem., 86:307. Olds, D., and VanDemark, N. L. 1957. Luminal fluids of bovine female genitalia. J. Am. Vet. Med. Assoc., 131:555. Ostle, B. 1963. Statistics in Research, 2nd ed., The Iowa State University Press, Ames, Iowa. Paredis, F. 1950. Verhandelingen von de Kloninklyke. Vlaamse Acad. vor Geneeskunde, 12:296. Parlow, A. F. 1961. Bio-assay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human Pituitarijonadotropins, A. Albert, Ed., Charles C. Thomas Co., Springfield, Illinois, p. 300. Parlow, A. F., Anderson, L. L., and Melampy, R. M. 1964. Pituitary follicle-stimulating hormone and luteinizing hormone concentrations in relation to reproductive stages of the pig. Endocrinology, 75:365. Plotka, E. D., Erb, R. E., Callahan, C. J., and Gomes, W. R. 1967. Levels of progesterone in peripheral blood plasma during the estrous cycle of the bovine. J. Dairy Sci., 50:1158. Porter, J. C., Siiteri, P. K., and Yates, C. W., Jr. 1967. Secretion of progesterone by the ovary of the rat (Abstract). Fed. Proc., 26:533. Prockop, D. J., and Udenfriend, S. 1960. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem., 1:228. Raeside, J. I. 1963. Urinary oestrogen excretion in the pig at oestrus and during the oestrous cycle. J. Reprod. Fert., 6:421. Rakha, A. M., and Robertson, H. A. 1965a. Changes in levels of follicle stimulating hormone and luteiniz- ing hormone in the bovine pituitary gland at ovu- lation. J. Endocrinol., 31:245. 134 Rakha, A. M., and Robertson, H. A. 1965b. The sequence, time and duration of the release of FSH and LH in relation to oestrus and to ovulation in the sheep. J. Physiol., 183:67. Ramirez, V. D., and McCann, S. M. 1964. Fluctuations in plasma luteinizing hormone concentrations during the estrous cycle of the rat. Endocrinology, 74:814. Ramirez, V. D., and Sawyer, C. H. 1965. Fluctuations in hypothalamic LH-RF (luteinizing hormone-—re1easing factor) during the rat estrous cycle. Endocrinology, 76:282, 1965. Reece, R. P. 1939. Lactogen content of female guinea pig pituitary. Proc. Soc. Exp. Biol. Med., 42:54. Reece, R. P., and Turner, C. W. 1937. The lactogenic and thyrotropic hormone content of the anterior lobe of the pituitary gland. Mo. Agr. Exp. Sta. Res. Bul. 266. Robertson, H. A., and Hutchinson, J. S. M. 1962. The levels of FSH and LH in the pituitary of the ewe in relation to follicular growth and ovulation. J. Endocrinol., 24:143. Robertson, H. A., and Rakha, A. M. 1965. The timing of the neural stimulus which leads to ovulation in the sheep. J. Endocrinol., 32:383. Rosa, C. G., and Velardo, J. T. 1959. Histochemical observations of oxidative enzyme systems in the uterus and vagina of the rat. Ann. N. Y. Acad. Sci., 75:491. ROSS, Go To , Odell, W. De ’ and RanOI'd, Po Lo 1967. Luteinizing hormone activity in plasma during the menstrual cycle. Science, 155:1679. Rothchild, I. 1966. The nature of the luteotrophic process. J. Reprod. Fert., Suppl. 1, p. 49. Saldarini, R. J., and Yochim, J. M., 1967. Metabolism of the uterus of the rat during early pseudopregnancy and its regulation by estrogen and progesterone. Endocrinology, 80:453. 