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Hormonal induction of dehydrogenase enzymes in mammary gland in vitro
GENERAL
AND
COMPARATIVE
17, 319-326 (1971)
ENDOCRINOLOGY
Hormonal
Induction
of Dehydrogenase
in Mammary E. &I. RIVERA Depwtment
of
Zoology.
Michigan
Gland AND
State
Enzymes
in Vitro
E. P. CCMMINS University,
Received December
East
Lansing,
Michigan
.@8%
9. 1970
Mammary explants from midpregnant mice undergo functional differentiation when cultivated in vitro with insulin, prolactin, and corticosterone. The activities of two enzymes of the pentose phosphate pathway for glucose metabolism, glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, increase by a-fold after cultivation with insulin and prolactin, and this increase can be maintained by the further addition of corticosterone. Maximal induction occurs by 48 hr after an initial lag period. Actinomycin D and cycloheximide prevented enzyme increases when present from the start of culture. Cycloheximide was largely ineffective when added during the late phase (44 hr) of induction, whereas actinomycin D was still 50% inhibitory at this time. This suggests that the essential peptide elongation is virtually completed by 44 hr, despite a continued need for RNA synthesis. Colchicine and cytosine arabinoside, whether added initially or after the explants had been exposed to hormones for 24 hr. had relatively little effect on enzyme induction. Hence, DNA synthesis and cell division do not appear essential for dehydrogenase increases. Fructose can replace glucose in the culture medium. Increased concentrations of glucose in the medium neither replace nor enhance hormonal effects. Hormones must be present in the medium for at least several hours to effect maximal induction at 48 hr. After this time, the continued presence of hormones is not required to maintain enzyme activity over the following 24 hr.
The functional development of the mammary gland is accompanied by marked increasesin the activities of several enzymes (Bartley et al., 1966; Baldwin and Milligan, 1966), among which are glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two enzymes of the pentose phosphate pathway for glucose metabolism. Since these enzymes are associ.ated wit’h the development of a hormone-dependent organ, an analysis of their regulation by hormones should provide a useful approach to the study of hormoneind.Jced differentiation. Studies of t’he mammary gland in viva and in vitro have shown that glucose-6phosphate and 6-phosphogluconate dehyd.rogenases are indeed responsive to hormones. Prolactin and cortisol increase their activit,ies in the mammary glands of hy319
pophysectomized or pscudopregnant animals (Baldwin and Martin, 1968, 1969; Heitzman, 1969). Prolactin and corticosterone also increase dehydrogenase activity in mammary organ cultures from pregnant mice (Rivera, 1969; Rivera and Cummins, 1969; Jones and Forsyth, 1969; Leader and Barry, 1969), although the effects of these hormones in vitro are dependent upon the presence of insulin. This latter observation is in accord with the requirement, for all three of the above-mentioned hormones (insulin, prolactin, and adrenal corticosteroid) for the induct,ion of several other developmental changes in mammary explants (Rivera, 1964; Juergens et al., 1965; Lockwood et al., 1966). Inasmuch as growth and functional act’ivity appear to be temporally regulated by hormones in mammary cultures (Stock-
320
RIVERA
AND
dale and Topper, 1966; Lockwood et al., 1967; El-Darwish and Rivera, 1970)) it was of interest to analyze the time sequence of hormone action vis iL vis the induction of dehydrogenase activity. Information on the combined and individual effects of hormones on enzymatic activity and the duration and nature of these effects would be of obvious value in understanding the hormonal mechanisms underlying mammary differentiation. Accordingly, the present study is concerned with the sequence of effect and the interactions of insulin, prolactin, and corticosterone on the activities of glucose-6-phosphate and 6-phosphogluconate clehydrogenases in organ cultures of the mouse mammary gland. MATERIALS
AND
METHODS
Orgnn cultzires. Mammary explants were derived from nulliparous pregnant (12-14 days) Swiss albino mice and cultivated as organ cultures as previously described (El-Dar\%-ish and Rivera, 1970). Stock solutions of insulin (I), corticosterone and prolactin (PL) were prepared in (B), medium 199 as before (El-Darwish and Rivera, 1970) and were added in various combinations to yield final concentrations of 5 pg/ml of the protein hormones and 1 @g/ml of the adrenal corticosteroid. Penicillin G was added to all media at a concentration of 50 III/ml. The cultures were maintained for 4, 24, 48, and 72 hr. Three to five experiments were run for every hormonal combination for each time interval studied. For each experiment, 100 mammary explants were