ROLE OF HORMONES III CGNTROLLING RIBONUCLEIC ACID SYNTHESIS DURING LACTOGENESIS Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY HARVEY W. MOHRENWEISER 1970 s... o. .. v- . r; >;-—~_ W“ wwd. Irv-'v - . ..' ' L" M i" ' LIBRARY I"? Michigan State University This is to certify that the thesis entitled ROLE OF HORMONES IN CONTROLLING RIBONUCLEIC ACID SYNTHESIS DURING LACTOGENES IS presented by Harvey W. Mohrenweiser has been accepted towards fulfillment of the requirements for __Ph..ll._degree inhinnhemiatry and Dairy Major professor Date June 3, 1970 0-169 L "IRA-V IINHFRQ __ _ if r\ PM‘ K C. fag-«Tug :2. ' - (#3471323? ABSTRACT ROLE OF HORMONES IN CONTROLLING BIBONUCLEIC ACID SYNTHESIS DURING LACTOGENESIS By Harvey W. Nohrenweiser The onset of lactation, lactogenesis, is associated with a rapid increase in the RNA, DNA and protein content of the mammary gland and concurrently, the mammary epithelial cell acquires the ability to synthesize the various caseins, whey proteins and lactose. Mammary eXplants from midpre¢_ nant mice have two phases of develOpment. The first phase, which involves epithelial cell division may require only insulin and hydrocortisone. The second phase, which is dependent upon prolactin acting upon cells previously incu- bated with insulin and hydrocortisone, is the acquisition of the functions of a lactating cell. Prolactin enhances general RNA synthesis in eXplants and in endocrinectomized lactating rats while cortisol acts in a Specific manner in endocrinectomized rats. A 12-153 RNA fraction having prOperties associated with mRNA has been observed and several Species of RNA are detected in differentiated mam- mary tissue which are not present in mammary tissue from virgin mice. This report deals with the Species of RNA synthesized during lactogenesis and the role of hormones in regulating RNA synthesis in mammary tissue slices. Incorporation of 3H-uridine into trichloroacetic Harvey W. Nohrenweiser precipitated RNA during the last hour of 10 hour incubations was about 8-fold higher than the incorporation into RNA fractions isolated by phenol extraction reflecting differ- ences in the fractions of RNA isolated by the two methods. There were also differences in the RNA synthesized during long and Short periods of incorporation. More 32F than 3H-uridine was incorporated into Specific fractions of RNA, probably reflecting differences in the Specific activity of the precursor pool. The incorporation of 32F per g of tissue was 15% less but the incorporation per mg of DNA was 30% higher 3 days prepartum than 3 days postpartum when corrections were made for differences in pool size. The RNA synthesized per gland is twice as much postpartum com- pared to prepartum rats. Doubling the glucose concentration and increasing the amino acid concentration 2.5 fold stimulated RNA synthe- sis 25-50%. The combination of prolactin, hydrocortisone, and insulin stimulated 32F incorporation to the same extent in the enriched and normal medium 199. Histological exam- ination of the tissue after 12 hours of incubation indicated necrosis and degeneration in those samples incubated without hormone while evidence for maintenance of cellular integrity and enhanced secretion was observed in the samples incubated with the 3 hormones. Actinomycin D inhibited protein syn- thesis 70-80% within 12 hours indicating that some Species' of RNA in mammary tissue turned over quite rapidly. Harvey w. Nohrenweiser The incorporation of 3H-uridine and l"IO-amino acids into trichloroacetic acid precipitated material during one hour after the tissue had been preincubated for 0-9 hours was difficult to interpret because it did not agree with the histological examinations. The no hormone control incorporated the most precursor during several time periods. In lactating tissue the incorporation rate declined during the last 3 hours of incubation and insulin was the only hormone treatment which enhanced RNA synthesis compared to the no hormone control. Insulin or insulin, hydrocortisone and prolactin stimulated incorporation in nonlactating tissue. 32 The effect of hormones on P incorporation into phenol extracted RNA was somewhat different from that on 3H—uridine incorporation into RNA precipitated with tri- chloroacetic acid. Prolactin plus insulin, with or without hydrocortisone, stimulated 32? incorporation into phenol extracted RNA in prepartum tissue while insulin plus hydrocortisone enhanced incorporation only at early times. In postpartum tissue, insulin plus hydrocortisone stimulated 32F incorporation and the addition of prolactin was without an additional effect. Separation of the various RNA Species by polyacryl- amide gel electrOphoreSis did not reveal any fraction from either pre- or postpartum tissue with a Specific activity higher than the mRNA fraction, even though a fraction of Harvey w. hohrenweiser approximately 158 could be separated and detected. Similarly, no unique fraction reSponded to hormonal stimulation but rather the incorporation into all fractions of RNA was enhanced to the same extent. Thus, no Species of RNA with the prOperties eXpected of mRNA could be detected with the techniques employed in these eXperiments. ROLE OF HORNONES IN CONTROLLING RIBONUCLEIC ACID SYNTHESIS DURING IACTOGENES IS By Harvey w. Nohrenweiser A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Departments of Biochemistry and Dairy 1970 (Sr-c5699 /-.J'7v 7/ ACKNOWLEDGMENTS The author wishes to eXpress his sincere apprecia— tion to Dr. w. A. Wood and Dr. Roy S. Emery for their advice, guidance and assistance throughout all phases of preparation of this manuscript. The contributions of Dr. Evelyn Rivera are also gratefully acknowledged. The author wishes to pay Special tribute to his wife, Elizabeth, for her ever helpful encouragement and assistance. A portion of this work was accomplished with fin— ancial assistance from the Department of Health, Education and Welfare, grant number 5-FOi-GN-36,h51. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . LIST OF APPENDIX TABLES . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . Physiological Changes During Initiation of Lactation . . . . . . . . . . . . . . . . . . . Changes in DNA Content . . Changes in RNA Content . . . . . . . . . . . Changes in Protein Content . . . . . . . . . Changes in Enzyme Activity . . . . . . . . . Changes in Metabolite Concentration . . . . Structural and Organizational Changes During Initiation of Lactation . . . . . . . . . . . . Hormonal Treatments Affecting Initiation of IIaCtat 1 on O O O O O O O O I O O O O O O O O I 0 Hormonal Requirements for Maintaining Lactation RNA and Protein Synthesis by Mammary Tissue slices 0 I I I I O O O O O O I O O O O O O O O Mammary Tissue Differentiation, i3 vitro . . . Protein Synthesis in EXplants . . . . . . . Role of Hormones in Controlling RNA Synthesis . . . . . . . . . . . . Role of Hormones in Controlling DNA Synthesis . . . . . . . . . Sequence and Role of Hormones Role of Insulin . . . . . . . Role of Hydrocortisone . . . . Role of Prolactin . . . . . . Mammalian RNA Metabolism . . . . . . . . . . . 111 Page vi viii ix CDO\O\UI\D \D \O 11 13 17 20 21 22 24 24 24 26 27 29 Pathway of RNA Synthesis . . . . . . . . . . Rapidly Labeled RNA . . . . . . . . . . . . General Mechanism of Hormone Action . . . . . . Translational Control . . . . . . . . . . . Transcriptional Control . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . Materials . . . . . . . . . . . . . . . . . . . methOds O O O O O O O O O O O O O O O O O O O 0 Tissue Slices . . . . . . . . . . . Incubation . . . . . . . . . . . Total Isolation of RNA and Protein . Extraction of RNA . . . Polyacrylamide GelE lggtrOphoresis of RNA Specific Activity of . . . . . RESULTS AND DISCUSS ION O O O I O O O O I O O O O 0 Development of Systems . . . . . . . . . . . . Development of Incubation Techniques . . . . Composition of Pre- and Postpartum Tissue . Changes in.PhOSphate Pool Size . . . . . Histological Evidence of Changes in Tissue During Incubation . . . . . . . . . . Characterization of RNA in Pre- and Post- partum Tissue O O O O O O O O O O O O O 0 Evidence for mRNA . . Discrepancies in RNA Synthesis as Assayed by Two Procedures . . . . . . . . . . . . . Results of Hormone Treatment . . . . . . . . . Effect of Hormones Upon RNA Synthesis . . . Role of Hormones in Regulating Amino Acid Incorporation in Tissue Slices . . Integration of Results from Several Methods of Assessing Hormonal Effects on Mammary Tissue Slices . . . . . . . . . . . . . . Calculation of Amount of RNA Synthesized by Iammary Tissue Slices . . . . . . . . . . . . . Calculation of Amount of Protein Synthesis . . iv Page 29 30 32 32 35 39 39 39 39 to 1+3 1+6 1&7 5o 50 so 59 co 63 73 76 85 85 92 97 90. 103 DISCUSSION SUMMARY BIBLIOGRAPHY APPENDIX Page 105 110 111 119 Table 10 11 12 LIST OF TABLES Tissue Slice Incubation Medium for Total RNA Isolation EXperiments (Medium 199-H) . . . . Tissue Slice Incubation Medium for Phenol Extracted RNA Experiments (Medium 199-E) . . Relationship of Amount to RNA in Sample to Area of Absorbance in Various Peaks . . . . Effect of Buffgr Systems and Metabolite Con- centration on H-Uridine Incorporation into RNA by Slices from Lactating Bovine Mammary G land 0 O O O O O O O O O O O O O O O O 0 I 0 Effect of Added Glucose and Amino Acids on Precursor Incorporation in RNA by Mammary Tissue Slices . . . . . . . . . . . . . . . Protein, RNA and DNA Content of Mammary Tissue of Rats, 3 Days Prepartum and 3 Days Postpartum . . . . . . . . . . . . . . . . . Specific Activity of Free PhOSphate in Mammary Tissue from Lactating and Nonlactat- ing Hats 0 O I O O C O I O O O O O I l O I I Effect of ength of Labeling Period Upon Amount of H-Uridine Incorporated into RNA by Mammary Tissue Slices . . . . . . . . . . Effect of Assay Method and IsotOpe Upon Calculated Amount of RNA Synthesized by Mammary Tissue Slices . . . . . . . . . . . Effect of Actinomycin D on Incorporation of Precursor into RNA by Mammary Tissue Slices 32? Incorporation into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rate 2 Days Postpartum . . . . . . . . . . . . . Effect of Various Hormones Upon 3H-Uridine Incorporation into TCA Precipitated RNA by Tissue Slices from Mammary Glands of Rats . vi Page 41 42 48 52 53 57 77 79 81 84 86 Table 13 14 15 16 17 19 Page Effect of Various Hormones Upon 3H-Uridine Incorporation into TCA Precipitated RNA by Mammary Tissue Slices . . . . . . . . . . . . 88 Effect of Various Hormones Upon 32F Incor- poration into Phenol Extracted RNA by Mammary Tissue Slices . . . . . . . . . . . . 90 Effect of Various Hormones Upon 1LI’CmAmino Acid Incorporation into TCA Precipitated Protein by Mammary Tissue Slices . . . . . . 93 Effect of Actinomycin D on 1“(L-Amino Acid. Incorporation into TCA Precipitated Protein by Mammary Tissue Slices . . . . . . . . . . 95 Calculation of Amount of RNA Synthesized by Lactating and Nonlactating Rammary Tissue . . 100 Relation of RNA Synthesis to the Increased RNA Content of Postpartum Tissue . . . . . . 102 Calculation of Amount of Protein Synthesized by Tissue Slices from Mammary Glands of Rats 3 Days Prepartum and 3 Days Postpartum . . . 104 vii Figure LIST OF FIGURES Scheme of Phenol Extraction of RNA . . . . . Photomicrograph of Lactating and Nonlactat- ing Mammary Tissue Slices Incubated in the Presence and Absence of Hormones . . . . . . ElectrOphoretic Analysis of a Mixture of E. coli Ribosomal RNA and Total RNA from 'LacEatIng Mammary Tissue . . . . . . . . . . Sucrose Density Gradient of Mammary RNA . . Effect of Sample Size and RNA Concentration on Separation Obtained by ElectrOphoresis . Electrophoretic Analysis of RNA from Prepar- tum and 6-18 Hours Postpartum Mammary Tissue ElectrOphoretic Analysis of RNA from Mammary Tissue of Rats 6-18 Hours Postpartum Labeled With BH-Urldine. o o o o o o o o o o o o o 0 viii Page 44 61 64 67 71 7t Table LIST OF APPENDIX TABLES Page Composition of Medium 199 . . . . . . . . . . 120 Carnoy-Hematoxin Method for Preparing Samples for Histological Examination . . . . . . . . 122 Effect of Various Hormones Upon 33F Incor- poration into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rats 3 Days Prepartum o o o o o o o o o o o o o o o o o o 123 Effect of Various Hormones Upon 32F Incor- poration into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rats 2 Days Prepartum O O O O O O 0 O O O O 0 O O 0 O O O 1214' Effect of Various Hormones Upon 32P Incor- poration into Phenol Extracted RNA by Tissue Slices from Mammary Tissue of Rats 6-18 Hours Postpartum . . . . . . . . . . . . . . 125 Effect of Various Hormones on 32F Incor- poration into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rats 72—84 Hours Postpartum . . . . . . . . . . . . . . 126 Effect of Various Hormones Upon 33P Incor- poration into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rats 48-60 Hours Postpartum . . . . . . . . . . . . . . 127 ix INTRODUCTION The onset of lactation, lactogenesis, is associated with the rapid appearance of several components which are unique to milk and the mammary gland. Characteristics of lactating mammary cells are the ability to synthesize and secrete the various caseins, whey proteins and lactose while the nonlactating epithelial cell, which has a histo- logical appearance distinctly different from the lactating cell, does not synthesize these compounds. From the beginning of pregnancy until attaining the lactational state, changes in the mammary cell seem to involve two distinct phases. The first involves epithelial cell division to form the lobuloalveolar structure while the second is the acquisition of the functions of a lactat- ing mammary cell. These changes have been extensively studied recently in mammary gland SXplants from the mid- pregnant mouse and the hormonal requirements for deveIOpment and differentiation have been outlined. These SXperimentS also suggest that at least 2 phases are involved in trans- forming the tissue derived from the mammary gland of a mid- pregnant mouse into tissue with the functional characteris- tics of the lactating gland but thus far it has been impos- sible to study the second phase independently of the cell division phase. Both DNA and RNA synthesis are required 1 2 for completion of phase 1 while phase 2 can be completed without concurrent DNA synthesis. Many hypothesis have been proposed concerning the involvement and role of several hormones in lactogenesis. Although all of the hypotheses involved direct hormone action on the mammary gland, they differ with rSSpect to the function of the hormone and the ultimate site of con- trol. Extensive increases in the RNA content of the mam- mary gland occur at the time of parturition. Hormone therapy in endocrinectomized animals indicated that several distinct classes of RNA may be synthesized in reaponse to different hormones. In other tissues also it is unclear whether hormones stimulate the synthesis of all classes of RNA or act in a very Specific manner by enhancing the synthesis of an RNA Species which could be called messen- ger RNA. Thus studies to ascertain the alterations in RNA synthesis which occur during lactogenesis were initiated. It was anticipated that investigations into the classes of RNA synthesized by pre- and postpartum mammary tissue slices and the influence of hormones upon synthesis during short- term incubations (which would be independent of the role of hormones in controlling cell division) could further elucidate the mechanism of lactogenesis and give additional insight into the mechanism of hormone action. LITERATURE REVIEW hysiological Changes During Initiation of Lactation Chapges in DNA Content The initiation of lactation is associated with the newly acquired ability of the mammary gland to secrete milk and this is reflected in changes in many of the measurable parameters of the tissue. The initiation of lactation in the rat is accompanied by an increase in the amount of tissue as well as cell numbers, total DNA, RNA and protein in the mammary gland. Kuhn and Lowenstein (1967) and Baldwin and Milligan (1966) reported a 30-50% increase in wet tissue weight between 2 and 3 days pre- partum (5 g) and 1 and 3 days postpartum (8 g). Tissue weights measured by Greenbaum and Slater (1957) were lower and the increase did not occur until the 3rd day of lacta- tion. During this same time period, the water content of the tissue increased from approximately 40%-3 days prepar- tum-to 65%-5 days later. Conversely, the percent of fat in the tissue decreased by 50% and thus the fat-free dry tissue remains relatively constant at 10-11% of the total wet weight (Kuhn and Lowenstein, 1967). The DNA content of mammary cells is quite constant (Mumford, 1964) and thus total DNA is routinely utilized as 3 Li a measure of total cell numgers and mammary deve10pment. The total DNA increase appeared quite variable, ranging from a 50% increase occurring sometime between 3 days pre- partum and 2 days postpartum in 3 separate eXperimentS (Kuhn and Lowenstein, 1966), to a 3-fold increase between the 15th day of pregnancy and the first day lactation (Thibodeau and Thayer, 1967). Baldwin and Milligan (1966) observed a doubling of total DNA between the 20th day of pregnancy (10 mg) and 48th hour of lactation (20 mg) as well as a 3-fold increase in the number of nuclei. Tucker and Reece (1963a, 1963b) reported a 2-fold increase in total DNA between the 12th and 18th day of pregnancy and additional 10% increases between the 18th day of gestation and the first day of lactation and also between days 1 and 4 of lactation. Two other techniques have been used to assay DNA synthesis. Traurig (1967) injected a mouse with 3H- thimidine one hour before sacrificing and counted the per- cent of intralobular-epithelial alveolar cells which were labeled, assuming that only these cells were undergoing mitosis. Only on day-2 (11%), day-3 (8%) and day-5 (3%) of lactation were more than 1% of the cells labeled. Stellwagen and Cole (1969) assessed cell division by measuring 