1967. Salisbury, G. W., and VanDemark, N. L. 1961. Physiology 9f Reproduction and Artificial Insemination of Cattle. W. H. Freeman 8 Company, San Francisco. 135 Santolucito, J. A., Clegg, M. T., and Cole, H. H. 1960. Pituitary gonadotrophins in the ewe at different stages of the estrous cycle. Endocrinology, 66:273. Schmidt, G., and Thannhauser, S. J. 1945. A method for the determination of deoxyribonucleic acid, ribo- nucleic acid and phosphproteins in animal tissues. J. Biol. Chem., 161:83. Schwartz, N. B. 1964. Acute effects of ovariectomy on pituitary LH, uterine weight, and vaginal cornifi- cation. Am. J. Physiol., 207:1251. Schwartz, N. B., and Bartosik, D. 1962. Changes in pituitary LH content during the rat estrous cycle. Endocrinology, 71:756. Schwartz, N. B., and Caldarelli, D. 1965. Plasma LH in cyclic female rats. Proc. Soc. Exp. Biol. Med., 119:16. Simkin, B., and Arce, R. 1965. Prolactin activity in blood during the normal human menstrual cycle. Proc. Soc. Exp. Biol. Med., 113:485. Simpson, M. E. 1959. Role of anterior pituitary gonado- tropins in reproductive processes. In Reproduction in Domestic Animals, H. H. Cole and P. T. Cupps, Eds., Vol. I, Ch. 3, Academic Press, New York. Simpson, M. E., van Wagenen, G., and Carter, F. 1965. Hormone content of anterior pituitary of monkey (Macaca mulatta) with Special reference to gonado- trophins. Proc. Soc. Exp. Biol. Med., 91:6. Sinha, Y. N. 1964. The normal mammary gland development of rats 10 to 100 days of age. M.S. Thesis. Michi- gan State University, East Lansing, Michigan. Sinha, Y. N., and Tucker, H. A. 1965. Udder growth of dairy heifers between birth and 12 months of age (Abstract), J. Dairy Sci., 48:800. Sinha, Y. N., and Tucker, H. A.- 1966. Mammary gland growth of rats between 10 and 100 days of age. Am. J. Physiol., 210:601. Smith, 0. W., and Kaltreider, N. B. 1963. Collagen con- tent of the nonpregnant rat uterus as related to the functional responses to estrogen and progesterone. Endocrinology, 73:619. 136 Snook, R. B., Brunner, M. A., and Saatman, R. R. 1967. Effect of antibovine luteinizing hormone in the cyclic heifer (Abstract). J. Dairy Sic., 50:1000. Soliman, F. A., and Nasr, H. 1962. Variation in gonado- trophic hormone-level during oestrous cycle. Nature, 194:154. Stockard, C. R., and Papanicolaou, G. N. 1917. The existence of a typical oestrous cycle in the guinea pig--with a study of its histological and physio- logical changes. Am. J. Anat., 22:225. Sutter, M. 1921. Cyclic changes in the mammary gland of the rat associated with the oestrous cycle. Anat. Rec., 21:59. Talwalker, P. K., and Meites, J. 1961. Mammary lobule- alveolar growth induced by anterior pituitary hor- homes in adreno-ovariectomized and adreno-ovariecto- mized-hypophysectomized rats. Proc. Soc. Exp. Biol. Med., 107:880. Telfer, M. A. 1953. Influence of estradiol on nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uterus. Arch. Biochem. Biophys., 44:111. Thibault, C. 1966. Luteal maintenance in hypophysectomized and hysterectomized sheep. J. Reprod. Fert., Suppl. 