dissected from each of two mice and randomly distributed among four groups of culture media. One-half of the explants in each group was used for the enzyme assays and the remainder for DNA determinations. The medium was changed at 48 hr in the cultures maintained for 72 hr. Enzyme nssnys. Explants were ground in an all-glass homogenizer with cold 0.15 M KC1 and a 105,OOOg supernatant was prepared by centrifugation for 1 hr. Glucose-6-phosphate dehydrogenase (n-glucose+phosphate:NADP oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-n-gluconate :NADP oxidoreductase, EC 1.1.1.44) activities were determined spectrophotometrically as described before (Rivera, 1969). The reaction mixture contained 1 mM of substrate (either glucose 6-phosphate or 6-phosphogluconate), 0.2 mM of NADP (nicotinamide adenine dinucleotide phosphate) , 10 mM MgCh, and 56 mM of Tris buffer, pH 8.0,
CUMRIINS
in a final volume of 1.0 ml. The reaction at 25% was started by addition of mammary supernatant, and the initial rate of extinction at 340 rnF was determined on triplicate aliquots of the supernatant. Because of the cell proliferation that occurs in mammary cultures (Stockdale and Topper, 19661, enzyme activities were expressed as micromoles of NADPH formed per minute per 10 mg DNA. In the experimentas with inhibitors, enzyme activities were expressed on the basis of tissue weight, since we found that actinomycin D and cycloheximide inhibited DNA synthesis by the mammary explants. DNA assays. Explants were homogenized in cold 10% trichloroacetic acid and the precipitate subjected to lipid extraction with methanol: chloroform (2:1), absolute ethanol, and ether. The precipitate was then incubated with 0.3 N KOH at 37’C for 3 hr, and the resulting digest was acidified with 10% perchloric acid (PCA) and placed at O’C for 1 hr. The acid-insoluble fraction was washed twice with cold 10% PC.4, and DNA was extracted by incubation with 5% PCA at 70°C for 30 min. Extracts were combined from three washes of acid-insoluble fraction and analyzed for deoxyribose by t.he diphenylamine reaction (Burton, 1956) using calf thymus DN.4 standards. Hormones and chemicals. Culture medium 199 was purchased from BBL Division, Bioquest, Maryland. Bovine insulin (24 IU/mg) was a gift from Dr. 0. K. Behrens, Eli Lilly and Company; nonesterified crystalline corticosterone, a gift from the Upjohn Company; ovine prolactin (P-S7. 24.3 IU/mg), a gift from the National Institutes of Health; and actinomycin D was a gift from Merck, Sharp and Dohme. Sodium penicillin G was purchased from Nutritional Biochemicals, and glucose-6-phosphate, 6-phosphogluconate, NADP, Tris, cycloheximide. colchicine, and cytosine arabinoside purrhascd from Sigma Chemicals.
RESULTS
Initial
Enzyme
Activities
of the Explants
Freshly dissected mammary tissues from 12- and 13-day pregnant mice showed similar dehydrogenase activities (Table 1). However, between the 12th and 14 th day of pregnancy, glucose-6-phosphate dehydrogenase activity increased by 63%, 6-phosphogluconate dehydrogenase by 43%, and total dehydrogenase (the sum of the activities of the two enzymes) increased by
HORMONES
AND
MAhlRlARY
TABLE
ENZYhfES
in
321
vifr0
1
INITLIL ENZYME ACTIVITIES OF MAMMARY EBPL.INTS
PMolesNAI>PH/min/lO mg l>NA lkys
of pregnancy 1’2 1:; 14
G-R-PDH
(a)
4..54 .5.54
* 0.51 *
0 71
7.&L
*
0 5:s
6-PGIIH 3.28 3.02 4.70
(b) f 0.56 * 0.21 + 0.26
Tot’al (a + b) 7.83 S.56 1’2.12
*
0.8'2
+ 0.86 + 0 77
activity, which was particularly apparent at 24 hr but which remained near baseline levels thereafter. Prolactin and corticosterone were required for the augmentation and maintenance of this basal, insulin-dependent induct.ion of enzyme activity, although these hormones alone were ineffective in the absence of insulin (Fig. 1). In 24-hr cultures, insulin Injluence of Hormones on Enzyme and prolactin increased total dehydrogenase .4ctizlity of Explanh in V&o by 70% over the initial levels, and by 48 During the first 4 hr of culture total hr this hormone combination was sufficient dehydrogenase activity was maintained at to cause maximal induction of total enzyme initial levels even without hormones (Fig. (161% over the initial control values), The 1). The absence of hormones was not maintenance of this induced lcvcl through ap larent until 24 hr and was reflected by 72 hr required corticosterone in addition a progressive decline in enzyme activity to prolactin, that is, the full combination to below initial values. Insulin alone caused of three hormones. a small increase in total dehydrogenase It is notewort.hy that the responses to 53%. The changes induced in vitra did not rlii’cr in magnitude according to the stage of pregnancy. For comparison of differential hormonal effects, the results were expressed as the change in activity relative to that of the initial explants at time 0 of culture. Explants were taken from 13-day pregnant mice for most experiments.