3H-thimidine incorporation into nuclear DNA. The rate of incorporation (dpm/ug DNA) gradually declined during the period-6 days prepartum to 6 days postpartum- except for a 3-fold increase on the first day of lactation 5 which is consistent with a burst of cell division immedi- ately after parturition. The rate of histone biosynthesis and the relative amount of the various classes of histones were unchanged during late pregnancy and early lactation, thus detracting from the concept of alteration of classes of histones controlling synthesis of unique Species of RNA and lactogenesis (Stellwagen and Cole, 1968, 1969). Changes in RNA Content The total RNA content of the mammary gland was highly correlated (r>o.93) with liter weight gain (Tucker, 1966) and thus has been used as an index of protein synthe- tic activity and secretory ability. The RNA to DNA ratio in mammary tissue of 15—18 day pregnant rats was approxi- mately unity but during the first 2-4 days postpartum a doubling of the total RNA content (15 mg increase) occurred and the RNA-DNA ratio approached 1.5 (Baldwin and Milligan, 1966; Tucker and Reece, 1963a, 1963b; Kuhn and Lowenstein, 1967). The incorporation of radioactive precursors (glycine and orotic acid) per gland into acid insoluble ribonucleic acid increased approximately 6-8 fold between the 18th day of gestation and the end of the first day of lactation when the rate of incorporation had reached a maximum (Wang and Greenbaum, 1962). The rate of incorporation declined as lactation progressed. Changes in Protein Content Changes in total protein content followed closely the changes in DNA and RNA content with a 2-3 fold increase occurring between the 20th day of pregnancy and the 3rd day of lactation (Tucker and Reece, 1963a, 1963b; Greenbaum and Slater, 1957; Slater and Planterase, 1958). Changggin Enzyme Activity The rapid increase in metabolic activity associated with lactation was reflected in changes in the various enzyme activities of the cells. Baldwin and Milligan (1966) reported that the patterns of enzyme activity in mammary tissue from rats 3 days and 15 days after parturi- tion were Similar. They concluded that the cells acquired an enzyme complement characteristic of fully lactating tissue soon after their formation and furthermore that the apparent changes in enzyme activities between tissue from midpregnant animals and lactating animals were reflections of the low levels of some enzymes in nonsecretory cells, which predominate in tissue from midpregnant animals. Generally, the enzyme activity increased dramatically dur- ing the first 3 days of lactation whether eXpressed as units per g of tissue or per mg of extractable protein. Some enzymes increased in activity after the 3rd day of lactation but these increases could largely be accounted for by the increased extractable protein and remained constant when eXpressed on a Specific activity basis. Kuhn 7 and Lowenstein (1967) reported similar results for enzymes associated with the biosynthetic pathways for major milk components. Thibodeau and Thayer (1967) reported that aSpartate transcarbamylase, the first unique enzyme in pyrimidine biosynthesis, increased slightly faster but to the same extent as did the RNA content of the gland at the time of parturition. Also the activity of 5'-nucleotidases were at a minimum during early lactation, indicating less degra- dation of nucleotides when they are in demand for synthetic processes (Wang, 1962), The enzymes associated with lactose synthesis are particularly interesting because although lactose could not be detected in mammary tissue until the 20th day (2 days prepartum) (Yokoyama, 1969) or 21th day (Kuhn and Lowenstein, 1967) of gestation, the enzymes and substrates necessary for substantial levels of lactose synthesis were present several days prepartum (Clock and McLean, 1954; Kuhn and Lowenstein, 1967). Palmiter (1969) recently demonstrated that the method of assaying lactose synthetase activity, the rate limiting enzyme in lactose biosynthesis, dramatically influenced the detected enzyme activity. Employing a tissue culture assay (whole cells of mouse mammary tissue) the enzyme activity was only 4 times back- ground on the 18th day of pregnancy. A 24- fold increase in activity occurred between the 18th day of pregnancy and R the 6th day of lactation. Enzyme assays in crude homogen— ates of mammary tissue indicated significant levels of enzyme were present 10 days prepartum with activity beginning to increase at that time. This was also reported by Kuhn and Lowenstein (1967) and would contradict the evidence that the rate limiting B subunit (d-lactalbumin) does not increase until the time of parturition (Turkington g£_gl,, 1968; Kuhn, 1968). This may indicate that com- partmentalization of cellular components is important in controlling lactose synthesis and regulating other meta- bolic parameters. Changes in Metabolite Concentration Independent of changes in enzyme activity, changes in metabolite levels can dramatically alter cellular metabolism and productivity. Baldwin and Cheng (1969) concluded that prepartum levels of energy substrates and milk precursors in the mammary gland were adequate to sup- port lactation, although some changes in metabolite levels did occur postpartum. The ratio of high energy phOSphate bonds to the total adenine pool actually decreased after parturition although the level of adenine nucleotides increased. Wang (1962), although using a limited number of animals, reported a 3-fold increase in ATP and total nucleotides between the 18th day of pregnancy and the first day of lactation but the percent high energy phOSphate bonds was unchanged. The molar ratio of ATP to (ADP plus 9 AMP) reported for mammary tissue decreased from 1.1 pre- partum to 0.5 during late lactation. This is much lower than the 3:1 ratio reported for rat liver (Williamson gfi‘gl., 1969) and chick brain (Kozak and Wells, 1969) indicating a low energy state in the tissue or possibly that anoxia had occurred during the time required to obtain the sample. Similarly the pyridine nucleotide pool increased 4-fold early in lactation but the TPNHzTPN+ ratio increased only slightly at parturition and the DPNH:DPL+ ratio decreased (Baldwin and Milligan, 1966). Glucose concentrations average 0.7-1.2 umoleS/g wet mammary tissue and do not change at the time of parturition (Baldwin and Cheng, 1969; Kuhn and Lowenstein, 1967). Stellwagen and Cole (1969) reported a 60% increase in the free amino acid pool between the 20th day of pregnancy and the first day of lactation (10.27 umoles/g wet tissue). Cell proliferation, secretory cell development, changes in enzyme activity and metabolite levels occur during lactogenesis. The latter two are influenced by hormones and have a role in develOpment of the mammary gland to the lactational state, but changes in the metabo- lite and enzyme levels do not appear to be the trigger for initiating milk secretion. Structural apd Organizational Changes During Initiation of Lactation During lactogenesis the cellular structure of alveolar cells undergo marked morphological changes which can be 10 observed by both the light and electron microscope. Mam- mary tissue from midpregnant mice had small alveoli. Cellular vacuolation was small and the amount of stainable material in the alveolar lumina was small or totally absent, but lipid droplets were evident. Most fully lactating cells had vacuoles, the alveoli were distended and abundant secretion in the lumen was evident when examined with the light microscOpe (Elias, 1957; Nandi, 1959). The use of the electron microscope has increased the resolution of the morphological changes associated with lactogenesis many fold. The nonlactating alveolar cell was characterized by a minimal content of endOplasmic reticulum, a small Golgi apparatus and a minimal number of mitochondia scattered throughout the cytOplasm. Only occasionally were milk protein dr0plets in evidence but several large milk fat drOplets were observed in the lumen (Wellings, 1969). Wellings (1969) summarized the ultrastructural changes associated with the initiation of lactation as follows: a) dramatic increases in the grandular endOplasmic reticu- lum, b) hypertrophy of the Golgi apparatus and movement to the apical region of the cell, c) increase in number but a diminution in size of the cytoplasmic fat droplets and d) an increase in the number of mitochondia. A 4-fold increase in the relative area of the endOplasmic reticulum and the Golgi apparatus and the number of mitochondria 11 occurred between the 19th day of pregnancy and 4th day of lactation in C3H/Crgl strain of mice. Hollman (1969) reported similar observations except for mitochondial volume which increased 2.5-fold per cell during the first 5 days postpartum. This increase in the grandular endo- plasmic reticulium is in agreement with the data of Gaye and Denamur (1969) where 75% of the polyribosomes in the pseudOpregnant rabbit mammary gland are not membrane-bound but prolactin which induces lactation, caused a substantial increase in the aggregation of the polysomes, the amount of microsomes, and their protein synthesizing capacity, Hormonal Treatments Affecting Initiation of Lactation Lyons (1958) hypOphysectomized and hypOphysectomized- ovariectomized-adrenalectomized rats during late pregnancy in experiments to determine the hormonal requirements for initiation of lactation. Prolactin and cortisol acetate together were able to initiate and subsequently sustain a level of lactation equal to 50% of normal (Bintaringsih ggig}., 1958). Similar results were obtained by Cowie and Tindal (1961). Ovariectomization of rats during midpregnancy resulted in induced mammary secretion (Lin and Davis, 1967). A rapid elevation of the RNA content of the gland was evident after 8 hours and new proteins were detected within 24 hours, associated with an increased rate of 1LIC—leucine incorpora- tion. The level of DNA remained constant indicating that 12 no extensive cell proliferation was necessary for initia- tion of lactation. The authors suggested that the lack of milk secretion prepartum was due to the inhibitory influ- ence of progesterone which was removed by ovariectomy or parturition. Similarly, estrogen administration to the lactating mouse blocked lactation and protein and RNA synthesis by mammary tissue Slices lg 31332 declined to a level compar- able to that observed in the 14th day postweaning mouse (Sirakov and Rychlik, 1968). Talwaker gt_gl. (1961) administered hydrocortisone acetate during the 5th-9th or 10th-13th days of pregnancy and were able to induce lactation. No other hormone was active alone or enhanced the activity of hydrocortisone. They concluded that low levels of active adrenal glucocor- ticoids were reSponSible for the absence of lactation. Injection of cortisol into the pregnant mouse initi- ated lactation and slightly stimulated the rate of RNA synthesis by tissue slices but the incorporation of 14C- leucine into protein was less than in tissue from pregnant mice (Sirakov and Rychlik, 1968). Changes in the free amino acid pool as observed by Stellwagen and Cole (1969) could bias the measurements. Cowie (1969) has outlined several of the general hypotheses for the initiation of lactation. First, the secretory activity of the mammary epithelium may be 13 inhibited by high levels of estrogen and/or progesterone. Also the release of prolactin from the anterior pituitory could be inhibited by these hormones. The decline in estrogen and progesterone at parturition releases the inhibition. Secondly, high levels of prolactin may be reSponsible for initiating lactation. High levels of oxytocin during parturition could cause a large amount of prolactin to be released from the anterior pituitory. Thirdly, during pregnancy the level of active adrenal corticoid is too low and either the level or activity is increased at parturition. Hormonal Requirements for Maintaining Lactation Earlier, Cowie (1957) had hypOphysectomized rats on the 4th day of lactation in order to study the hormones required for maintenance of lactation. The combination of adrenalcorticotrOphic hormone and prolactin was able to maintain lactation at approximately 50% of normal. High levels of prolactin alone had a small effect while adrenal- corticotrOphic hormone alone was ineffective. Talwalker 33.2}. (1960) administered adrenal glucocorticoids to intact rats daily from the nth-18th day of lactation and were able to increase molk secretion. Baldwin and Martin reported the effects of hypo- physectomy on the first (1968a) or 10th day of lactation (1968b) and subsequent hormone therapy upon various 1n parameters of mammary metabolism. Mammary gland weight, RNA and DNA per g of tissue, 32F incorporation into nucleic acids and 1”Culeucine incorporation into protein were decreased by hypOphysectomy on the first day of lactation. Mammary gland weight and RNA and DNA content were increased by administration of either cortisol or prolactin but both cortisol and prolactin were required to increase these parameters to levels comparable to sham Operated controls. Prolactin alone was as effective as the combination of cortisol and prolactin in stimulating 32F incorporation into all of the various nucleic acid fractions. It was concluded that prolactin was controlling DNA synthesis and stimulated RNA synthesis in a general manner while cortisol, which only affected 32P incorporation into a RNA fraction defined as DNA-like, acted in a Specific manner. Cortisol did not enhance amino acid incorporation into total extract- able protein while both cortisol and prolactin were neces- sary for retention of the capacity for casein synthesis. Two types of enzyme activity reSponse were noted. The activity of most enzymes was independent of hormone treat- ment and was a reflection of cell numbers, while glucose-6-P dehydrogenase reSponded differently in that the combination of cortisol and prolactin caused a h-fold enhancement of activity. HypOphysectomy on the 10th day of lactation caused decreases in mammary gland weight but the RNA and DNA con- 15 centration were affected only slightly (Baldwin and Nartin, 1968b). Prolactin and prolactin plus cortisol administra- tion increased the RNA and DNA content of the tissue to almost 30% above sham-operated controls. Similarly pro- lactin increased 32P incorporation into nuclear RNA also Z-fold over control values. Other results were similar to those obtained with rats hypOphysectomized on the first day of lactation. Additionally, it should be noted that although lL'C-leucine incorporation into casein by the prolactin-cortisol groups was comparable to the sham Operated control group, the low growth rate of the pups in the hormone group indicated a drastic reduction in synthetic activity. Baldwin 3392;. (1969) investigated the effects of endocrinectomy on the fifth day of lactation and subsequent hormone therapy for 5 days upon RNA synthesis by isolated mammary gland nuclei. The results are outlined as follows: 1) alloxan diabetes did not affect either of the RNA poly- merase activities (high or low salt) or the type of RNA synthesized as analyzed by nearest neighbor frequency and polyacrylamide gel electrophoresis, 2) ovariectomy followed by estrogen administration did not effect RNA synthesis but progesterone treatment caused alteration of the base composition and nearest neighbor frequency in the synthe- sized RNA which is consistent with the results of Lin and Davis (1967), 3) adrenalectomy and treatment with hydrocor- tisone did not effect RNA polymerase activity, RNA base 16 composition or nearest neighbor frequency but did alter the pattern of RNA synthesis as analyzed by gel electro- phoresis, thus supporting the concept of a Specific effect of cortisol upon RNA synthesis (Baldwin and Martin, 1968a, 1968b), 9) both cortisol and prolactin administration to hypOphysectomized rats increased RNA polymerase activity in the low salt assay and changed the base composition and nearest neighbor patterns of the RNA synthesized. The addition of hormone in 21332 did not affect the extent or class of RNA synthesized. The fractions of RNA synthesized in 32232 appeared to correSpond to those characterized by Sirakov 32 21° (1968) as being messenger RNA. The authors concluded that these data, in conjunction with the observa- tion that prolactin and cortisol had Specific effects on several synthetic enzymes (Kursrud and Baldwin, 1969), were further support that cortisol and prolactin exert Specific regulatory influence on the rates and patterns of RNA synthesis in mammary secretory cells. HypOphysectomy causes drastic changes in cell number thus the changes in patterns of RNA synthesis could have resulted from changing cell pOpulation. This change in cell pOpulation was used to explain differential changes in enzyme activity in pre— and postpartum tissue (Baldwin and Milligan, 1966). 