1, p. 63. Tucker, H. A. 1964. Influence of number of suckling young on nucleic acid content of lactating rat mammary gland. Proc. Soc. Exp. Biol. Med., 116:218. Turner, C. W. 1939. The mammary glands. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth, and E. A. Doisy, Eds., Ch. 11. The Williams and Wilkins Co., Baltimore. Turner, C. W., and Gomez, E. T. 1933a. The normal develop- ment of the mammary gland of the male and female albino mouse. Mo. Agr. Exp. Sta. Res. Bul. 182. Turner, C. W., and Gomez, E. T. 19330. The normal develop- ment of the mammary gland of the male and female guinea pig. Mo. Agr. Exp. Sta. Res. Bul. 196. Turner, C. W., and Gomez, E. T. 1936. The development of the mammary glands of the goat. Mo. Agr. Exp. Sta. Res. Bul. 240. 137 Velardo, J. T. 1959. Steroid hormones and uterine growth. Ann. N. Y. Acad. Sci., 75:441. Van Dyke, H. B., and Chen, G. 1936. Observations on the biochemistry of the genital tract of the female Mocaque, particularly during the menstrual cycle. Am. J. Anat., 58:473. Van Rees, G. P. 1964. Interplay between steroid sex hor- mones and secretion of FSH and ICSH. In Major Problems in Neuroendocrinology, E. Bajusz and G. Jasmin, Eds., p. 322. The Williams and Wilkins Co., Baltimore. Velle, W. 1958. Undersokelser over naturlig forekommende oestrogener hos droutyggere og gris. Thesis. Institutt for seksualfysiologzOg seksualpathologie. Norges Veterinaeshogskole, Oslo, Norway. White, W. D., and Browing, H. C. 1962. Evidence for the periodic release of pituitary luteotrOpin during the estrous cycle of the mouse. Texas Rep. Biol. Med., 20:484. White, A., Handler, P., and Smith, E. L. 1964. Principles of Biochemistry, 3rd ed., McGraw-Hill Book Company, Inc., New York. Young, W. C. 1941. Observations and experiments on mating behavior in female mammals. Quart. Rev. Biol., 16:135. Young, W. C. 1961. The mammalian ovary. In Sex and Internal Secretions, 3rd ed., William C. Young, Ed., Vol. 1, Ch. 7. The Williams and Wilkins Co., Baltimore. I‘ll-rill inlilj APPENDICES 138 APPENDIX 1 HEIDENHAIN'S FIXATIVE PROCEDURE AND COMPOSITION OF BOUIN'S FLUID 139 Heidenhain's Fixative Reagents: Water 90 ml Potassium dichromate 1.8 g Mercuric chloride 4.5 g Glacial acetic acid 4.5 ml Formaldehyde (40%) 10 ml 1. The fixative should be prepared immediately before use or as two solutions, one containing the acetic acid and formaldehyde and the other the remaining ingredients. Fixation.should take place in the dark. Tissue should be washed with 4% formaldehyde in the dark. Soak tissue over night in Lugol's Iodine. Lugol's Iodine: mix 1 g potassium iodide with'0.5 g of iodine. Add 2-3 ml of water and shake.until dissolved. Then dilute to 50 ml. Transfer to several changes of 70% aloohol until no further color comes out. Proceed with routine alcohol dehydration. 