24
hrr.
48 hrs.
72
ha.
FIG. 1. Changes in total dehvdmgenase activity of mammary explants iu response to different coml)itlniions of inslllin (I), prolactin (PL), and corticosteroneCB).Resultsare ex-pressed aspercentagec,hnllge~II ettcyme activity from that. at. time 0 of culture.
322
RIVERA
AND
prolactin and corticosterone were not immediate. A similar lag of enzyme response was observed in the mammary glands of pseudopregnant rabbits given prolactin and cort~isol (Heitzman, 1969). In t,hese cultures, no enzyme increases could be attributed to either hormone over the first hours in vitro (Fig. I), although t’hey were present from the start of culture in media that contained t,hem. The activity of prolactin was expressed before that of corticosterone, inasmuch as prolactin and insulin caused t’he highest increase in total dehydrogenase activity at 24 hr, whereas corticosterone and insulin had no effect above that obtained with insulin alone (Figs. 1 and 2). Moreover, prolactin but not corticosterone was essential for maximal enzyme induction in 4%hr cultures. The proportion of total dehydrogenase activity due to glucose-6-phosphate dehydrogenase was always greater than that due to 6-phosphogluconate dehydrogenase in the cultured as well as in the fresh ex-
G-6-
CU.\IilIINS
plants (cf. Table 1 and Fig. 2). However, the magnitude of response to hormones was always greater with the slower enzyme, 6-phosphogluconate dehydrogenase. The maximum increase in the activity of the latter enzyme was 248% over initial values, whereas that of glucose-6-phosphate dehydrogenase was 193% over initial values. It had previously been shown that insulin was necessary for continued glucose uptake by mammary explants (Rloretti and DeOme, 1962). Although insulin’s effect on glucose transport can sometimes be duplicated by increasing glucose concentration (see review by Wool, 1965), enzyme activity in mammary cultures could not be increased by raising t’he concentration of glucose to five or ten times that normally used, 1 mg/ml (Table 2). On the contrary, it appeared that higher glucose levels were slightly inhibitory whether hormones were present or absent from the medium. Such an effect could have resulted nonspecifically from increased osmot’ic pressure rather than
PDH
6- PGDH TOTAL
cl
24
I
DH
hrs.
I*B
48
I+Pl
I*B.PL
FIG. 2. Differential effects of hormones activity in the presence of insulin-containing tions involving 3-5 cultures *SE.
I
hrs.
72
I
I+B
on glucose hormone
6-phosphate combinations.
hrs.