17 RNA and Protein Synthesis by_Nammary Tissue Slices Sirakov and Rychlik (1968) reported that mammary tissue slices from 16 day lactating mice incorporated 56 umole of 1LPG-Jeucine per mg DNA per hour into trichloro- acetic acid-precipitable protein. Tissue from 18 day pregnant mice was only 30% as active and was only as act ve as normal resting mammary tissue from in day postweaning mice. Incorporation per mg dry weight increased 10-fold between the pregnant and lactating periods (609 umole/mg dry weight). Expressed as incorporation per mg RNA the mammary tissue from pregnant mice was as active as tissue from lactating mice (16 uumole/mg RNA). The incorporation of 3H-uridine into trichloroacetic acid-precipitated material increased 7-fold in the lactat- ing tissue compared to tissue from pregnant mice. The incorporation per cell doubles between the pregnant and lactating state but the Specific activity of the RNA syn- thesized by tissue slices from pregnant mice was 50% higher (0.9 uumole/mg RNA) than RNA from lactating tissue. The lactating tissue slices maintained a linear rate of 3H-uridine and 1L‘C-leucine incorporation for only 90 minutes, while the incorporation in slices from nonlactat— ing glands was linear throughout the total incubation period (150 minutes). Addition of various hormones in vitro did not affect the rate or extent of incorporation. Mayne 33 al., (1966) reported that insulin stimulated 18 1uC--adenine incorporation into RNA in tissue slices from 12-17 day pregnant rats during the first 3 hours of incu— bation. The reSponse of tissue slices from 12-15 day lactating rats was very variable (3-40% stimulation) and in 2 of 6 eXperiments the insulin stimulation was not statistically significant. Insulin also stimulated 1“C- leucine incorporation (38%) into protein by slices from midpregnant rat mammary tissue but no consistent stimula- tion was detected in similar eXperiments with slices from lactating tissue. Actinomycin D inhibited RNA synthesis 97% in mammary slices from pregnant rats and inhibited lac-leucine incorporation 25% but did not prevent the insulin stimulation of protein synthesis, thus suggesting that insulin does not stimulate protein synthesis only via increased RNA synthesis. Sirakov g£_gl. (1968) characterized the RNA synthe— sized by mammary tissue slices from pregnant and lactating mice during a 90 minute 1E.Xl££2 incubation. Very little radioactive material was isolated from the cytOplasm of lactating tissue. Analysis of the cytoplasmic RNA from nonlactating tissue on methylated-albumin (MAX) columns indicated the SRNA had the highest Specific activity with a broad radioactive region correSponding to the rRNA being of lower Specific activity. The RNA which bound tightly to the column had only 25% the Specific activity of the rRNA fractions (calculated from Figure l-b) even though 19 the tightly bound RNA was referred to as rapidly labeled, DNA-like-RNA. Contrary to the author's conclusions all of the nuclear RNA separated by NAK column chromatography are equally labeled if the Specific activity of each frac— tion is calculated rather than being eXpressed as total radioactivity per peak. Sucrose gradient analysis of cytOplasmic RNA from nonlactating tissue indicated that a heterodiSperse fraction, slightly smaller than 188 and largely overlapping the SRNA peak, was extensively labeled. The ability of isolated RNA to stimulate amino acid incorporation in a bacterial cell-free protein-synthesiz- ing system has been used as an assay for mammalian mRNA but it has been shown that rRNA also has this property, presumably because it inhibits ribonuclease degradation of mRNA by acting as an alternative substrate (Hunt and Wilkinson, 1967). Sirakov gt 3;. (1968) employed this technique to further characterize the fractions of RNA isolated from mammary tissue. Crude preparations of nuclear RNA from nonlactating tissue had the greatest stimulatory ability and cytOplasmic RNA from lactating tissue had the lowest activity (3%). Conversely, assay- ing the fractions isolated by sucrose density centrifuga— tion indicated the 12-158 fraction of cytOplasmic RNA from lactating tissue had the greatest stimulatory activ— ity. A similar fraction from nonlactating tissue was much less active. Several very high molecular weight Species 20 of nuclear RNA from nonlactating tissue had stimulatory activity which was absent in comparable fractions from lactating tissue. Thus the authors concluded that the 15S RNA fraction of lactating tissue was the major Species of RNA synthesized in the nucleus but it did not enter the cytoplasm as measured by Specific activity of this frac- tion in the cytoplasm and therefore cytoplasmic mRNA turned over very slowly and had a long half life. Palmiter (1969a) observed that within 1 hour of the addition of insulin, hydrocortisone and prolactin, the incorporation of uridine, amino acids and galactose into TCA precipitated material was enhanced in midpregnant mouse mammary slices which had been previously incubated for 17-21 hours without hormones. Insulin alone was par- tially active indicating it may have a passive role by simply restoring normal metabolic function. Mammary Tissue Differentiation In Vitro Since the early eXperiments of Elias (1957), demon- strating the maintenance of cell features, and Elias and Rivera (1959), demonstrating the induction of secretory reSponse in eXplants (mammary epithelial tissue from a mouse 10-14 days pregnant) incubated in a chemically defined medium containing prolactin, insulin and hydro- cortisone, many advancements have been made in elucidating the hormonal reSponse mechanism. 21 Protein Synthesis in Explants During the first 3 hours of incubation, mammary tis- sue from the midpregnant mouse incorporates 32F into a protein which is identical to authentic mouse casein (Turkington gt_gl., 1965). This synthesis appears to occur in some cells which have already differentiated into a lac- tating type cell. EXplants incubated without insulin, hydrocortisone and prolactin lost most of the ability to synthesize casein while eXplants cultured in the presence of the 3 hormones for 48 hours synthesized casein at a 3-5 fold stimulated rate and incorporated 32? into noncasein phOSphOprotein at a 50% increased rate (Juergens 33 g}.. 1965) . Insulin alone was able to maximally stimulate the synthesis of soluble nonmilk protein while all 3 hormones were necessary for the induction of d-lactalbumin and B-lactoglubulin synthesis (Stockdale 32 gl., 1966). The synthesis of the whey proteins was not coordinate but after 48 hours of culture the ratio of whey protein to casein approached that observed in mouse milk. Mayne and Barry (1970) reported insulin and prolactin stimulated 32P incorporation into casein 4-fold within 24 hours and 5-fold at 48 hours over the level attained with eXplants incubated in the presence of insulin or insulin and corticosterone which were only Slightly more effective than no hormone. Corticosterone acted synergistically 22 with the other 2 hormones to stimulate 32F incorporation an additional 50-100%. The incorporation of 1I‘l'C-lysine into total protein reSponded in a similar but less dramatic manner to the various hormone combinations. Role_of Hormones in Controlling RNA Synthesis Total RNA synthesis during the last 4 hours of a L8 hour incubation was increased 4-fold when eXplants were cultured for 48 hours in the presence of all 3 hormones. An initial basal rate of 3H-uridine incorporation was main- tained with no hormone addition while insulin, insulin plus prolactin and insulin plus hydrocortisone were as effective as the 3 hormone combination during the first 24 hours. No increase in RNA synthesis was observed with these latter combinations though during the second 24 hours (Stockdale 33- 3;” 1966). Mayne gt El° (1966) and Mayne and Barry (1967) noted a decreased rate of incorporation (68%) of lac-adenine into RNA within 3 hours in SXplants incubated in the absence of insulin and an increasing rate of decline in incorporation in total RNA during the first 12 hours of incubation. Mayne §£_gl. (1968) reported that all 3 hormones were neces- sary to maintain a basal level of RNA synthesis for 48 hours. The RNA content of explants incubated 24 and 48 hours in insulin and corticosterone containing medium declined 3.5 and 22.2% and they incorporated 14.1 and 56.4% less 1LPG-adenine during the last 3 hours of the reSpective 23 incubation periods than during the initial measurements. The RNA content of explants cultured in the presence of all 3 hormones was increased 8.0 and 10.3% at 24 and 48 hours and the rate of lLl'C-adenine incorporation was enhanced 20 and 18%. Further investigation (Mayne and Barry, 1970) indicated insulin plus prolactin and insulin, prolactin and corticosterone stimulated 1Lia-uridine incorporation 2-fold during 48 hours of incubation when compared to insulin alone or insulin and corticosterone. Time course studies indicated that insulin-stimulated RNA synthesis reached a maximum after 12 hours of incubation and then declined while RNA synthesis by eXplants incubated in the presence of all 3 hormones was stimulated an additional 50% during the next 12 hours. Recently, Green and T0pper (1970) reported that hydrocortisone does not stimulate RNA synthesis. Prolactin stimulated RNA and casein synthesis in epithelial cells previously incubated with insulin and hydrocortisone but also stimulated RNA synthesis (as measured by uridine incorporation into TCA precipitated material) in eXplants incubated with insulin which are unable to synthesize casein. Turkington (1970) using RNA-DNA hybridization tech- niques detected Species of RNA in eXplants incubated in presence of 3 hormones which were not detected in virgin mouse mammary epithelial cells. 24 Role of Hormones in Controlling DNA Synthesis DNA synthesis by mammary epithelial cells was reported to be maximally stimulated by insulin alone (Turkington and TOpper, 1967). Contrary to this report, Mayne and Barry (1970) observed that insulin plus prolac- tin and insulin, prolactin and corticosterone stimulated 3H-thymidine incorporation into DNA 3- and 4-fold, reSpec- tively, when compared to insulin alone or insulin and cor- ticosterone. Time course studies indicated that insulin stimulated DNA synthesis for only 12 hours before it returned to basal levels while DNA synthesis remained at the 12 hour level for 24 hours if prolactin and cortico- sterone were also present. Sequence and Role of Hormones Lockwood 32 El! (1967) were able to partially resolve the sequence of hormone requirements for eXplant differen- tiation. Insulin appeared to be involved in epithelial cell proliferation but cells formed during the influence‘ of insulin alone would not synthesize casein after prolactin addition unless one cycle of cell division occurred in the presence of hydrocortisone. The data supporting this sequence and the role of each hormone will be outlined. RQle of Insulin Insulin stimulated glucose uptake (2x) and the con- version of 1“'Cmglucose to 1”C02 (10x) and fatty acids (10x) 25 by mouse mammary eXplants although the extent of conversion both with and without insulin decreased 50% during 48 hours of incubation (Mayne and Barry, 1970). Insulin stimulated amino acid incorporation into protein (Wool and Manchester, 1962) and also enhanced amino acid incorporation in the presence of actinomycin D (Mayne 32 31., 1966). Glucose—6-P dehydrogenase activity was increased in eXplants incubated 22 hours in insulin-containing medium (Leader and Barry, 1968). Further addition of other hormones was without effect at 22 hours but the addition of prolactin or serum from lactating mice was stimulatory in cultures continued for 45 hours. Inhibitor studies indi- cated RNA synthesis was not necessary for the insulin ~Stimulation and RNA and protein synthesis were not necessary after 3.5 and 12 hours reSpectively for the increased activ- ity measured at 22 hours. Turkington and Ward (1969) measured RNA polymerase activity in nuclei isolated from mammary eXplants. Insulin caused a 3-fold increase in activity while hydrocortisone was without a direct effect. Insulin also promoted forma- tion of daughter cells within the alveolar epithelium which were ultrastructurally indistinguishable from parent cells (Mills and TOpper, 1969, 1970). The daughter cells still contained a minimal amount of endoplasmic reticulium and only a few mitochondria. 26 Role of Hydrocortisone Addition of hydrocortisone to the insulin medium resulted in cells with an intermediate level of ultrastruc- tural deveIOpment. The rough endOplasmic reticulium increased extensively and the lateral paranuclear Golgi apparatus increased (Mills and TOpper, 1969, 1970). Most of the remaining evidence concerning the role of hydrocortisone is indirect in that it is dependent upon subsequent action by prolactin. Only the prolactin stimu- lation of RNA synthesis occurs in cells which have been previously incubated in insulin alone as well as insulin and hydrocortisone even though casein synthesis does not occur in the insulin treated cells (Green and TOpper, 1970), thus the exact role of hydrocortisone is unclear. Hydrocortisone had an effect on induction of RNA polymerase activity in that only covertly differentiated cells (cells previously incubated in insulin and hydrocor- tisone) reSponded to prolactin stimulation. Colchicine inhibited enzyme activity only if added during the period of covert differentiation (Turkington and Ward, 1969). Corticosterone may stimulate glucose metabolism in the presence of insulin (Mayne and Barry, 1970). This could indicate that at least part of the action of the adrenal corticoids is to stimulate metabolism rather than to directly affect cell replication. 27 Role of Prolactin The addition of prolactin to postmitotic, covertly differentiated mammary cells stimulated RNA synthesis 2- fold and resulted in casein synthesis (Turkington, 1968). Either colchicine or hydroxyurea added at zero time pre- vented the stimulatory effect of prolactin added 12 hours later indicating that prior cell division and DNA synthesis are necessary before prolactin may act (Mayne and Barry, 1970). Prolactin could initiate the secretory reSponse in covertly differentiated cells even though colchicine was added simultaneously, indicating that prolactin acted after hydrocortisone and insulin and also that the action of prolactin did not require DNA Synthesis (Turkington 33 gl., 1967; Turkington and Ward, 1969). The addition of actino- mycin D before or simultaneously with prolactin resulted in complete inhibition of casein synthesis (Mayne and Barry, 1970). Addition of actinomycin D 16 hours after prolactin addition (Turkington, 1968) or during the last 8 hours of the 48 hour incubation in the presence of all 3 hormones (Stockdale gtwgl., 1966) depressed casein synthesis indi- cating that a rather short-lived RNA Species is involved in casein biosynthesis. Inhibition of protein synthesis at the time of prolactin addition, also inhibited casein syn- thesis (Nayne and Barry, 1970; Turkington and Ward, 1969). The prolactin stimulation of casein synthesis in the 28 presence of insulin and corticosterone did not occur during the first 12 hours of incubation but the addition of pro- lactin within the first 12 hours of incubation to eXplants incubated in corticosterone and insulin-containing medium resulted in a stimulation of casein synthesis 24 hours later. Prolactin was without effect if not added during the first 12 hours of incubation. Prolactin addition resulted in completion of the ultrastructural differentiation of hydrocortisone-insulin. treated cells. There was translocation of the rough endOplasmic reticulium, the Golgi apparatus and the nucleus and the appearance of secretory granules in the cytoplasm (Mills and TOpper, 1970). EXplants incubated in the presence of insulin and hydrocortisone and subsequently transferred to prolactin- containing medium exhibited dramatically increased lactose synthetase activity (Palmiter, 1969) and Specifically increased the level of a-lactalbumin which is the rate- limiting subunit of lactose synthetase (Turkington 32 gl.. 1968). Thus it would appear that acquisition of the lacta- tional state was dependent upon prolactin acting on cells previously incubated in insulin and hydrocortisone. The events which occur during lactogenesis can be briefly summarized as follows: 1) most parameters measured, including DNA, RNA, enzyme activity and metabolite concentra- tion increased; 2) the data from endocrinectomized rats 29 indicated that hydrocortisone and/or prolactin were neces- sary for initiation and maintenance of lactation; 3) mam- mary explants required insulin, hydrocortisone and prolac- tin for differentiation into lactating cells. The exact stimulus for initiation of lactation is still unknown. Mammalian RNA Metabolism Several features of mammalian cells give rise to unique problems in RNA synthesis. The majority of the cellular DNA is located in the nucleus while the bulk of the RNA is in the cytOplasm, thus the relationship of transcription to translation is much more complex than in bacterial systems. Secondly the slow turnover of mammalian cells indicates that control of RNA and protein degradation may be an important regulatory phenomena. Over 90% of the radioactive precursor incorporated into RNA by HeLa cells during 30 minutes of incubation was retained in the nucleus (Perry, 1962), Evidence exists that this rapidly-labeled material was composed of: 1) larger molecules which were precursors to ribosomal RNA (r-pre-RNA); 2) a class of RNA comprising about 1% of the total RNA which was constantly being synthesized and degraded within the nucleus (HnRNA) (Darnell, 1968). Pathway of RNA Synthesis Darnell (1968) has summarized the pathway of ribosomal RNA formation as follows: 1) within the nucleus a 458 30 ribosomal RNA precursor (r-pre-RNA) is synthesized which is subsequently cleaved to a 413 and then to a 368 molecule. The 458 r-pre-RNA molecule is a polynucleotide which contains sequences destined to become 288 and 16S ribosomal RNA and some unmethylated regions of unknown function (Brown, 1968); 2) the 36S particule gives rise to an 188 ribosomal RNA molecule in the nucleOplaSm and a 328 particle, probably still in the nucleus; 3) the 32S par- ticle is degraded to yield the 28S ribosomal RNA Species; 4) the 28S and 18S ribosomal subunits are tranSported into the free ribosomal subunit pool of the cytoplasm; 5) a SS ribosomal RNA molecule is associated with the 453 precursor molecule and is processed in conjunction with the 28S ribosomal RNA molecule although a free cytOplasmic pool of SS molecules exists (Knight and Darnell, 1967); 6) ribosomal proteins appear to become associated with the 458 RNA molecule either during or immediately after its synthesis (Warner and Soeiro, 1967): and 7) about 50% of the 458 r-pre-RNA is precursor to the 288 and 188 ribosomal RNA molecules but the remainder appears to be degraded within the nucleus (Jeanteur gt_gl., 1968; Weinberg and Penman, 1970). Rapidly—Labeled RNA Recently it has been suggested that eukaryotic cells transcribe