140 ‘Reagents: 141 Bouin's Fluid Picric acid (saturated aqueous solution) 75 ml Formaldehyde (40%) 25 ml Acetic acid (glacial) 4 ml APPENDIX II COMPOSITION OF THE RAT FEED 142 The rat feed was composed of the following ingredients: Ground shelled corn (1/8 inch screen) Soybean oil meal, 50% protein Alfalfa meal, 17% protein Fishmeal, 65% protein Dried whey Pro-strep 20, 0.54% penicillin and 2.72% streptomycin Pro-Gen, 20% arsanilic acid Vitamin A, 10,000 units/g Irradiated yeast, 9,000 units/g Choline chloride D, Ca Pantothenate Riboflavin Nicotinic Acid (niacin) Vitamin B-12 (0.1% mannitol trituration) DL alpha tocopherol acetate, 250 IU vitamin E/g Menadione (vitamin K) DL methionine Limestone Dicalcium phOSphate Iodized salt Manganous sulphate (MnSOu-H2O) 32.5% manganese Ferrous sulfate (FeSOu-7H2O) 20.9% iron 143 607.0 lb 280.0 lb 20.0 lb 25.0 lb 25.0 lb 4.0 oz 0.5 lb 364.0 38.0 318.0 2.5 1.5 15.0 3.0 8.8 1.0 GQOQOQOQOQOQOQOQOQOQ 227.0 16.01b 17.5 lb 5.0 lb 168.9 g 215.2 g 144 Calcium carbonate (CaCO3) 40.4% calcium 83.8 g Zinc carbonate, basic (ZnCO3) 56.0% zinc 40.2 g Cupric sulfate (CuSOu-SHZO) 25.45% copper 12.9 g Cobalt chloride (CoCl2-6H2O) 24.77% cobalt 4.7 g Potassium iodide (KI) 76.45% iodine 2.2 g This ration gave the following analysis: protein 21.2% fat 3.9% crude fiber 2.5% productive net energy 902 C/lb APPENDIX III COMPOSITION OF SOLUTIONS FOR PROTEIN ANALYSIS 145 Biuret Reagent Cupric sulfate (CuSOu-5H2O) 1.5 g Potassium sodium tartrate (KNaCuHu06.4H2O) 6 g Dissolve in about 300 ml of water Add slowly 300 m1 of 10% sodium hydroxide solution to the above mixture and l g of potassium iodide and bring the volume to 1000 ml. 146 APPENDIX IV COMPOSITION OF SOLUTIONS FOR HYDROXYPROLINE ANALYSIS 147 Resin-charcoal Preparation Cation-exchange resin (AG l-X8, 200-400 mesh, chloride form) 20 g Norit A 10 g Wash the mixture several times with 6N HCl in a course sintered-glass funnel. Dry with ethanol and ether to a fine powder. Borate Buffer Boric acid 61.84 g Potassium chloride 225.0 g Distilled water 800 ml Adjust pH to 8.7 with lON KOH. Make final volume to 1000 ml. Alanine Solution Alanine DL alpha 10 g Distilled water 90 ml Adjust pH to 8.7 with lON KOH. Make final volume to 100 m1. 148 (a) (b) (c) 149 Ehrlich's Reagent Sulfuric acid (concentrated) 27.4 ml Alcohol (absolute) 200 m1 Add acid to alcohol slowly. p-Dimethyloaminobenzaldehyde 120 g Alcohol (absolute) 200 m1 Mix in another beaker. Add (a) into (b) slowly while stirring. APPENDIX V PROCEDURE FOR ASCORBIC ACID ANALYSIS OF OVARIES IN LUTEINIZING HORMONE BIOASSAY 150 (a) (b) (C) (0) Reagents Metaphosphoric acid solution: 2.5% 2,6-dichlorophenol indophenol solution: dissolve 20 mg of 2,6-dichlorobenzenoneindophenol (Eastman sodium) in distilled water and bring the volume to 500 ml. Sodium acetate solution: dissolve 22.65 g of sodium acetate-3H2O in 500 m1 of distilled water, adjust pH to 7.0 with 0.6 ml of 6% acetic acid. Indophenol-acetate solution: mix equal volumes of (b) and (c). Analytical Procedure The trimmed ovary is homogenized with 2.5% meta- phosphoric acid in the dilution of 10 mg/ml and filtered through Munktell's No. 00 filter paper. 3 ml of the filtrate is added to 5 ml of the indophenol acetate solution and the color development is read be- tween 20 and 60 seconds in a Beckman DB Spectrophoto- meter at 515 mu against a distilled water blank. The amount of ascorbic acid in the ovaries is calcu- lated from a standard curve obtained by using U.S.P. Reference Standard Ascorbic Acid and is expressed as mg ascorbic acid per g of ovarian tissue. 