I+ B
and 6-phosphogluconate Each bar is the mean
I+PL
I*B.PL
dehydrogenase of 3-5 observa-
333 TABLE EFFECTS
OF GLUCOSIC
.IND
2
FRUCTOSE
ON ENZYME
ACTIVITIW
Relative Hormone :j ddi tion I None None None I + I iI + I + I + I i-
Gliwose
(mg/ml)
Fruct.ose
(mg/ml) -
B + PL B + PL B + PI, PL PL PL
’ Control
activities
of tot,al
dehydrogenase
24 Hr
48 Hr
10Oa
-
6.5
-
-
56 31
-
-
-
-
1OOa 81 82
1OoC 1
-
99
06
5
activities
for each grollp
were
adjusted
increased glucose per se. It is interesting, however, that glucose can be replaced by fructose with similar results even with fructose levels five times the concentration of glucose used normally (Table 2). To determine whether protein and RNA synthesis are required for enzyme induction, and actinomycin D were cycloheximide, tr:&ed for their effects on dehydrogenase acativity. Actinomycin D, 5 pg/ml, inhibited the incorporation of uridine-14C into mammary explant RNA by 96% within 4 hr; cycloheximide, 10 pg/ml, inhibited the incorporation of 14C-labeled amino acid mixt’ure into mammary explant protein by 87% in 4 hr. Explants cultured wit,h insulin 3rd prolact’in were exposed to inhibitor during the early (0 hrj and late 144 hrj periods of enzyme induction, and assays performed at 48 hr. Cycloheximide and actinc.mycin D almost completely inhibited enzyme increases when administered from the start of culture (Table 3). When addition was delayed until 44 hr, cyclolrcximide caused only 13% inhibition, wllercas actinomycin D caused about 50% inhibition of enzyme activity. To explore the relationship of DNA svnthesis and enzyme induction, the effects of colchicinc and cytosine arabinoside were examined. Colchicine, 0.05 Kg/ml, inhibited thymidine-3H incorporation into mammary expIant DNA by 48% in 24 hr ; cytosine nrabinosicle. 5 ,-cg/ml, inhibited incor-
to 100%.
poration by 80% in 24 hr. The inhibitors were administered to the medium at 0 hr and at 24 hr to explants continuously exposed to insulin and prolactin for 48 hr. Inhibitory effects were less pronounced than with RNA and protein inhibitors. Colchicine prevented enzyme induction by 24% when present from the start of culture (Table 3) ; its effect was even less when administered at 24 hr. Cyt’osine arabinoaide had only negligible effects regardless of the time of administrat,ion. To determine whether hormones were continuously required for enzyme induction. explants were exposed to hormones for 0.5 hr, 4 hr, or 48 hr after which they were washed in several changes of hormone-free TABLE >:FFECTS
OF INHIBITORS
3 ON
RTLYME
Inhibitor
Time of addition (hr)
Cycloheximide Cycloheximide Actinomycin D Bctinomycin D Colchicine Colchicine Cytosine arabinoside Cytosine arabinoside
0 44 0 44 0 24 0 24
ACTIYITIBR~
9;) Inhibition of enzyme act,iGity 91 13 93
55 24 13 10
6
a All media contained insulin and prolactin. The results represent the means of duplicate experiments.
324
RIVERA
AND
medium and cultured again without hormones. Transferral to hormone-free medium after 0.5 hour exposure to insulin and prolactin resulted in 89% inhibition of enzyme induction at 48 hr. Delaying transferral to hormone-free medium for 4 hr resulted in 61% inhibition. When explants were cultured with insulin, prolactin, and corticosterone for 48 hr, washed, then cultured for an additional 24 hr without hormones, enzyme activity was essentially the same as that of the controls which had been continuously exposed to hormones for 72 hr.
CUMMINS
may be the formation of new cells which have the capacity to undergo differentiation in vitro in responseto appropriate hormonal stimuli. Insulin also initiates RNA synthesis (Mayne et al., 1966, 1968; Turkington and Ward, 1969; Green and Topper, 1970; El-Darwish and Rivera, 1971)) which may be a prerequisite for enzyme induction in mammary tissue (Leader and Barry, 1969). In addition, insulin’s role may be to maintain a base-level of dehydrogenase activity (Rivera and Cummins, 1971), the amplification of which requires synergistic action with at least prolactin. The interaction of insulin, prolactin, and DISCUSSIOK corticosterone appears to be manifested These results support previous obser- in a specific sequence and is independent vations of a triple hormonal requirement of whether hormones were all present in for mammary differentiation in vitllo. The the medium from the start of culture. For enzymatic increases found here are in example, the highest increases in total deaccord with the results of Bolton and hydrogenase activity did not occur ur.til 48 hr and was achieved with insulin and Bolton (1970)) who reported a preferential stimulation of the oxidation of glucose prolactin, regardless of whether corticostercarbon atom 1 over carbon atom 6 in one was present or absent. However, this response to the triple-hormone combination peak in total enzyme could be maintained in explants of rabbit mammary gland. In for 72 hr only when all three hormones this study, enzyme induct’ion followed a were