more genetic information than is translated into protein (Church and McCarthy, 1970) and that this extra RNA 31 is associated with a class of RNA, DNA-like in composition, which is confined to the nucleus (Attardi 33 21., 1966; Penman gt a&., 1968). After 20 minutes eXposure to 32F over 75% of the radioactive material in the nucleus was associated with this fraction rather than the r-pre-RNA (Soeiro §2_2l., 1968). Aronson and Wilt (1969) have cal- culated that only 6% of the nuclear RNA synthesized was transferred out of the nucleus. Soeiro 33,2}. (1968) and Church and McCarthy (1967) calculated that less than 10% of the nuclear RNA was precursor to cytOplasmic RNA. Indications are that 60-90% of this material was not trans- ferred to the cytoplasm even hours after it was synthesized, but eventually decayed to the acid soluble pool (Amaldi and Attardi, 1968; Attardi 33 21., 1966). The half life of this RNA fraction in HeLa Cells was calculated to be less than 3 minutes (Soeiro 33 31., 1968) while the half life of mRNA in HeLa cells was greater than 3 or 4 hours (Penman 33 al., 1963). Most suggestions as to the function of this RNA fraction indicate a possible relationship to mRNA and regulation of cell function but the reasons for a rapidly turning over Species confined to the nucleus and how it functions in regulation are not evident. These observations, that much of the rapidly labeled RNA never became involved in protein synthesis and also that most of the rapidly labeled RNA in the cytOplasm was in the soluble RNA fraction (Latham and Darnell, 1965), would 32 suggest that data implying that DNA-like-RNA and rapidly labeled RNA were synonymous with mRNA, needs to be reeval- uated. General Mechanisms of Hormone Action It has often been observed that many hormones can regulate the rate of protein synthesis in their reSpective target organs but are without effect when added directly to a cell-free protein synthesizing system.inlzi££g. Studies with inhibitors have implicated RNA synthesis in the mechanism of hormone action. These observations gave credence to a hypothetical mechanism, based upon the repressor-inducer model of bacteria, involving hormonal modulation through selective control of mRNA synthesis (Karlson, 1963). Tata (1968) has outlined several observa- tions which are inconsistent with this being the only mechanism of hormone action: 1) ribosomal RNA is the major product of gene transcription; 2) there is a high degree of selective restriction of RNA to the nucleus; and 3) much of the mRNA in the animal cell is long-lived sug- gesting that the entire protein-synthesizing unit may turn over at similar rates (0mura 32 gl., 1967). Thus several theories concerning the mechanism of hormone action have been preposed. Translational Control The anabolic action of some hormones such as insulin 33 (Wood and Cavicchi, 1967) and adrenalcorticotrOphic hormone (Garren.gt.gl., 1965) may be the result of a form of trans- lational control of protein synthesis, namely increased metabolic rate rather than a direct effect on RNA synthesis. Recent reports that insulin acts only at the cell membrane and is involved in membrane tranSport are consistent with a nontranscriptional role for insulin (Minimura and Crofford, 1969; Kono, 1969). Means and Hamilton (1966a, 1966b) detected increased RNA synthesis by rat uterus when estrogen was injected but also noted increased 3H-uridine uptake in the presence of estrogen. Agarwal 22.31- (1969) measured a 20% increase in the acid soluble nucleotide pool of rat liver 3 hours after cortisone treatment and 30 minutes after 3H-orotic acid injection. Billing et al. (1969) were unable to detect any increased RNA synthesis by the immature rat uterus until at least 5 hours after estradiol administra- tion when corrections were made for increased uptake of RNA precursors. Thus it would seem that one mechanism of hormone action involves tranSport across the cell membrane and increased metabolite concentration. A more elaborate translational control mechanism involving a translation repressor has been pr0posed by Thompson 23 El' (1966) to explain the superinducibility by actinomycin D of dexamethasone-induced tyrosine transamin- ase. Gelehrter and Tompkins (1967) were unable to detect 34 any increase in total cellular RNA or its rate of synthesis from labeled precursors by hepatoma cells in tissue cultures when dexamethasone was inducing a 3-15 fold increase in enzyme activity suggesting that only very small changes in RNA synthesis are required for hormone action. Reel and Kenney (1968) have suggested that the superinduction was a result of actinomycin D inhibiting transaminase degradation before it affected the rate of synthesis, thus the existence and nature of translation repressors are unclear and controversial. Another possible source of translational control revolves about changes in cellular ultrastructure. Palmiter 23 El' (1970) used physical and electron micro- scOpe assays to observe organization of ribosomes into polysomes in the chick oviduct when either estrogen or progesterone was administered. The early assembly of polysomes occurred from preexisting ribosomes, before new ribosomes entered the cytOplasm. This was similar to the observations of Gaye and Denamur (1969), that prolactin caused increased polysome assembly in rabbit mammary tis- sue (and induced lactation), and of Mills and TOpper (1970) detailing ultrastructural changes in reSponse to hormones in differentiating eXplants. It has been proposed that membrane bound ribosomes are more active than free ribo- somes (Henshaw 33 gl., 1963) or may actually be a mechanism for controlling rates and types of proteins synthesized (Pltot, 1969). 35 Another type of nontranscriptional control involves tranSport of RNA from the nucleus to the cytOplasm. Spirin (1967) prOposed that mRNA may be tranSported from the nucleus to the cytoplasm complexed with ribonuclear pro- tein as particles which have been termed informsomes and which have the potential to control translation. Church and McCarthy (1967) reported a modification of this selective tranSport of nuclear RNA in regenerating rat liver. Willems 32 El. (1969) observed an increased transfer of nuclear RNA into the cytoplasm during compensa- tory renal growth. Total RNA synthesis was not accelerated but the quantity of heterodiSperse rapdily turning over RNA in the nucleus declined. Church and McCarthy (1970) using RNA—DNA hybridization techniques were able to detect many Species of RNA molecules which were confined to the nucleus of the uterus of both estrogen treated and control rabbits. Some RNA molecules restricted to the nucleus of uterine cells from control animals were detected in the cytoplasm of uterine cells from treated animals. The Spectrum of molecules retained in the nucleus seemed more dramatic in animals before hormone treatment. These observations would suggest that increased protein synthesis could occur via increased processing of RNA into the cytoplasm rather than increased RNA synthesis. Transcriptional Control The most often observed result of hormone administra- 36 tion is a dramatic increase in RNA synthesis. The type of RNA synthesized during hormonal stimulation remains a point of contention. Tata (1968) views hormone action as resulting from synthesis of all types of RNA and eSpecially new-highly active ribosomes while others (Kidson and Kirby, 1964) argue for hormone action through selective stimulation of new mRNA. The data supporting the first theory are 2—fold. Namely, no increase in synthesis of an RNA fraction known to be mRNA has been observed without similar increases in all classes of RNA (Jackson and Sell, 1968), thus suggest- ing mRNA and rRNA increase in a parallel manner, and secondly, in many hormone-target organ interactions includ- ing the effect of estrogen on rat uterus, growth hormone and hydrocortisone on rat liver and thyroid hormone on toadpole liver, newly formed microsomes have been isolated which have increased protein synthesizing ability (Tata, 1968). Much data has also been accumulated indicating mRNA is synthesized in reSponse to hormone administration (Kidson and Kirby, 1964; Fujii and Vallee, 1967; Drews and Brawerman, 1967). The RNA which is rapidly labeled (Kidson and Kirby, 1964), extracted by hot detergent phenol (Greenman.gt‘gl., 1965) or high pH (Drews and Brawerman, 1967), is DNA-like in composition and/or has the ability to stimulate amino acid incorporation in a cell-free system 37 (Cartouzou.g£’gl., 1965) has been implied to be mRNA. Hydrocortisone stimulated 32P incorporation into the hot phenol extracted RNA (Greenman.g£‘§l.. 1965) and 3H-orotic acid incorporation into the high pH extracted RNA (Drews and Brawerman, 1967). Schmid 32 22° (1967) demonstrated increased synthe- sis of RNA which had characteristics of mRNA when cortisol was added 2E.Z$332 to isolated nuclei from rat liver. The fraction of RNA extracted with phenol at 55° was most rapidly labeled and was more effective in stimulating amino acid incorporation in a cell-free system than a similar fraction from nonhormone stimulated nuclei. Subsequently, Lang gt 2l° (1968) were able to isolate an RNA fraction from livers of cortisol treated rats which increased the tyrosine-d-keto-glutarate transaminase activity when added to an in 31232 protein synthesizing system. Inhibitor studies indicated that the enzyme was synthesized in 32352 by 22.2212 synthesis. Recently, many investigators have employed the RNA- DNA hybridization technique to detect new RNA Species syn— thesized during hormone influence. O'Malley and McGuire (1968, 1969) were able to detect unique nuclear RNA Species in the chick oviduct 6 hours after progesterone administra- tion and before avidin synthesis. Hahn 23 g}. (1969) detected new rapidly labeled RNA Species in the liver of the oviparous lizard prior to estrogen induced vitellinogenesis. 38 The nature of the new RNA Species are unknown although the observations are consistent with a Specific early hormone effect on mRNA synthesis. But with the large amounts of RNA synthesized having no known function, it is not possible to make a priori assumptions as to the nature of the new RNA Species. MATERIALS AND METHODS Materials Primiparous Sprague Dawley rats were obtained from Spartan Research Animals Incorporated, Haslett, Michigan. Uridine-5-3H, L-leucine-luC, L-amino acid-luC-mixture, and 32 H3 Hydrocortisone acetate, insulin (bovine pancreas-26 units/ POu-carrier free-were obtained from New England Nuclear. mg), actinomycin D, lactalbumin hydrolyzate (tissue culture grade) and bovine liver robosomal RNA were from Mann Research Laboratories. Prolactin (ovine-B grade) and agarose (B grade) were from Calbiochem. Medium 199-Hanks base (10x) was purchased from Difco Laboratories and Medium 199-Earles base (10x) was from Baltimore Biological Laboratories. Deoxyribonuclease (bovine pancreas-electro- phoretically purified) was obtained from Worthington Bio- chemical Corporation. Tri-iso-prOpylnaphthalene sulfonate and m-cresol were obtained from Eastman Organic Chemicals. Sodium lauryl sulfate was from Sigma. Acrylamide and N, N'-methylene-bis-acrylamide were prep grade and obtained from Canalco Industries. Methods Tissue Slices Rats (normally 3 per eXperiment) were usually 39 40 sacrificed within 30 minutes after the pups were removed (if lactating). The tissue from the inguinal glands was removed as rapidly as possible and placed in cold Krebs- Ringer bicarbonate buffer (DeLuca and Cohen, 1964) con- taining 0.1% glucose and 0.1% lactalbumin hydrolyzate. The tissue was washed several times, out free-hand into 10-25 mg pieces and washed several more times with buffer. The slices were quickly blotted and weighed before being transferred to the incubation flasks. Incubation Tissue slices were incubated in a modified Medium 199 (Table 1 and 2) at 37° under an atmOSphere of 95% 02- 5% 002. The flasks were gently shaken (40 cycles/minute) in a Dubnoff shaker. The incubations were stOpped by removing the tissue from the incubation medium and either freezing or adding 10 ml of acetone. Total Isolation of RNA and Protein Total RNA and protein were isolated by the methods of Mayne gt 9;. (1966) with some modifications. The slices were washed in 10 ml of acetone, homogenized in a minimal amount of H20 and reprecipitated with 20 ml of acetone. Two subsequent TCA (7%) washes were done before the pre- cipitate was dissolved in 1 ml of 1 N NaOH and then pre- cipitated twice more with TCA. The precipitate was dried by washing with ethanol, ethanol-ether (1:1) and finally 41 Table 1. Tissue Slice Incubation Medium for Total RNA Isolation Experiments (Medium 199-H) Usually 350 mg of tissue slices were incubated in 12 ml of buffer under an atmOSphere of 95% 02-5% co2 at 37° in a shaking H20 bath. -v '- — ——— —— —— w— 1 part Medium 199-Hanks base (10x) w/o NaHCO3 Appendix Table 1 9 part Autoclaved triple distilled H20 0.01 M N-Z-Hydroxyethyl Piperazine-N-2- Ethane Sulfonic acid (HEPES) 0.035 M NaHC03 0.1% Glucose 0.15% Lactalbumin Hydrolyzate 50 ug/ml Penicillin G, 1585 units/mg 50 ug/ml Streptomycin Sulfate PH 7.4 42 Table 2. Tissue Slice Incubation Medium for Phenol Extracted RNA Experiments (Medium 199-E) One g of tissue slices was incubated in 25 ml of buf— fer under an atmOSphere of 95% 02-5% C02 at 370 in a shaking H 0 bath. 2 250 ml Modified Krebs-Ringer Bicarbonate buffer 75 ml Medium 199-Earles base (10x) w/o NaHCOB 675 ml Autoclaved triple distilled H20 2.2 g NaHCO3 50 ug/ml Penicillin G, 1585 units/mg 50 ug/ml Streptomycin Sulfate 0.15% Glucose 0.15% Lactalbumin Hydrolyzate pH 7.4 Modified Krebs-Ringer Bicarbonate Buffer: 80 mM NaCl, 5 mM KCl, 2 mM CaClZ. 2.5 mM MgSOu 43 ether. The dried precipitate was dissolved in 0.5 N NOR and aliquots taken for determination of RNA, DNA, protein and radioactivity. RNA and DNA were determined by the orcinol and diphenylamine methods reSpectively (Schneider, 1957) and the method of Lowry §t_al. (1951) was used for protein determination. Radioactivity was determined with a 3 channel Packard Tri-Carb liquid scintillation counter employing the XDC scintillation solvent of Burno and Christian (1961). Efficiency (18% for 3H and 39% for 14 C) was determined by the "Spiking" technique of 0kita.g£ g}. (1956) and correction was made for overlap of 140 into the 3H channel (12% of 14C). Control experiments indicated less than 0.5% of the uridine was incorporated into the nonbase hydrolyzed (DNA) fraction, thus RNA and DNA were not separated. Extraction of RNA RNA was extracted by modification of the methods of Scherrer and Darnell (1962), Penman (1966) and Loening (1967). Phenol was distilled and washed with 0.001 M EDTA shortly before use. Bentonite was prepared by the method of Frankel-Conrat and Singer (1961). The homogen- ization buffer was 0.05 M sodium acetate 0.01 M EDTA, 0.1 M KCl at pH 6.0. This isolation scheme is shown in Figure 1. The tissue was gently homogenized in 10 volumes of buffer containing 6 mg of purified bentonite per ml. Twelve ml of phenol containing 10% m-cresol and 2% tri- 44 Figure 1. Scheme for Phenol Extraction of RNA 1 g tissue homogenized in 10 ml of buffer containing 6 mg/ml bentonite 1) add phenol plus detergent 2) stir Phenolrfraction- interphase-residue 1) add buffer and phenol 2) heat r’ , remainder aqueous phase (discard) 1) add phenol plus detergent 2) heat r_f phenol phase aqueous phase (discard 1) add phenol 2) heat r—' ’ ‘1 phenol phase aqueous phase (discard) 2) heat rt 1 CHCl phase aqueous phase (discard) 1) add CHCl3 2) heat '— j CHCl phase aqueous phase (discard) 1) 2% NaAC 2) 2 vol ETOH, -200 \V RNA 45 iso-prOpylnaphthalene sulfonate was added and the sample stirred for 20 minutes at room temperature. The samples were cooled to #0 and the phases separated by centrifuqa- tion for 2 minutes at 8,000 X g. Fifty mg of tri-iso-prOpynaphthalene sulfonate was added to the aqueous phase after it was removed. Five ml of buffer and 5 additional ml of phenol were added to the remaining phases which were then heated to 550 for 4 minutes. The phases were separated as before and the aqueous phases combined. The combined aqueous phase was extracted twice with 12 ml of phenol at 550 for 2 minutes. The phases were separated by centrifugation at 4° for 2 minutes at 8,000 x q. The resultant aqueous phase was extracted twice with chloroform at 550 for 2 minutes. Each time the phases were separated by centrifugation at 500 x g at room temper- ature for 30 seconds. The final aqueous phase was made 2% with sodium ace- tate (pH 5.1) and the RNA precipitated by the addition of 2 volumes of ethanol at .200 for 8 hours. The precipitate was collected by centrifugation, dissolved in several ml of 2% sodium acetate (pH 5.1) and reprecipitated with cold ethanol. After centrifugation, the pellet was dissolved in enough gel buffer to yield an RNA concentration of 1-2 mg per ml. RNA was determined by the method of Warburg and Christian (19h2) and recovery was usually 80% of eXpected and the 260/280 ratio was about 2.0. 