151 APPENDIX VI PITUITARY LUTEINIZING HORMONE CONTENT OF RATS AND DATA FOR INDIVIDUAL ASSAYS 152 153 .mommzpcmnmo CH councfipsfi mammmm mo hopes: map mo owmpo>m cm ma msam>,nomm* am.m 00.0 0m.m m0.: mmmnmsa nm.m Amvma.m Amvm>.m Amvam.m Amvma.m m Hm.m AmVHH.m Amva0.H Am0H0.m A000~.m 0 Hz.m Amvmm.m Amvmw.m Amvmm.m Amvmw.m m H0.m Amvm0.m Amvma.m Amvflm.a Amvmm.m m HH.N Amvma.m AH000.H Aflvm0.H Aflvma.m H mumpHSpHQ\m: mappmmfio manpmmpmz mssumm magpmooum .oz mwmpm>< macho smflomo msoppmm mo owmpm msonpmm .mmaozo wzonpmo m>fim pmpfim map wcfipsu mums mo pcopcoo ocoeso: msfiufizfimpsa humpHSBHmII.H mqmm cm ma mzam> comm: =HH.o mm:.o mmfl.o wam.o mwmnm>< oum.o Amvmza.o Amvmma.o Amvzmm.o Amvuam.o m mmm.o Amvemfl.o Amvmom.o Amvmmm.o Amvmmm.o : omm.o Amvmmo.o Amvmzm.o Amvama.o Amvmom.o m mmm.o Amvmaa.o Amvmwm.o Amvmmo.o Amvzam.o m m:m.o Amvmoo.o Amvam:.o Amvomo.o Amvam:.o H .mnmpfispfia\pH moppmmda manpmmpmz manpmm moppmmonm .oz mwmam>¢ macho :mflomu msoppmm no mwmum msoppmm .mmaomo msonumm m>am ummfim on» weapso mama mo pompcoo cauowaoao unapflsufimll.a mqm<9 159 TABLE 2.--Pituitary prolactin data of rats for individual assays. Cycle Stage of Pituitary Prolactin gigggagg gsénggggéiegge Lambda No. Cycle. Pool No.. Potency Potency Potency vs/ms l P 1 3.35 3.09 0.91 - 15.12 0.79 2 7.49 9.44 1.76 — 66.20 0.90 E 1 0.39 0.58 0.03 — 1.82 0.96 2 0.25 0.38 0.02 - 1.10 0.90 M l 1.61 ' 1.46 0.39 - 6.17 0.79 2 7.39 7.46 2.12 - 42.74 0.78. D 1 0.43 0.62 0.03 - 1.99 0.96 2 0.80 0.93 0.11 - 3.46 0.90 2 P 1 3.21 209“ 0.87 - lue3l 0079 2 2.03.' 1.76 0.53 - 7.77 0.77 E 1 0.21— 0.37 0.01 - 1.00~ 0.96 M 1 13.88 16.33 3.83 -108.64 0.79 2 1032‘ 1016 0.32 - “.7“ 0.77 D 1 0.53' 0.74 0.05 - 2.48 0.96 2 lens 1059 0026 "' 6089 0090 3 P 1 3.08 2.81 0.83 - 13.54 0.79 2 0.29 0.29 0.05 - 0.92 0.71 2 0.62 0.53 0.14 - 1.91 ' 0.71 M 1 7.60 7.85 2.15 - 45.57 0.79 2 1.03 0.83 0.27 - 3.24 0.71 D 1 0.41 0.61 0.03 - 1.92 0.96 2 0.46 0.42 0.09 - 1.44 0.71 4 P 1 0.57 ' 0.58 0.10 - 2.03 0.79 2 3.72 3.32 1.06 - 16.43 0.77 E 1 0.46 0.66 0.04 - 2.13 0.96 2 1.90 1.64 0.49 - 7.16 0.77 3 5039 ' 6039 1.25 "" “0.36 0.90 M 1 2.18 -1.96 0.56 — 8.83 0.79 2 1.78 1.54 0.46 - 6.63 0.77 3 7.09 8.84 1.67 - 60.88 0.90 D l 0.44 0.64 ‘ 0.04 - 2.05 0.96 2 2.32 2.01 10.63 - 9.12 0.77 3 0.75 0.89 0.10 - 3.26 0.90 5 P 1 7.93 8.26 2.24 - 48.42 0.79 2 0.2“ 0.25 0.0" — 0.80 0071 E 1 1.18 1.43 0.16 - 6.03 0.96 2 0.15 0.07 ’ . 0.02 - 0.51 0.71 3 3.40 3.01 0.96 - 14.61 0.77 M 1 1.24 ' 0.98 0.34 - 3.97 0.71 2 1058 1037 0039 "' 5079 0.77 3 1.23 1.37 0.20 - 5.66 0.90 D l 0.30 0.30 0.05 - 0.96 0.71 2 1.19 1.05 0.28 - 4.20 0.77 3 1081 1096 0.3“ - 9.02 0.90 T v— 'P - proestrus, E - estrus, M - metestrus, D - diestrus. 160 a.mm m.Hm m.mm a.mm mmmnm>¢ m.am N.o> w.o> m.:n H.Hw m m.>m m.mw m.mm m.mm m.mw : m.mm m.Hm 3.00 5.Hw m.Hm m m.mm m.mm m.mm 5.2m o.m: m m.m: m.mm :.m: w.:: m.m: H 3m m ooa\we manummfia monpmmumz monpmm monummonm .oz mwmpm>< macho macho msoppmm go mwmpm msoapwm .mmaomo «3000mm m>Hm ompfim on» weapso mama mo pnwfios ocmaw momeems omuummmo com UmEEHnBII.m mqm¢e