46 Polyacrylamide Gel Electrophoresis of RNA The procedure described is a modification of the sys- tem of Weinberg gt a}, (1967) and Peacock and Dingman (1969). Gels of 2.2 and 2.4% acrylamide and .6% agarose were rou- tinely used. The gels were cast in round, high quality quartz tubes (0.5 cm I.D. x 10 cm). The gel buffer was 40 mM Tris base, 20 mm sodium acetate and 3 mm EDTA at pH 7.6. Ten ml of aceylamide solution, adequate for 4 gels, were prepared at one time. A mixture of 60 mg of agarose, 7.5 ml of buffer and 0.5 ml of glycerol was heated until the agarose was in solu— tion. Two hundred and forty (or 220/mg of acrylamide, 10 mg N,N'-methylene-bis-acrylamide and 8 ul of N, N, K', N'-tetra-methylethylenediamine were dissolved in 2 ml of buffer. The temperature of the solutions was adjusted to 400 and they were mixed. Six mg of ammonium persulfate was added and the gels were cast. The gels were cooled at 40 for 2 minutes in order to assure that the agarose would set before the acrylamide polymerized. After 1 hour of polymerization, a small layer of material which had formed above the gel prOper was removed, leaving a gel 9.5 cm long, with a flat surface and little 260 mu absorbance at the tOp. The gels were preelectro- phoresed for 1 hour at 5 mA per tube. The electrOphoresis buffer was the same as the gel buffer except it contained 0.3% sodium lauryl sulfate. a? The sample (10-80 ug of RNA) containing 5% sucrose was layered on the gel and a current of 3 mA per tube was applied for 10 minutes followed by a current of 5 mA per tube for an additional 80 minutes. Some samples contained larger than normal amounts of DNA and were incubated with an equal volume of deoxyribonuclease solution (200 ug of enzyme/ml of 200 mm NaCl, 1 mm KHZPOg and 3 mh MgClz at pH 7.0) for 30 minutes at 37° immediately before being electrOphorised. The gels were scanned without being removed from the tubes at 260 mm with a Gilford recording Spectrophoto- meter equipped with a linear tranSport attachment. The area of the various peaks was measured with a plamineter. The repeatibility of the area determination and the rela- tionship of area to amount of RNA applied to the gel is shown in Table 3. Gels containing 32F were cut into 2 mm slices and counted using a scintillation fluid of 7 g of 2,5-diphenyl- oxazole and 0.6 g of 1,4-bis-2-(5-phenyloxazolyl)-benzene per liter of toluene. Determination of 32F in gel slices by Cerenkov radiation (suggested by L. L. Beiber) indicated very little quenching of 32F by the acrylamide. Gels con- taining 3H were counted according to the techniques of Peacock and Dingman (1967). Specific Activity_of 32? Acid soluble material for 32? Specific activity 48 Table 3. Relationship of Amount of RNA in Sample to Area of Absorbance in Various Peaks The amount of RNA in several samples was determined by the method of Warburg and Christian (1942). The area of the peaks was determined with the use of a plamineter. Several samples containing only sucrose were electrOphoresed so the base line absorbance could be accurately determined. A ‘ n L ___._ _- ug of RNA in Sample Area Under Peaks sq cm/lO mg 30 18.1 6.03 37.5 23.3 6.21 37.5 24.5 6.53 45 27.7 6.15 48 31.1 6.47 52.5 31.0 5.90 Average 6.21 sq cm/10 ug of RNA. 49 determination was prepared by the method of Baldwin and Cheng (1969). The tissue was powdered under liquid nitro- gen and then homogenized in 4 volumes of cold perchloric acid—ethanol-water (8:50:42). After centrifugation at 40 for 10 minutes at 35,000 x g the supernatant was removed and neutralized with 3 N K2C03 in 0.5 N triethanolamine. The KClOu was removed and the supernatant analyzed for free phOSphate. The phOSphate determinations were by the method of Peter (1963). Two ml of isobutanol-benzene (1:1), 4 ml of acid molybdate (1.5 g of ammonium molybdate in 100 ml of 0.5 N H2804) and 1 ml of sample were vigorously shaken for 30 seconds before the phases were separated. The absorbance of the upper layer was determined at 410 mu. Radioactivity was measured with a Nuclear Chicago low background planchet counter at 30% efficiency. RESULTS AND DISCUSSION The results will be presented in two major sections. The first section will deal with develOpment of the various systems. The Optimal incubation conditions for RNA synthe- sis were develOped and changes in the composition of the tissue between 3 days prepartum and 3 days postpartum were examined. Also in this section, characterization of the. RNA by polyacrylamide gel electrOphoresis and the discrep- ancies between RNA synthesis as assayed by phenol extrac- tion and TCA precipitation will be discussed. The second section will treat the effect of hormones upon RNA and protein synthesis and will also discuss the interrelationship of the several methods of assessing hor- monal effects. Throughout these sections the abbreviations, I for insulin, P for prolactin, and NC for hydrocortisone acetate will be used in the tables. DevelOpment of Systems Development of Incubation Techniques Preliminary eXperiments to define the Optimal Operat- ing parameters of the systems utilized bovine as well as rat mammary tissue. Bovine tissue, which had been frozen in several different buffer systems including glycerol, never incorporated radioactive precursors as well as unfrozen 50 51 tissue which was subsequently used in all eXperiments. The first eXperiments involved develOpment of the buffer system necessary for maximum rate and extent of incorporation. The results presented in Table 4 indicate that both buffering capacity and metabolite levels are important in maintaining maximal levels of incorporation in bovine mammary tissue slices. The enhanced incorpora- tion in the incubation systems containing higher buffer concentrations is evident within the first hour of incuba- tion. The effect of the higher buffer concentration is undoubtedly a reflection of the low sodium bicarbonate level used in the Hank's base medium and the resultant inability to maintain prOper pH when large amounts of very metabolically active tissue was incubated rather than a stimulatory effect of bicarbonate. The higher buffer concentration was able to maintain pH 7.4 as was the Medium 199-Earles base used in later eXperiments. The inclusion of additional glucose and amino acids in the complex incubation medium had an even greater stim- ulatory effect on 3H-uridine and 32F incorporation into RNA by tissue slices from rat mammary gland (Table 5) than was observed with bovine mammary tissue. A stimulatory effect of additional amino acids and glucose was not observed with bovine tissue during the first hour of incu- bation but the combination of glucose and amino acids stimulated 3H-uridine incorporation during the first 30 minutes in rat mammary tissue slices. 52 Table 4. Effect o Buffer Systems and Netabolite Concentra- tion on H-Uridine Incorporation into RNA by Tissue Slices from Lactating Bovine Mammary Gland One g of bovine mammary tissue slices were incubated in 10 ml of Medium 199 (Appendix gable 1) with the indicated additions and 10 no of uridine-5- H (24.7 C/mmole as outlined in Methods. RNA was precipitated with TCA as outlined in N et hod s . dpm/mg RNA Buffer System 60 Min 180 Nin Medium 199 + 35 mg NaHCO3 1000 2330 Medium 199 + 350 mg NaHCO3 1300 3325 Medium 199 + 35 mg NaHC03 and 0.01 w HEPES1 1070 1955 Nedium 199 + 350 mg NaHCO3 and 0.01 M HEPES 1370 2610 Medium 199 + 350 mg NaHCOB-0.01 N HEPES and 0.1% glucose 1420 3135 Medium 199 + 350 mg NaHCO -0.01 N HEPES— 0.1% glucose and 0.1% lactal- bumin hydrolyzate 1390 4325 1N-Z—Hydroxyethyl Piperazine-N-Z-Ethane Sulfonic acid 53 Table 5. Effect of Added Glucose and Amino Acids on Incor- poration of Precursor into RNA by Mammary Tissue Slices a) 300 mg of mammary tissue slices from rats 72—84 hours postpartum were incubated in 5 ml of Medium 199-H (Table 1) with and without added amino acid and glucose and containing 5 no of uridine-5-3H (24.7 C/mmole. The RNA was precipitated with TCA as outlined in Methods. Uridine Incorporated, dpm/iOO cg RNA Treatment 30 Min 60 Min 180 Nin Medium 199 282 487 1842 Medium 199 + 0.1% glucose + 0.1% lactalbumin hydrolyzate 732 2346 4135 b) One g of tissue from mammary glands of rats 3 days prepartum and 72-84 hours postpartum was incubated in Medium 199-E (Table 2) or Medium 199 (Appendix Table 1) with 50 do of 3 P as outlined in Methods. RNA was extracted and frac— tionated and the Specific activity of the 308, 188, and sRNA determined. Insulin (I), hydrocortisone (HC) and prolactin (P) were present at a concentration of 40 ug/lOO mg of tissue. _i_.i 2 3 P Incorporated, Treatment opm/10 ug RNA/12 hours Prepartum Medium 199-E 3713 Medium 199 2255 Medium 199—E + HC-P-I 4518 Medium 199 + HC-P-I 2686 Postpartum Medium 199-E 823 Medium 199 545 Medium 199-E + HC-P-I 1166 Medium 199 + HC-P-I 667 54 Similarly the increased level of amino acids and glucose stimulated the incorporation of 32F into phenol extracted RNA from both lactating and nonlactating slices. The data in Table 5b is biased in that the Specific activity of the 32p in the Medium 199-Earles base (Appendix Table 1) medium is approximately 20% lower than in the Medium 199-E (enriched medium, Table 3) but the data in Table 7 indicates that the Specific activity of the tissue phOSphate pool is pr0portional to the Specific activity of the 32F in the incubation medium, thus approximately 25% of the increased incorporation would be due to increased Specific activity of the precursor pool. Based on this assumption, the increased amino acids and glucose caused a 50% increase in the amount of RNA synthesized. The addition of hydrocorti- sone, prolactin and insulin did not affect the reSponse of the tissue to added metabolites. Previously, Wool and Manchester (1962) had observed that amino acid incorporation by lactating mammary tissue slices was stimulated by the addition of oxidizable sub- strates or insulin, but the addition of oxidizable substrates prevented the insulin stimulation of incorporation which occurred when slices were incubated in minimal medium. This suggested that insulin increased the available energy supply to the tissue. Although no direct effect of glucose alone was detected, the increased level of glucose in the incubation medium was retained because of the observation 55 of Wool and Manchester (1962) and also because of the report that glucose (10 um/ml) prevented many of the extreme mor- phological modifications observed when mammary tissue was dissociated with trypsin (Pitelka 33 31.. 1969). The level of glucose in the enriched incubation medium in the eXperiments in Tables 4 and 5 is 11 um/ml while a level of 14 um/ml as indicated in Tables 2 and 3 was used in all subsequent eXperiments. The glucose concentration of the buffer was 2.5 fold higher than normally observed in rat blood (Long, 1961) and 10 times the level reported for mammary tissue (Kuhn and Lowenstein, 1967; Baldwin and Cheng, 1969). Schingoethe gt 2;. (1967) reported that the addition of amino acids, including levels 10 times higher than nor- mally contained in Medium 199, stimulated the synthesis of d-lactoglubulin and B-casein. The addition of 0.15% lac- talbumin hydrolyzate increases the amino acid concentration of the incubation medium 2-fold (final concentration 2.2 mg/ml) compared to normal Medium 199. This is approximately 2.5-fold higher than the free amino acid level in blood (Long, 1961) but only 40% and 120% higher than the free amino acid concentration in lactating and nonlactating rat mammary tissue. The stimulation of synthesis by added metabolites would indicate that although sufficient levels of metabolites for substantial amounts of synthesis are available, the 56 metabolite level of the cell may limit productivity. Baldwin and Cheng (1969) reported that the level of metabo- lites in prepartum mammary tissue was adequate to sustain milk synthesis but that at high levels of production, metabolite concentration may become limiting. Similarly Kuhn and Lowenstein (1967) detected all the precursors for lactose several days before lactose was detected. Thus, although metabolite level may be important in determining the level of production it would not appear to be the key to initiation of lactation. It is also possible that in 31333 concentrations are the result of a decline in metabolite level during preparation of the tissue slices or simply that uptake is more limiting 12.21232 than in vivo. Composition of Pre- and Postpartum Tissue The relative changes in RNA, DNA and protein content of the tissue as reported in Table 6 are similar to those reported by other workers (Baldwin and Milligan, 1966; Tucker and Reece, 1963a, 1963b) but the content per 100 mg of wet tissue is 20-30% less. This is probably a reflection of the amount of buffer weighed out with the tissue but may also result from a lower RNA content of the gland. The protein content of the tissue increased 28%, the PNA con- ‘tent, which indicates cell number, increased 50% and the PiNA content increased 125%. The RNA-DNA ratio increased 535% indicating increased synthetic activity per cell as VTell as an increased number of cells. A. 1- axidu 57 Table 6. Protein, RNA and DNA Content of Mammary Tissue of Rats 3 Days Prepartum and 3 Days Postpartum The RNA, DNA and protein content of the TCA precipi- tated tissue was determined as outlined in Methods. The values are the average of 20 observations from 6 postpartum rats and 10 observations from 3 prepartum rats. Content per 100 mg Wet Tissue Ratio1 3 Day§_Prepartum 'Protein 2.46 mg 3.7 RNA 87 0g 1.3 DNA 66 :1g 1.0 3 Day§gPostpartum Protein 3.17 mg 3.2 RNA 193 mg 2.0 DNA 7 99 0g 1.0 1Ratio of mg of protein and mg of RNA to ug of DNA per 100 mg of tissue. 58 Changes in.Phosphate Pool Size The phOSphate pool size in mammary tissue (Table 7) increases 50% between 3 days prepartum and 2 days postpar- tum which is reflected in a 30% decrease in the Specific activity of the free phOSphate pool after 12 hours of incubation. The difference in Specific activity between the samples incubated with and without hormone is probably an indication of the eXperimental error of the method. The ratio in 32? Specific activity between the lactating and nonlactating tissue was quite repeatable (77 and 72%) in two experiments. In each experiment, the Specific activity of the phOSphate pool in lactating tissue was 40% of the Specific activity of the buffer while in nonlactat- ing tissue it was 60% of the Specific activity calculated for the 32p in the buffer. Similar changes in pool size at the time of initia- tion of lactation have been reported for total nucleotides and uridine nucleotides (Wang, 1962) adenine nucleotides (Baldwin and Cheng, 1969) and amino acids (Stellwagen and Cole,_1969). The Specific activity of the intracellular phOSphate pool is only 32% at one hour and 70% at 3 hours of the maximum attained at 12 hours indicating that incorporation (during short periods of incubation would be low because of the low Specific activity of the precursor pool. Also any Geffect on uptake which results in increased Specific activ- 1.ty of the precursor pool, particularly during short term 59 .woapmnsosa mo maze: m pmma mQHHSp maco pzmmmam mmmt llllr ill moa M H.N Iii aSpamemoa mason omim: moa x H.m 111 fiSpHmemoQ wagon 0H moH N ©.N Iii coHpmpmow go amp Spma sodmeSosH uo masomlmfi HHH moH x H.m m.m Himivm + mason NH moa x m.o m.m wagon NH moa x :.m :.m mados m cofipmpomm do amc USN HH moa a m.a S.H *Hnmnom + mason NH moa x m.m ©.H amazon NH moa M m.HH u.a Himiom + maso: NH moa x m.oH 0.H mados NH moa N m.m w.H ago: H acapdumou mo.NdQ Spwa H Hm oaoaj\aao mammaa pm: m\am mace: ll”) .oamamm mean was So coma msoapmgaaampmo N mo ommaobm can can mosam> HH< .Aoaoaj\aap OH x :.Hv pm cm mm: o: m.NH .HHH pcmEdaoaNc SH pad Aoaoa:\aao wma N 3.0V AH ommmdz ma ooHv mimma azapcz mo HE mm on pocpm mm: m m mo 0: om HH bum H meEaaoaNo SH .mpOSSoz Ca occaauso mm popmanca mm: mammap ho m 020 mpmm wcdpmpomHst pad wsapmpomq 80am mammae mamfiamz SH cpmzamonm comm ho hpabfipo< capaooaw .m manna 60 incubations will be immediately reflected in increased incorporation into RNA, which, if changes in Specific activity of the precursor pool were not accounted for, would be interpreted as increased RNA synthesis. Several previous reports of hormonal stimulation appear to be due to increased uptake of precursor. heans and Hamilton (1966a) concluded that the early increases in uterine RNA synthesis in reSponse to estrogen was due to increased uridine uptake and Billings gt a}. (1969) could detect no increased RNA synthesis in uterine tissue until 5 hours after estrogen administration if corrections were made for changes in the Specific activity of the uridine pool. The changes in Specific activity of the intracellu- lar pool may be more important with 3H-uridine than with 32P because of the observation that uptake is the rate limiting step in the incorporation of uridine into acid soluble phOSphorylated products (Plagemann and Roth, 1969). Histological Evidence of Changes in Tissue During Incubation Histological examination was undertaken to determine the extent to which gross morphological changes were occurring during the extended periods of incubation. Photomicrographs of the tissue are shown in Figure 2, The tissue incubated for 9 or 12 hours without hormone exhibited more necrosis than tissue incubated with insulin, hydrocortisone and prolactin. The tissue incubated without hormone was often grossly degenerated, did not have well 61. Figure 2. Photomicrograph of Lactating and Nonlactating Nammary Tissue Slices Incubated in the Presence and Absence of Hormones Upper, left: Tissue slice from rat mammary gland 3 days pre- partum incubated 9 hours without hormones. (x400) Upper, right: Tissue slice from rat mammary gland 3 days pre- partum incubated 9 hours in presence of insulin, hydrocortisone and prolactin. (x400) Lower, left: Tissue slice from rat mammary gland 3 days poitpartum incubated 12 hours without hormone. (x 00) Lower, right: Tissue slice from rat mammary gland 3 days postpartum incubated 12 hours with insulin, hydrocortisone and prolactin. (X400) 0 62 63 defined alveoli, and more pycnotic nuclei were observed. Nore stainable material was normally observed in the lumen of tissue slices incubated with the 3 hormone combination. Lesser amounts of stainable material were observed in the alveolar lumen of nonlactating tissue incubated 12 hours in the presence of hormones than in lactating tissue slices under similar conditions. In general, the 3 hormone combination appeared to maintain the tissue for 12 hours in a condition similar to that observed at the beginning of the incubation period. Characterization of RNA in Pre- and Postpartum Tissue In order to investigate the possibility that Specific minor fractions of RNA are synthesized in reSponse to hormones or that the profile of RNA Species synthesized changed at the time of parturition, a polyacrylamide gel electrOphoresis system was develOped for separating the various RNA Species according to size. SirakOV'gt a}. (1968) identified a 12-158 RNA Species as mRNA and Baldwin.§£ a}. (1969) reported that isolated mammary nuclei $2.1i232 synthesized mainly RNA'S of 12—158 and thus presumably mRNA. In order to ascertain that the electrOphoresiS system would separate a 12-158 RNA Species from the 18S rRNA Species, a mixture of rRNA from Escherichia 0011 (268 and 16S) and RNA from lactating mammary tissue was electrOphorised. It is evident from Figure 3a that a 158 Species of RNA would be readily resolved by the electro- phoresis system employed. ElectrOphoresis for longer 64 .nne mm Hagenanepm gm pom oomaaozaoapomac mm: Adv CH Esosm mm How 05mm 059 AD .mHmw omoamwm No.0 1 opasmammaod mdlm SH gas om How pmmoaosaoapooam mm; mzm mamfiEda pad “Hoe .m mo magpxas ems .mpospoz SH poCHHpso mm mammap appends wgdpmpoma Soak oopomapxm was «zm Am mammab hamaamz wgapmpomq 80am «zm Hmpoe pad 42m Hmaomopam aaoo .m 90 macaw“: m wo mammadg< oapmaogmoapooam .m madmam 65 H ,3 j J -N .0 l l l l (D V’ _m I!) —<0 U) (D co ‘2 - | ‘1 e- -— ‘ fl "V 3% ———=: r” R «N 0 J l l l o. in. 0. in. N - — O m“ 098 ‘BONVBHOSBV DISTANCE (cm) 66 periods of time (Figure 3b) resolved a peak which was slightly smaller than 168. The peaks were designated as 308, 188 and SRNA on the basis of their migration rela- tive to the values of Peacock and Dingman (1968). The individual peaks were identical to apprOpriate fractions isolated by sucrose density gradient centrifugation (Figure 4) and to liver rRNA purchased from Mann Research Labora- tories. The separation attainable with large amounts of RNA from lactating tissue is shown in Figure 5a and the effect of applying dilute samples is illustrated in Figure 5b. The same material was applied to both gels except a smaller amount of material which was 4-fold diluted was applied to the second gel. Better separation Was generally achieved with small (<50 ug) concentrated (2 mg/ml) samples. The resolution of RNA Species from nonlactating tissue (Figure 6a) was normally not as good as with RNA from lactating tissue (Figure 6b) although the separation was adequate. This poorer resolution did not appear to be due to degradation of the RNA but was possibly a result of DNA contamination. The peak denoted as DNA was the only one to disappear when the sample was incubated with DNase. The peak migrating faster than SRNA was only observed in 'those samples previously incubated with DNase. A similar 'peak was observed to migrate Slightly faster than sRNA, When ATP or ADP were added to the sample. Thus, material migrating faster than SRNA was normally not assayed. 67 Figure 4. Sucrose Density-Gradient of Nammary RNA The sample was the total phenol extracted RNA from lactating tissue. The gradient was 5-30% (w/v) sucrose in triS-acetate buffer (0.01 N tris, 0.01 N sodium acetate, 0.14 M KCl). The volume was 12 ml and was run for 7 hours at 30,000 rpm at 170 in the SB 283 rotor of the 3-60 International ultracentrifuge. Analysis was with a Gilford recording Spectrophotometer equipped with a flow cell. 2.0 I5 :5. E O (D (\l 8” Z l.O <12 m (I. O CD CD <[ 0.5 68 308 IBS J l I 4 6 8 ml ELUTED “SRNA IO l2 69 .Hom sedan m Spflz oobhomno mocmppomnw unschwxomn on» ma mQHH dogmmo one .maow mmonmmm mm.o I moaamHmhow Rm.m ca me n pm SHE om hou ommHHOSQOHpomHo mm: mammap mamaema wsaumpoma Soak Aaj\mj u.ov 42m mo m: om An .mHow mmopmww mm.o n modemamnom &dlm ma ma m pm :aa om pom omwaQOSQOHpomHm was mammap mamfifima wzapwpoma 809m AH1\M3 NV 42m mo mm mm Am mawmhosmoupooflm mp oosawpp< :oflpmammom no :oapmspsmonoo ¢zm and mmam capsmm mo pommmm .m opswfim ABSORBANCE, 260 my 2.0 "on 0.5 7O I I ’6 ‘~ \ ~~ f' e 2 ' DISTANCE (cm) 0) 71 .Hmm xsmap m spas oophomno mocmnaomnm osmoswxomn oSp ma mafia dogmmo one .Hom omoammm &o.o I modamazhom &:.m ma as m pm Eda om Mom oomahona Iompomao mm: Eduamapmoa maso: mHIo mums mo mammap mamaama Bosh AQOapmo IHHQQm op HOHHQ odeQ Spas oopmnsocav Aaj\mj o.mv «am 90 m: ma an .Hmm mmoammm mw.o I mofiemampom &:.N 2H ma m pm SHE om Mom oomahosponpomao mm; .moonpmz ma omQHHpSO mm adppmamhm mmmo MIN mush mo mammap mamaama anm ompomhpxo Aaj\wj m.av «2m mo my om Am commas mamBEmz advamapmom mpsom mHIw Ugo Ezphmamhm Eopm 42m mo mammamc¢ capoMOQQOHpomHm .0 mpsmam 72 E8 moz<._.m_o N m m <20 8858 _ _\|lr ‘ I I \ I, (Eu m6 0; 0. ON “L“ 092 ‘BONVBHOSBV 73 A peak absorbing at 260 mu always migrated faster than the 188 rRNA Species and as shown in Figure 3a and 3b, it was somewhat smaller than 168. Thus, a Species with precedent for being mRNA-like can be resolved, and was observed in both pre- and postpartum mammary tissue. A similar Species was also detected in RNA isolated from mammary tissue of the midpregnant mouse, (E. Cummins, personal communication). The coincidence of the 3H—uridine labeling pattern and the absorbancy profile is illustrated in Figure 7. Similar profiles were obtained when 32? labeled RNA was electrOphorised. There was no evidence of any radioactive material being excluded from the gel. Evidence for mRNA Two lines of evidence indicate that the 153 RNA Species isolated by the techniques utilized in these eXperi- ments was not the mRNA proposed by Sirakov gt 3}. (1969) and Baldwin.g£_§l. (1969). The amount of 158 RNA present (although because the peak was small, accurate quantitation was difficult) relative to rRNA was constant between mid- pregnancy and late lactation. The data of Gaye and Denamur (i969) and Mills and TOpper (1970) indicated the number of polysomes increased during lactogenesis thus suggestinq an increased amount of mRNA postpartum. Secondly, as presented in.Appendix Tables 3-7 no Species of RNA was detected, which had a Specific activity 74 Figure 7. ElectrOphoretic Analysis of RNA from Mammary Tissue of Rats 6-18 Hours Postpartum Labeled with 3H-Uridine One 3 of tissue slices was incubated in hedium 199-E with 50 me of BH-uridine (Sp. Act. 24.7 C/umole) for 7 and 12 hours. The RNA was extracted as outlined in methods. Twenty ug of RNA was electrOphorised for 90 min at 5 ma in 2.2% acrylamide - 0.6% agarose gel. 75 ABSORBANCE, 260 mu [‘5 '— 0 I2 hour incubation 7 300 o 7 hour incubation - 250 I .O — I —- 200 i + a .50 0.5 -i IOO .4 5O I O Ltd-1-4” 6 8 DISTANCE (cm) RADIOACTIVITY (dpm) 76 higher than rRN , with the possible exception of SRNA from prepartum tissue. This increased rate of SRNA synthesis prepartum is consistent with Turkington's (1969) observa- tion that the amount of SRNA relative to rRKA increases during late pregnancy. Also hormones did not significantly affect the 15S RNA Species in any manner distinct from the other classes of RNA as might be eXpected if hormones have a Specific effect on mRNA synthesis. Thus, utilizinm the techniques available, no Species of RNA with characteris- tics associated with mRNA or uniquely influenced by hor- mones could be detected. Discrepancies in RNA Synthesis as Assayed by_$wo Procedures Two distinctly different methods were used to assess the extent and rate of incorporation of labeled precursor into RNA. The TCA precipitation method measured 3h— uridine incorporation into all acid insoluble, base-labile products while the phenol extraction method measured the incorporation of 3R-uridine or 32F into 3 defined Species of RNA which accounted for over 90; or the radioactivity observed, namely, the 308, 183 and sRNA fractions. The time period during which the radioactive precur- sor was present influenced the calculated level of synthe- sis (Table 8). It would have been anticipated that the calculated cummulative synthesis would have been less, rather than greater as was observed. The reason for this being, that when the isotOpe was present for only the last 77 Table 8. Effect of Length of Labeling Period Upon Amount of H-Uridine Incorporated Into RNA by Nammary Tissue Slices 350 mg of tissue from the mammary land of rats were incubated in hedium 199-H (Table 1) with H-uridine1 as out- lined in Methods. The RNA was precipitated with TCA and the radioactivity determined. i —f -_7 L7 ’1 -——-——-r_ _r H Uridine Incorporation2 uumole/mg RNA 5 Hrs 7 Hrs 10 Hrs Prepartum Zero time3 170 232 _-- Cummulativeu 254 372 -__ Postpartum Zero time3 182 226 458 CummulativeL'L 333 497 644 1 Prepartum tissue - Uridine-5-3R - 15 uc/incubation (Sp. Act. 143 uc/umole). Postpartum tissue - Uridine-S-BH - 15 uc/incubation (Sp. Act. 125 uc/umole). aCalculations based on Specific activity of uridine in the incubation medium. 3IsotOpe was added at beginning of incubation (zero time). “IsotOpe was present only for last hour of incubation, thus isotOpe added at the end of 4, 6, or 9 hours of preincuba- tion. The values were obtained by summina the series of such incubation. 79 hour of incubation (cummulative), the time required to establish a labeled precursor pool should have been mawni- fied because durinm each hour a new precursor pool was established rather than only once as when the isotope was added at zero time. Instead these results indicate a rapid burst of 3H-uridine incorporation immediately after the precursor is added. It is also possible that non— Specific binding of the isotOpe was occurring or that large amounts of uridine were being incorporated at the end of existing polynucleotides. It does not seem to involve a decrease in the Specific activity of the precur— sor pool during longer incubation periods either because the rate of incorporation in these eXperiments is quite linear. Also the calculated uptake of uridine, based upon RNA synthesis is less than 0.1% of the uridine in the medium. The method of isolating the REA and labeled precur- sor used also influenced the calculated levels of synthesis (Table 9). The TCA precipitation method indicated over 7 times more uridine was incorporated than was detected by the phenol extraction method. This would indicate that the fractions of RNA isolated by the two methods were quite dissimilar. The amount of 32F incorporated into phenol extracted RNA is almost ZOO-fold higher than the amourt of 3H-uridine incorporated (assuming that RNA is 25% uridine, Baldwin and Nartin, 1969a, 1968b) which is undoubtedly a 79 Table 9. Effect of Assay Method and IsotOpe Upon Calculated Amount of RNA Synthesized by hammary Tissue Slices Values for phenol extracted uridine are from Figure 7, for TCA prggipitated uridine are from Table 9, and for phenol extracted P are from Table 14. All calculations are based on the Specific activity of the precursor in the incubation medium. Precursor Incorporated, unmole/mg RNA During Incubation Period 7 Hrs 10 Hrs 12 Hrs Phenol extracted uridine 3o -_- 50 Total precipitated uridine 226 458 _-_ Phenol extracted 32F 21,000 --- 30.000 80 reflection of the Specific activity of the intracellular precursor pool. The extent to which actinomycin D inhibits incorpor- ation was also dependent upon the assay method (Table 10). The incorporation of 3H-uridine into TCA precipitated RNA was only inhibited about 65% by actinomycin D while 32F incorporation into phenol extracted RNA was almost completely inhibited in lactating tissue and in nonlactating tissue during 12 hours but not 3 hours of incubation. The lack of complete inhibition of uridine incorporation is not in agreement with the results of Mayne and Barry (1967) where actinomycin D (at slightly higher concentrations) completely inhibited incorporation. The almost complete lack of 32F incorporation into phenol extracted RNA (Tables 11 and 14) during one hour of incubation was noted. Although only one experiment is reported utilizing 3H—uridine incorporation into phenol extracted RNA (Figure 7) the same lack of incorporation is noted indicating that differences in uptake of isotOpe would not eXplain all of the discrepancies between the two systems. Also, preliminary eXperiments indicated signifi- cant levels of 32P were incorporated into TCA precipitated RNA during the first 30 and 60 minutes of incubation. This would again indicate that the fractions of RNA isolated by the two methods are not identical. Yet the recovery of RNA by the phenol extraction method was almost equal (90%) to 81 Table 10. Effect of Actinomycin D on Incorporation of Precursor Into RNA by Mammary Tissue Slices a) 350 mg of tissue from mammary glands of rats 2-3 days prepartum or 3 days postpaftum were incubated in Medium 190—H (Table 12) with 3H-uridine as outlined in Methods. The RNA was precipitated with TCA and the radioactivity determined. w ‘-'__ ———— .— Uridine IncorporatioB uumole/mg RNA/60 Min __ Treatment 1 Hr 3 Hrs 5 Hrs 7 Hrs Prepartum No addition 36 —- 67 58 + 7.1 ug actinomycin D3 27 (74)“ -- 22 (33) 19 (33) + 4.3 ug actinomycin D -- -- 22 (33) 21 (36) Postpartum No addition 49 -- 60 90 / + 12 ug actinomycin D 43 (89) 30 27 (39) 27 (34) lPrepartum tissue - Uridine-S-BH - 15 uc/incubation (SD. Act. 143 uc/umole) Postpartum tissue - Uridine-5-3H - 10 uc/incubation (Sp. Act. 125 uc/umole) 2Isotope was only present during last hour of incubation. 3Actinomycin D was resent at the concentration/100 mg tissue as indicated (4.3 ug 100 mg = 1.2 ug/ml). 3% of normal. 82 Table 10. (Continued) b) One g of tissue from mammary glands of rats 3 days prepartum or 72-84 hours postpartum was incubated in Medium 199-E (Table 2) with so do of 33p as outlined in Methods. RNA was extracted and fractionated and the Specific activity of the 308, 188 and SRNA fractions determined. 32F Incorporation com/10 um RNA Treatment 3 Hrs 12 Hrs Prepartum No addition 543 3713 + 10 ug actinomycin D 423 80 Postpartum No addition 131 881 + 10 ug actinomycin D 34 21 13 hour sample was preincubated for 1 hour with actinomycin D before 33F was added. 2Actinomycin D was present at a concentration of 10 ug/100 mm tissue (4'ug/m1). 83 the total precipitated RNA and the recovery of phenol extracted material would probably be higher except for the relatively large losses (percentage) due to small sample size. Thus, the difference must be due to a small frac— tion of RNA which turns over very rapidly. In order to ascertain that the difference observed between the short term 3H-uridine incorporation eXperiments and the longer labeling periods with 32F were not associated with the fractionation of the RNA Species on acrylamide gel an isolation precedure involving phenol extraction followed by TCA precipitation was used (Table 11). The RNA extracted by this procedure was comparable to the fraction I-RNA (rRNA) of Baldwin and Nartin (1968a, 1968b). The small amount of tissue used in these eXperiments prevented accurate measurements of the Specific activity of the fraction II-RNA although generally it was similar to the Specific activity of the fraction I-RNA. The lack of incorporation during the first hour is in agreement with the data obtained when the phenol extracted RNA was frac- tionated by electrOphoreSis. This would indicate that the isotOpe incorporated during a one hour labeling period is predominatedly into an RNA fraction which is not isolated by the two phenol extraction methods employed. The discrepancies can be eXplained by the observations of Perry (1962) that most of the radioactive precursor incor- porated into RNA during Short incubation periods is retained 84 Table 11. 32p Incorporation Into Phenol Extracted RNA by Tissue Slices from Mammary Glands of Rats 2 Days Postpartum 400 mg of tissue from mammary gland of rats 3 dagg prepartum were incubated in Medium 199-H with 50 uc of P as outlined in Methods. RNA was twice extracted at room temperature with phenol containing 1% sodium deoxycholate and then precipitated with TCA as outlined in Methods. Insulin (I), prolactin (P) and hydrocortisone acetate (H0) were present at a concentration of 40 ug/100 m§~of tissue. 32P Incorporation Cpm/100 ug RNA 1 hour 0 3 hours 336 5 hours 012 7 hours 1724 10 hours 2137 1 hour + I 66 5 hours + I 895 10 hours + I 2129 1 hour + HC-P-I 30 5 hours + HC-P-I 1002 10 hours + HC-P-I 2989 113.5 ue/ml. 85 in the nucleus and that a large portion of this incorpora- tion is into a small class of RNA (HnRNA) (1% of total RNA) which is degraded within the nucleus with a half life of less than 3 minutes (Soerio 32 al., 1968). Thus it would seem that the 3H-uridine incorporated into TCA precipitated RNA was predominately into HnRNA while 32P incorporation into phenol extracted RNA was predominately a measure of cytOplasmic RNA synthesis. Calculations suggest that greater than 90% of the isotOpe incorporated into RNA does not enter the cytOplasm and at the present time has no known function (Church and NcCarthy, 1967; Aronson and Wilt, 1969). This is not to indicate that it has no function nor that the incorporation is not meaningful but until it can be shown that this syn- thesis is reflected in changes within the cytoplasm, these types of studies will be difficult to interpret. Thus, if most of the precursor was incorporated into the HnRNA frac- tion during short incubation periods and the HnRNA was not isolated by the phenol extraction method, this could eXplain the discrepancies between the two systems. Results of Hormone Treatment Effect of Hormones gpon RNA Synthesis The results of experiments to ascertain the effect of Short term incubation in the presence of hormone upon 3H_uridine incorporation are presented in Table 12. All of 86 Table 12. Effect of Various Hormones Upon 3H-Uridine Incor- poration Into TCA Precipitated RNA by Tissue Slices From Mammary Glands of Rats 400 mg of tissue from the mammary glands of rats 3 days postpartum was incubated in Medium 199-H (Table 1) with 10 uc of Uridine-5-3H (Sp. Act. 153 uc/umole) as outlined in Methods. The RNA was precipitated with TCA and the radio- activity determined. Insulin (I), hydrocortisone acetate (HC) and prolactin (P) were present at a level of 25 ug/100 mg of tissue (9 ug/ml). EXpressed as % of 3H-Uridine Incorporated by Slices Incubated Without Hormone1 Treatment 60 Min 180 Min No hormone2 100 100 + I 116 105 + HC 108 74 + P 128 80 + HC-I 112 110 + p-1 115 155 + HC-P-I 146 106 1dpm/ngNA + hormone 2100% = 25 uumole/mg RNA during 1 hour and 80 uumole/mg RNA during 3 hours. 87 the hormones stimulated 3H—uridine incorporation during the first 60 minutes but only the combinations containing insulin were stimulatory during 3 hours of incubation. The very large variations in incorporation, as were also observed by Nayne 33.21. (1966), made interpretation of the results qualitative at best. In an attempt to compensate for the variability dur- ing short term eXperiments, the incubation period was extended to 10 or 12 hours. The uridine incorporation during the last hour of these incubations is presented in Table 13. IrreSpective of the hormonal conditions, lactat— ing tissue slices incorporated less 3H-uridine during the last hour of a 10 hour incubation than during the last hour of a 7 hour incubation. Insulin was the only hormone treat- ment which was as effective as no hormone in lactating tissue while in nonlactating tissue, insulin alone, the 3 hormone combination, and prolactin plus twice the level of insulin and hydrocortisone stimulated uridine incorporation when compared to the no hormone control. The incorporation by lactating tissue slices incubated with insulin alone decreased between 5 and 7 hours and a significant decrease in incorporation between 5 and 7 hours was also observed in nonlactating tissue slices incubated with insulin or the 3 hormone combination indicating that tissue slices with the most synthetic activity during early incubation periods were unable to maintain that rate during extended incubation. 88 Table 13. Effect of Various Hormones Upon 3H-Uridine Incor- poration Into TCA Precipitated RNA by Mammary Tissue Slices 350 mg of tissue from the mammary glands of rats 2-3 days prepartum or 3 days postpaftum was incubated in Medium 199-H (Table 1) with H-Uridine as outlined in Methods. The RNA was precipitated with TCA and the radioactivity determined. Insulin (I), hydrocortisone acetate (HC) and prolactin (P) were present at a basal level of 50 ug/lOO mg of tissue (15 us/ml). —— .u- on. Uridine Incorporation uumoles/mg RNA/60 Min2 Treatment 1 Hr 3 Hrs 5 Hrs 7 Hrs 10 Hrs3 Prepartum4 No hormone 35 -- 59 54 -- + I -- -- 88 68 -- + P -- -- 57 55 -- + P-I -- -- 51 62 -- + P-HC -- -- 31 35 ~- + P-HC-I 35 -- 73 62 -- + 2X(HC-I)P -- -- 82 79 -- Postpartum5 No hormone 51 72 75 84 70 + I 47 70 95 85 71 + HC -- -- 74 71 63 + P-I -- -- 62 74 63 + HC-P-I 50 73 75 72 59 1Prepartum tissue - Uridine-5-3H - 15 uc/incubation (Sp. Act. 143 uc/umole). Postpartum tissue - Uridine—5-3H — 10 uc/incubation (Sp. Act. 125 uc/umole). 2Calculations were based on Specific activity of uridine in incubation Medium. 3IsotOpe was present only during last 60 minutes of incubation. 4Data was from only 1 experiment. 5Data is average of 2 or 3 experiments. 89 The combination of prolactin and hydrocortisone without insulin inhibits uridine incorporation 30-50% in nonlac- tating tissue. The effect of hormones upon 32P incorporation into phenol extracted RNA is summarized in Table 14. The incor- poration into various fractions of RNA is presented in Appendix Tables 3-7. Instead of being present for only the last hour of incubation as was the 3H-uridine, 32P was present during the entire incubation period. Contrary to the results obtained with 3H-uridine incorporation, several hormone combinations stimulated 32F incorporation into RNA when compared to the no hormone control. Generally differ- ences of less than 10% were considered insignificant because of variation in the Specific activity of the precursor pool and also errors in measuring the area of the various peaks and in slicing the gels. Some liberty was taken with these criteria when small but consistent differences were evident in several observations. Although only three direct measurements were made, insulin appears to stimulate 32P incorporation, both by itself and in conjunction with other hormones. The stimula- tion by insulin alone was about 10% while generally the stimulation by the 3 hormone combination was 20-50%. This indicates that at least one of the other two hormones had a stimulatory effect in the presence of insulin. Although only a limited number of observations were made; prolactin 90 ospm I-II :sma III III III HIcIom + mama IIII mmma III III III omosaos on H adphmapmom maaom mHIb WHmfi IIII III III III III Hlmlnvm + masm IIII III III III III ocosaon oz Bapamaoam mama N -I- been -- I- has I-I H-d + I-I- osmm -II III oms mam HIom + -III mmam -II wmm on: III HImIom + I-I- Hewa III mew ms: moa escapes oz Bapawaoam_mmmm m mam NH mam 0H mam m mam m mam m am H pnoapwohe «mm m: oa\aao soapwhoahoozH mmm .Aas\wa mHv mammap go we ooa\wa 0: mo Hoboa w as psomoha cams Amv :HpocHoaa pad Aomv mpwpoow oaomdpaoooapaz .AHV QHHSmsH .posfiaampop wzmm pad mma .mom can go mpa>apod oawaooam as» psd popmcoapomag .popomapxo mm; «Em .mpoSpoz SH posdapso mm .poms mm; on on cons H advawaoaa vacaahoaxm 2a paooxo m m mo 01 om Spas AN manned mImmH azapoz 5a popmnSosH mm: sapwoapmoa undo: :wlmm am mHIm no adphwaoaa mamp mIN mama mo upscam madaama 80am mammdp mo w oso mooaam mammas mamasma as «am popomapxm Hoconm OpQH cofipmaoaaoosH a Scab mosoaaom wSoHam> do pommmm . H manna mm s .cofimeSosa mo maze; m puma baaaap mfiso pflomoha was oaopomHN .onmeSocH do mason m puma msath maso pmomoha mm; oaopOmH 91 H I-I IIII -- I-I oma II- «HIE-om + IIII IIII III III mHH III mogoaaom o: mmm IIII III III III III a + mafia II- III III III -II HIom + NQHH IIII III mom III III HImIom + mam II- II- dam on II- osoawos 02 HH asbwocphom nwsom oo-m: II-I smma I-I I-I -I OH H-om + I-I I-I- I-I I-I ooa -II HIm + -I- Hood III III sea -I H + -- -- -- man was on H-d-on I IIII me III mma Hma o escape: 02 H sapamapmom unacm :mImm III- -- -- on: I-- II- HH + mpmm -II II- II- -- III HIoIom + comm -III I-- am: I-I III onosnoe oz HH Eduamapmom mHSOm mHIw 92 plus insulin were as active as the 3 hormone combination at longer incubation times with prepartum tissue, while at shorter times hydrocortisone and insulin stimulate synthe- sis. In postpartum tissue the combination of hydrocorti- sone and insulin were as stimulatory as the 3 hormone com- bination and enhanced incorporation to a greater extent than insulin alone. The 3 hormone combination stimulated 32P incorporation in all tissues examined, the enhanced rate of incorporation being detected at 5 hours in the lactating tissue but not until 10 hours in the nonlactating tissue. It is possible that this delay was due to the time required for cell division but it is more likely a reflection of the time required to deplete the endogenous hormone pool of the cell. The enhanced rate of synthesis in the presence of the 3 hormone combination was also observed if the sample was preincubated for 9 hours and the isotOpe was present for only the last 3 hours. Role of Hormpnes in Regulating Amino Acid Incorporation in Tissue Slices Amino acid incorporation by tissue slices from pre- partum mammary tissue measured during the last hour of incubation (after preincubation for various lengths of time) is enhanced by insulin alone or insulin plus prolactin and hydrocortisone (Table 15). Prolactin plus insulin was the only treatment which caused increased incorporation of amino acids during the 7th hour of incubation relative to 93 Table 15. Effect of Various Hormones Upon 1LPG-Amino Acid Incorporation Into TCA Precipitated Protein by Mammary Tissue Slices 350 mg of tissue from the mammary glands of rats 2-3 days prepartum or 3 days postp rtum was incubated in Medium 199-H (Table 1) with 1 no of 1 C-amino acid mixture (Sp. Act. approximately 0.5 uc/ug in each eXperiment) as outlined in Methods. The protein was precipitated with TCA and the radioactivity determined. Insulin (I), hydrocortisone acetate (HC) and prolactin (P) were present at a basal level of 50 ug/lOO mg of tissue (15 ug/ml). Amino Acid Incorporatign, ugl/mg Protein/60 Min Treatment 1 Hr 3 Hrs 5 Hrs 7 Hrs 10 Hrs Prepartum3 No hormone in -- 25 21 -- + I -- -- 25 25 ~- + P -— —— 21 20 -_ + P-I -- _- 22 26 -- + P-HC -- -- 15 19 -- + HC-I-P 12 _- 25 25 -_ + 2X(HC~I)P -— —- 28 25 -- Postpartum” No hormone 18 21 28 35 32 + I 18 32 32 35 28 + HC-I -- -- 27 34 27 + P-I -- —- 23 28 29 + HC-P-I 18 2h 25 24 20 1Calculations are based on Specific activity of amino acid incubation medium. 2IsotOpe was present only during last 60 minutes of incuba— tion. 3Data is from only 1 eXperiment. uData is average of 2 or 3 eXperiments. 94 the incorporation during the 5th hour of incubation. This was also observed when uridine incorporation in prepartum tissue was measured indicating that the reSponse by pre- partum tissue to prolactin may be delayed. Only insulin alone stimulated amino acid incorpora- tion in lactating tissue slices and then only at 3 and 5 hours of incubation (Table 15). Again prolactin plus insulin stimulated amino acid incorporation 10 hours rela- tive to the rate of incorporation during shorter periods of incubation. In no case was prolactin plus insulin more active than insulin alone, thus, indicating that the reSponse was a reflection of lower levels of incorporation during shorter times of incubation. Evidence that all of the hormone stimulation was not mediated via RNA synthesis was indicated in eXperiments involving actinomycin D inhibition of protein synthesis. As previous discussed RNA synthesis was inhibited 70-100% by actinomycin D depending upon the assay system and the extent of inhibition was not influenced by hormones. The results presented in Table 16 indicate that protein syn- thesis is also inhibited by actinomycin D, but in nonlac- tating tissue, hormones retarded the inhibitory effect of actinomycin D. The 3 hormone combination enhanced amino acid incorporation in the presence of actinomycin D approx- imately 30% compared to the no hormone sample with actino- mycin D. No effect of hormone in the presence of actinomycin D 95 Table 16. Effect of Actinomycin D on 1LPG-Amino Acid Incor- poration Into TCA Precipitated Protein by Mammary Tissue Slices 350 mg of tissue from the mammary glands of rats 2-3 days prepartum or 3 days postp rtum was incubated in Medium 199-H (Table 1) with 1 uc of 1 C-amino acid mixture (Sp. Act. approximately 0.5 uc/ug in each experiment) as outlined in Methods. The protein was precipitated with TCA and the radioactivity determined. Insulin (I), hydrocortisone acetate (HC) and prolactin (P) were present at a basal level of 50 ug/lOO mg of tissue (15 ug/ml). k .- _-—— __...._ _____ Amino Acid Incorporation ug/mg Protein/6O Min1 Treatment 1 Hr 3 Hrs 5 Hrs 7 Hrs Prepartum No hormone 14 -- 25 21 + HC-P-I 12 -- 25 25 + 7.1 actinomycin D2 11 (78)3 -- 9 (38) 6 (28) + 7.1 actinomycin D + HC-P-I 12 (97) -- 12 (49) 9 (36) + 4.3 actinomycin D -- -- 8 (33) 8 (36) + 4.3 actinomycin D + HC-P-I -- -- 12 (47) 12 (48) Postpartum No hormone 16 22 25 32 + 12 pg actinomycin D 20 1b (65) 10 (40) 6 (17) 1IsotOpe was present only during last hour of incubation. 2Actinomycin D was present at the concentration/100 ma tissue as indicated (4.3 ug/lOO mg = 1.2 ug/ml). 3% of normal. 96 was observed with tissue from postpartum rats. Since RNA synthesis did not appear to be required for this hormonal stimulation, some degree of hormonal regulation at a non- transcriptional level must occur. Several possibilities for control exist. Namely, increased uptake of precursor, increased translation of stable mRNA, decreased rate of RNA turnover (degradation) or possibly some combination of these events. Mayne g£_gl. (1966) observed a similar stim- ulation of the same order of magnitude when mammary tissue slices from prepartum rats were incubated with insulin and actinomycin D. The inhibition of protein synthesis by actinomycin D indicates that a Species of RNA (presumably mRNA) is turn- ing over quite rapidly. This Species of RNA turns over faster in lactating tissue, where amino acid incorporation is inhibited 83% after 6 hours preincubation, than in non- lactating tissue where amino acid incorporation is inhibited only 60-70%. This increased inhibition by actinomycin D would indicate that mRNA has a relatively short half life and the mRNA for the milk proteins may be degraded faster than mRNA for cellular proteins. This inhibition data contradicts the conclusions of Sirakov 33 El. (1966) that mRNA turns over very slowly in the cytoplasm of lactatinq mammary tissue. The extent of inhibition of protein syn- thesis is similar to that reported by Mayne gt_al. (1966) and Stockdale E£.§l° (1966). 97 Integration of Results from Several Methods of Assessing Hormonal Effects on Mammary Tissue 811033 The results of histological examination of both pre— and postpartum tissue indicated that during 9-12 hours of incubation, hormones were necessary for maintenance of cellular integrity. The Specific hormone combination necessary for maintenance was not ascertained because only the 3 hormone combination was compared to no hormone at all. Similar results were obtained if the incorporation of labeled precursor during 5-12 hours of incubation into phenol extracted RNA was measured. When incorporation of 1LLC-amino acids was measured during the last 3H-uridine or hour of 5, 7 and 10 hours of incubation, only in nonlactat— ing tissue were hormone combinations containing insulin able to stimulate synthesis. In the same type of exueriment with lactating tissue slices insulin alone was most effec- tive in maintaining uridine incorporation at a level compar- able to the no hormone control. Insulin alone stimulated amino acid incorporation by lactating tissue slices at 3 and 5 hours but could not maintain incorporation at a level comparable to the no hormone control at 10 hours. At least two possible eXplanations occur for the seemingly paradoxical inhibition of RNA and protein synthe— sis by hormones which are necessary for preventing necrosis of the tissue. Haddad e£_al. (1969) have reported that increased RNA synthesis occurred in degenerated nerve ‘tissue compared to normal nerve tissue. Also isolated 98 tadpole tail pieces undergoing regression incorporated more amino acids into protein and uridine into RNA than similar tail slices prior to metamorphosis (Tata, 1966) but a decreased incorporation of leucine and no change in uridine incorporation were observed in in 3132 eXperi- ments (Tonoue and Frieden, 1970). Thus it would seem that 12.XEE£2' degenerating tissue incorporates more precursor into RNA and protein than does normal tissue. This could be a mechanism to retard or repair degeneration or possibly the permeability of the cell membrane is increased and the only change is in the Specific activity of the precursor pool and this does not occur in_zizg. The other possibility is that the rate of synthesis and accumulation of secretory products in hormone treated tissue is suffi- cient to either inhibit uptake of precursor or decrease the rate of synthesis as is observed in’zlzg_when intramammary pressure increases. The incorporation of uridine and amino acids into TCA precipitated products, because it was a short term incubation, would be very reSponsive to these influences. Thus the data in Tables 13 and 15 are probably a reflection of hormonal stimulation, state of degeneration and synthetic activity of the tissue. The incorporation when the isotope was present for longer periods of time would be less susceptible to the latter two influences because the precursor pool is probably well established before degeneration and synthetic activity become important 99 and also the rate of uptake of precursor is somewhat less important in the longer term eXperiments. Thus, 32F incorporation into phenol extracted RNA is probably a better measure of hormone influence on RNA syn- thesis than is the measurement of 3H-uridine incorporation into TCA precipitated RNA. Calculation of Amount of RNA Synthesized by Mammary TissuefiSlices The amount of 32P incorporated by tissue slices in 31232 into RNA per mg of tissue is greater in postpartum tissue than in prepartum tissue when corrections are made for the differences in Specific activity of the precursor pool and the increased RNA content (2.2-fold) of the tis— sue. The incorporation per mg of DNA is still greater pre- partum even when these corrections are made. The calculations based on the measured Specific activity of the isolated RNA, the Specific activity of the precursor pool and the RNA content of the tissue indicate 100% more RNA/gland is synthesized postpartum than prepartum (Table 17). Similar calculations of 3H-uridine incorporation indicated about 2.5 times more RNA was synthesized per g of lactating tissue than per g of nonlactating tissue if no corrections are made for changes in pool size. Sirakov and Rychlik (1968) reported that the Specific activity of the isolated RNA was higher from prepartum tissue than from postpartum tissue as was also observed in these eXperiments 100 Table 17. Calculation of Amount of RNA Synthesized by Lactat- ing and Nonlactating Mammary Tissue 18th Day of Gestation 300 0pm . 3.5 x 106 gpm 86 uumole Pi incorporated 10 ug RNA/hr ° umole Pi - 10 ug RNA/hr 87 ug RNA x 5 g mammary tissue x 86 uumole Pi incorporated 100 mg tissue rat 10 ug RNA/hr = .0374 umole Pi incorporated/gland/hr .0374 umole P 1 gland7hr x 339 (average NW of nucleotide monOphoSphate) = 12.7 g ug RNA synthesized/gland/hr ‘- 3rd Day_of Lactation 120 ppm L 2.5 x 106 opm _ 48 uumgle Pi incorporated 10 ug RNA/hr ' umole P1 10 11°; RNA/hr 193 ug RNA x 8 g_mamma:y tissue x 48 uumole Pi incorpprated 100 mg tissue rat 10 ug RNA/hr = .0741 umoles Pi incorporated/gland/hr .0741 umole Pi g1and7hr x 339 (average NW of nucleotide monophOSphate) = 25.1 mg RNA synthesized/gland/hr 101 if changes in precursor pool size and RNA content were not taken into account. The level of incorporation in their mouse mammary tissue slice system was about 1% of the level reported in Table 10. Wang (1962) reported the uridine nucleotide pool doubled at the time of the initia- tion of lactation. If this is reflected in a decreased Specific activity of the precursor pool, the RNA synthesis per g of tissue by lactating tissue could be 5 times higher than in nonlactating tissue. The calculations outlined in Table 18 suggest that differences in the amount of RNA syn- thesized per mammary gland between prepartum and postpartum tissue may account for about 5% of the increase in RNA con- tent of the gland although it could be probably 2—fold higher if the time required for the extracellular 32F to establish an intracellular precursor pool is accounted for. Also the lower amount of RNA/g of tissue (note Table 6) would also result in lower values, thus the calculated level of synthesis could possibly account for 25% of the increased RNA of the mammary tissue. The discrepancy between 25% and 100% would indicate that either ip‘zitgp synthesis is not an accurate measure of lg 3112 synthesis or the rate of RNA degradation ip_3132 decreases at the time of parturition. It is still impossible to separate the increased RNA content from the increases in DNA and tissue because they all increased, although to differing extents, during the initia- tion of lactation. 102 Table 18. Relation of RNA Synthesis to the Increased RNA Content in Postpartum Tissue 3rd day of lactation 15.7 mg RNA/rat mammary gland 18th day of gestation 4.4 mg RNA/rat mammary gland _~ 11.3 mg increase in RNA 3rd day of lactation 25.1 ug RNA synthesized/gland/hr 18th day of gestation 12.7 ug RNA synthesized/gland/hr 12.4 0g more RNA synthesized/hr 3 days postpartum 1) Assume there was an 156 hour time interval between measure— 2) 12.4 ug more RNA synthesized 3) ments. Also, assume that there was no change in amount of RNA degraded/hour, and the rate of increase was constant throughout interval. 2 (for average increase) x 156 hours = .97 mg increase in RNA Il2%_%§ = 8.6% of increase could come from increased rate of synthesis. 103 Calculation of Amount of Protein Synthesis Calculations pretaining to the amount of amino acids incorporated into protein are in Table 19. Assuming a 3 day postpartum rat secretes 10 ml of 12% protein milk (1.2 g of protein/day), the minimum rate of synthesis in tissue slices is 20% of the 12.2132 rate. Any difference in Specific activity between the free amino acid pool and the amino acids in the medium would increase the calculated amount of synthesis. Although the calculations on amount of synthesis are approximations, they indicated the tissue slices were very active during the period of incubation. 104 Table 19. Calculation of Amount of Protein Synthesized by Tissue Slices from Nammary Gland of Rats 3 Days Prepartum or 3 Days Postpartum 3 Days Postpartum 1) Amino acids incorporated = 35 ug/mg protein1 2) 3.17 mg protein/100 mg wet tissue 3) Thus 106 mg protein synthesized/100 mg wet tissue/hour 4) Assume mammary tissue of rat weighs 8 g 5) Thus 8.5 mg of protein produced/rat/hour = 204 mg of protein synthesized/day 3_Days Prepartum 1) Amino acids incorporated = 25 ug/mg protein1 2) 2.46 mg protein/100 wet tissue 3) 62 mg protein synthesized/100 wet tissue/hour 1Calculation based on assumption that specific activity of free amino acid pool in tissue is same as Specific activity of amino acids in incubation Medium, thus calculation is minimum value. DISCUSSION The results of this study which attempted to disso- ciate the process of cell division from initiation of lac- tation are consistent with other observations concerning a role for hormones during lactogenesis. The stimulation observed when the glucose and amino acid concentrations in the medium were increased would indi- cate that energy metabolism or general metabolite levels may have a role in controlling synthetic processes. The rather general effect of the combination of all 3 hormones in enhancing uridine incorporation during the first 3 hours of incubation as well as the stimulatory effect of insulin alone and the necessity for insulin to be present for other hormones to act could be the result of a general increase in metabolism and/or membrane permeability. Also the enhancement of amino acid incorporation by the 3 hormones in the presence of actinomycin D presumably occurs without RNA synthesis, and thus must reflect either increased uptake or increased metabolism. Nayne 33 21. (1966) reported that insulin alone had this same effect. Insulin has been shown to stimulate uptake and metabolism (Nayne and Barry, 1970; Louis and Williams, 1970) which would be compatible with it being stimulatory in the presence of actinomycin D. These data would be consistent with the suggestion 105 106 of Linzell (1970) that although increased blood flow does not cause the initiation of lactation, it is associated with it and may increase the flow of metabolites necessary for milk synthesis to the gland immediately after parturi- tion. The stimulation of synthesis by increasing metabo- lites would be compatible with the suggestion of Baldwin and Cheng (1969) that metabolites may be limiting synthesis at high levels of milk production. All of these observa- tions would be consistent with a stimulation of synthesis at the level of translation rather than transcription dur- ing lactogenesis. Nore definitive conclusions concerning the role of hormones can be reached by examination of the synthesis of RNA Species isolated by phenol extraction and electrophoresis. The longer time period of incorporation would mean a larger portion of the labeled RNA would be located in the cytOplasm while RNA isolated from samples incubated shorter times would give information about rapidly labeled RNA. Prolactin and insulin are effective in stimulating 32p incorporation into the 303, 18S and SRNA fractions iso- lated from mammary tissue of rats 2 days prepartum while hydrocortisone, except possibly during Short term incuba- tion, was without effect as also reported by Nayne and Barry (1970). Hydrocortisone and insulin were most effec- tive in stimulating RNA synthesis in lactating tissue slices. The addition of prolactin did not enhance incorporation. 107 The stimulatory effect of hydrocortisone is consistent with the report of Talwaker gt 3;. (1961) that hydrocortisone administration to lactating rats increased milk secretion. These combinations were also the most effective in main- taining amino acid incorporation into protein in the reSpec- tive tissue. The major Species of RNA appear to turnover at similar rates in lactating tissue while the Specific activ- ity of the SRNA is higher than the Specific activity of the rRNA in mammary tissue prepartum. This is consistent with increasing SRNA relative to rRNA during late pregnancy as was observed by Turkington (1969). The inability to detect any Species of RNA which was rapidly labeled relative to rRNA, even at short time inter- vals, was disturbing eXpecially Since the reports of Sirakov 33 El- (1968) and Baldwin.§£ a}. (1970) of RNAS with char- acteristics ascribed to mRNA being isolated from mammary tissue and the suggestion of Baldwin and Martin (1969a, 1969b) that hydrocortisone stimulates synthesis of Specific RNA Species. The few times when high Specific activity Species were observed in the 12-158 region it appeared to reflect the problem of accurately determining the area of the small peak rather than extremely high levels of radio- activity. Actually the calculations of Soeiro 33 El. (1969) indicate that the rate of synthesis of mRNA in HeLa cells, with a mRNA half life of 3 hours, would only be one sixth 108 the rate of synthesis of rRNA indicating that Specific activ- ity rather than rate of synthesis measurements are necessary for detecting rapidly labeled RNA Species. Also calculations indicate that over 75% of the 32F incorporated during the first 20 minutes of incubation of HeLa cells is into HnRNA, thus if the rates of RNA synthesis in mammary nuclei are similar to HeLa cells, less than 10% of the RNA synthesized in the studies of Baldwin.gt‘§l. (1969) could have been mRN . Similarly the observations of Sirakov 32 El. (1968) that much of the rapidly labeled material they called mRNA did not leave the nucleus would suggest it was actually HnRNA. The inhibition of protein synthesis in lactating tissue slices by actinomycin D is also inconsistent with the conclusion that cytOplasmic RNA turns over at a very slow rate (Sirakov, gt_§;., 1968). An RNA Species of 158 has been observed in all mam- mary tissue samples examined including midpregnant mouse and late pregnant rat. Although quantitation of the area of this peak is difficult no change in the ratio of the area of this peak to the ribosomal peaks was observed as would be eXpected from that data of Gayne and Denamur (1969) and Mills and TOpper (1970) that the number of polysomes increases during lactogenesis. Thus although a Species of RNA which has hitherto been described as having character- istic of mRNA could be isolated, characteristics such as rapid turnover, change in amount with functional chances of 109 the tissue or differential reSponse to hormone treatment could not be detected with the methods employed. Mammary tissue slices from rats in late gestation reSpond to increased metabolite availability and prolactin plus insulin administration by increasing the incorporation of labeled precursor into all classes of RNA while hydro- cortisone plus insulin and increased metabolite concentra— tions stimulate RNA synthesis in tissue slices from mammary glands of rats 2 days postpartum. The apprOpriate hormone combination complements the increased metabolite availabil- ity. These observations coupled with the inability to isolate Species of RNA which reSponded in a rapid and Specific manner to hormones would indicate that lactogenesis is an inherent prOperty of the differented mammary epithelial cell. SUNMARY Mammary tissue slices incubated 12,33332 incorporated labeled precursors of RNA and protein at a rate calculated to be comparable with rates of synthesis in 3122. Several discrepancies in rate of incorporation and reSponse to hormones are observed when defined RNA fractions are assayed rather than total precipitated RNA. Increased metabolite concentrations and insulin stimulated incorporation of both protein and RNA precursors. The combination of hydrocorti— sone, prolactin and insulin was also able to overcome a portion of the actinomycin D inhibition of protein synthesis, thus some regulation at the level of translation occurs in mammary cells. 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Composition of Medium 199 Ingredients Per Liter 1-Arginine HCl . . . l-Histidine HCl H20 1- Lysine HCl . . . 1-Tyrosine . . . 1-Tryptrophan . l-Phenylalanine 1-Cystine . . i-Methionine . 1—Serine . . . 1-Threonine . l-Leucine . . 1-Isoleucine . 1-Valine . . . 1-Glutamic Acid H20 1-ASpartic Acid . l-Alanine . . . . l-PI‘Oline o o o o i-Hydroxyproline . Glycine . . . . Ca—D-Pantothenate Biotin O O O O O Folic Acid . . . . Choline Chloride . Inositol . . . . . para-Aminibenzoic A Vitamin A Acetate Calciferol . . . . Menadione . . . Alpha TocOpherol Ph Ascorbic Acid . . Glutathione . . Cholesterol . . Sodium Acetate . l-Glutamine . . AdenosinetriphOSpha Adenylic Acid . . Ferric Nitrate 9H20 0 i O I O . O O O O O O O O O I O O O O O 0 O 0 . O O O O O m N coco.moooooooooogooooooooo00.0.00. mooooo'dooo{2.000900000000000000000000 N nag-00000000000000.0000. (I) :3‘ N d' Ribose . . . . 1- Cysteine HCl H2 Adenine Sulfate Guanine H01 . Xanthine . . . Hypoxanthine . Thymine . . . Uracil . . . Thiamine HC1 . 0000000000000000020.00.000.000000000.000.000.- ooooooooooooooo00000000000000.0000000000.00... OOOOOCOOOOOmOOOOOWOQOOOOOOOOOOOOOOOOOOOOOOOOOO H 00000000000 0 I. I HUIt-‘N owwwmw H mg m8 m8 U"; [118 mg m8 m8 m8 m8 m8 m8 m8 m8 m8 m8 mg mg m8 m8 mg mg m8 m8 m8 m8 m8 m8 mg mg m8 m8 m8 m8 m8 m8 m8 m8 m8 m8 m8 mg mg mg Appendix Table 1. 121 (Continued) Ingredients Per Liter Riboflavin . . Pyridoxine HCl Pyridoxal HCl Niacin . . . . Niacinamide . Deoxyribose . Tween 80 . . . Sodium Chloride . . Potassium Chloride . Calcium Chloride . . Magnesium Sulfate . Monosodium,PhoSphate Dextrose . . . . . . Sodium Bicarbonate . O O 0 O O O O Earle's Base 6.8 0.4 NHOOO N HON 9 2 ) 7 5 7 QIN‘D‘QOQW'NOQ 0.01 0.025 0.025 0.025 0.025 0.5 5 ms ms ms ms ms 45 ms Hank's Base 8 0.4 0.14 0.108 0.06 1 (0.35) E 8 8 8 8 RI g 122 Appendix Table 2. Carnoy-Hematoxin Method for Preparing 1) 2) 3) 4) Samples for Histological Examination L. r sample placed in Carnoy's fixative overnight H0AC-ET0H-CHC13 (1:6:3) sample stored in 70% ETOH transferred to eosin for up to 12 hours dehydrated for 1 hour each in 70% ETOH, 95% ETOH and 100% ETOH before transferring to toluene for 30 minutes 5) placed in Paraffin I (not over 65°) for 12 hours 6) placed in Paraffin II (not over 65°) for 2-12 hours 7) block and cut 8 u thick Staining: 1) Xylene I 5 min 2) 3) 4) 5) 6) 7) 8) 10) Xylene II 5 min Alcohol - 100%, 95%, 70%, 50% - 3 min in each, rinse well after each step Distilled H20 - rinse well Hematoxin - 4 min Tap H 0 - rinse off blue color for few minutes and examine with microscOpe - look at nuclei 0.17% ROI-70% ETOH - dip once and place immediately in tap H20 to complete destaining blue for 15-20 minutes Note: Check microscopically to determine if slices have been adequately destained or if they have been overstained Eosin - 3~5 min Alcohol - 70%, 95%, 100%, 100%, - 3-5 min in each, rinse well after each step Xylene I 3-5 min Xylene II 3-5 min Mount 123 Effect of Various Hormones Upon 32F Incor- poration Into Phenol Extracted RNA by Tissue Slices From Mammary Glands of Rats 3 Days Prepartum (EXperiment I). Appendix Table 3. One g of tissue slices was incubated in 25 m1 of Medium 199-E with 30 no of 3 P for periods of time indicated. RNA was fractionated as outlined in Methods. The basal level of each hormone was 40 ug/iOO mg of tissue. opm 32P Incorporated/10 ug RNA 288 185 153 SRNA 1 hour 111 100 125 105 1 hour + 2x(HC-I) 96 124 145 182 1 hour + HC-I 210 209 215 217 3 hours 410 511 813 513 3 hours + 2x(HC-I) 421 403 383 546 3 hours + HC-I 841 830 1068 615 3 hours + P-I 448 490 543 365 3 hours + HC-P-I 487 465 770 459 5 hours 769 760 608 1044 5 hours + HC-P-I 790 730 789 1055 10 hours 1695 1666 1681 2161 10 hours 2x(HC-I) 2420 2633 3597 3357 10 hours HC-I 2437 2041 1738 2331 10 hours P-I 4013 3571 4231 2903 10 hours P 3384 2936 3225 2925 10 hours HC—P-I 2055 2042 1477 2360 124 Effect of Various Hormones Upon 32F Incor- poration Into Phenol Extracted RNA by Tissue Slices From Mammary Glands of Hats 2 Days Prepartum (EXperiment II). Appendix Table 4. One g of tissue sliceg was incubated in 25 ml of Medium 199-E with 50 no of 3 P and hormones for periods of time indicated. RN was fractionated as outlined in Methods. Actinomycin D and 3 P were added at beginning of incubation to 12 hour sample. The basal level of hormone was 40 ug/100 mg of tissue. Seven ug of actinomycin/100 mg tissue was present. 32p Incorporation cpm/io ug RNA 288 188 158 SRNA 7 hours + HC-P-I 1845 1905 1507 2802 12 hours 3172 3091 3450 4877 12 hours + HC-I 2972 3331 3819 4688 12 hours + HC-P-I 3876 4100 4596 5577 12 hours + normal—1991 1973 2080 1433 2712 12 hours + normal-199 + HC-P-I 2799 2319 2806 2942 3 hours + actinomycin D2 304 305 359 660 12 hours + actinomycin D 26 20 26 220 1Normal-199 was Medium 199-Earles salts without added glucose and lactalbumin hydrolyzate. 2The 3 hours samgge mycin D before P was 4 hours. was preincubated for 1 hour with actino- was added, thus total incubation time 125 Effect of Various Hormones Upon 32F Incor- poration Into Phenol Extracted RNA by Tissue Slices From Mammary Tissue of Rats 6-18 Hours Postpartum Appendix Table 5. One g of tissue sliceg was incubated in 25 ml of Medium 199-E with 50 no of 3 P and hormones for periods of time indicated. RNA was fractionated as outlined in Methods. The basal level of each hormone was 40 ug/100 mg of tissue. 32p Incorporation Cpm/10 ug RNA 283 183 153 SRNA Experiment I 3 hours 159 236 138 200 7 hours 1501 1903 1703 661 7 hours + HC-P-I 1870 2552 2603 1109 12 hours 2247 2722 2093 1978 12 hours + HCaP-I 2637 3336 2077 1965 EXperiment II 5 hours 509 453 470 331 12 hours 2532 2516 780 2140 12 hours + HC-P-I 3178 3629 3119 2897 12 hours + 11 555 565 288 317 24 hours1 326 335 351 301 24 hours + 80.11 222 152 213 236 24 hours + HC-P-I1 211 209 291 238 lThe 32? was present only for the last 5 hours of incubation. 126 Appendix Table 6. Effect of Various Hormones on BZP Incor- poration Into Phenol Extracted RNA by Tissue Slices From Mammary Glands of Rats 72-84 Hours Postpartum (Experiment I) One g of tissue slice? was incubated in 25 ml of Medium 199-E with 50 no of 3 P and hormones (40 03/100 mg of tissue). RNA was fractionated as outlined in Methods. Ten ug of actinomycin D/100 mg tissue was present as indicated. 32? Incorporation Cpm/io ug RNA 288 188 158 SRNA 1 hour --- --- --_ --- 1 hour + HC-I 5 10 16 16 1 hour + HC-P-I 11 18 44 32 3 hours 103 99 187 192 3 hours + I 103 118 87 101 3 hours + P-I 158 174 145 238 3 hours + HC-P-I 113 118 103 114 5 hours 205 205 173 184 5 hours + HC-P-I 338 380 228 298 10 hours 883 860 1050 901 10 hours + I 1083 1227 860 964 10 hours + HC-I 1357 1157 1185 1158 3 hours + actinomycin D1 --- 17 5 85 10 hours + actinomycin D 5 7 9 50 1The 3 hour sample was preincubated i3 the presence of actinomycin D for 1 hour before the 2P was added. 127 Appendix Table 7. Effect of Various Hormones Upon 32F Incor- poration Into Phenol Extracted RNA by Tissue Slices From Mammary Glands of Rats 48-60 Hours Postpartum (EXperiment II). One g osztissue was incubated in 25 ml of Medium 199-E with 50 no of P and hormones (40 ug/lOO mg tissue). RNA was fractionated as outlined in Methods. 32F Incorporation opm/lo ug RNA 288 188 15s SRNA 3 hours 48 40 91 121 7 hours 217 155 194 417 7 hours + HC-P-I 419 304 315 911 12 hours 679 825 744 996 12 hours + HC-I 1105 975 1031 1435 12 hours + P 655 604 677 1198 12 hours + HC-P-I 1137 595 527 1401 12 hours + normal-.1991 474 524 606 648 12 hours - normal-1991 + HC-P-I 683 558 512 759 12 hours2 87 85 221 211 12 hours + Hc-P-I2 98 119 189 222 1Normal-199 was Medium 199-Earles salts without added glucose and lactalbumin hydrolyzate. 2The sample was preincubated for 9 hours and the isotope was only present for the last 3 hours. WTITI'Il‘Ijfiuifll‘fld'fll[11111111113111nyES