present. Thus, prolactin manifests its activity before corticosterone and plays a specific time course, comparable to that observed for nucleic acid and protein syn- dual role in the induction and maintenance thesis in mammary explants (Stockdale of enzyme activity. Although cort,icosterone and Topper, 1966; Lockwood et al., 1967; does not appear to be a strict requirement for enzyme induction, its presence in the El-Darwish and Rivera, 1970). The lack of any significant enhancement 72-hr cultures more effectively maintains of dehydrogenase activity during the first induced enzyme activity than do insulin hours in z~iiro indicates a lag or latent and prolactin alone. Comparable effects of corticosterone on nucleic acid levels (Elphase in enzyme induction. Although insulin alone caused a small rise in enzyme Darwish and Rivera, 1970, 1971) indicate activity, this effect remained relatively that the adrenal steroid may be more important for maintaining than for stimuconstant throughout the duration of culture. This finding is in keeping with insulin’s lating at least certain kinds of mammary capacity to maintain the viability of mam- responses in culture. On the other hand, mary explants (Elias, 1959; Rivera, 1964) maximum induction of milk protein synwhich otherwise deteriorate in the absence thesis appears to require the full triad of of insulin and the failure of insulin alone hormones (.Juergens et al., 1965; Lockwood to induce differentiative responses (Rivera, et al., 1966). 1964; Juergens et al., 1965). The increases in enzyme activity reported It has become increasingly apparent that here cannot be accounted for by mere insulin’s role encompassesmore than sus- increases in cell number, inasmuch as taining tissue survival. Since insulin stim- activities were corrected for DNA increases. ulates Dh’A synthesis (Stockdale and Moreover, most of the cellular proliferation Topper, 1966; El-Darmish and Rivera, occurs by 24 hr and requires only insulin 1970), the consequence of this activity (Stockdale and Topper, 1966; El-Darwish
and Rivera, 1970)) whereas peak enzyme induction occurs subsequently and requires prolactin as well as insulin. The observation that increased glucose concentration neither replaced the effect of insulin nor enhanced the combined effects of insulin, prolactin, and corticosterone indicates that the hormonal effects are probably not mediated through glucose transport. This is supported by t,he observation that fructose can replace glucose in thn hormonal induction of enzymes, as well as of milk protein (Voytovich and Topper, 1967). Actinomycin D and cycloheximide largely prevented rises in enzyme activity when present from the start. of culture. This nolild suggest, that both RNA and protein synthesis are involved in enzyme induction. Actinomycin D was still 50% inhibitory during the terminal phase of induction, whereas cycloheximide was only slightly so. It therefore appears that peptide elongation of the protein (or proteins) involved in the increase of enzyme activity occurs, for the most part, prior to 44 hr, whereas the need for RNA synthesis is continuous. Leader and Barry (1969) rcl:orted that actinomycin D failed to inhibit dehydrogenaee increases if added after 3.5 hr of culture and suggested that all essential RKA synthesis is completed by this time. The reasons for our difference in results are not known, but it ehould also he noted that casein synthesis in mammary explants is also sensitive to actinomycin D throughout 48 hr of culture (Stockdale anal Topper, 1966). The observation that colchicine prevented hormone-stimulated casein synthesis (Siockdale and Topper, 1966) led us to examine the relation of DNA synthesis ancl mitosis to enzyme induct’ion. The relatively minor effects of colchicine and cytosine arabinoside on enzyme increases suggests that DNA synthesis and cell division are less important for this funcGonal parameter than for milk protein synthesis. This is further supported by the ohservation that hydroxyurea also fails to prevent dehydrogenace increases in mammary explants (Leader and Barry, 1969).
Alt,hough the concentration of colchicinc used here inhibited DNA synthesis by only 50010, the extent of inhibition of enzyme activity was not proportional-only 24% when colchicine was present from the star: of culture. Cytosine arabinoside, which inhibited DNA synthesis by SO%, was even less inhibitory to enzyme induction. The results obtained by washing explants after prior exposure to hormones indicate that hormones must be present in the medium for at least the first several hours of culture. This may reflect, a critical time interval during which hormones initiate event’s leading to increased enzyme activity. After peak induction at 48 hr, washing and transferral to hormone-free medium had no effect on induced levels over the subsequent 24 hr. Thus, it appears that t.he events set in motion by hormones can be maintained after a critical period for at least 24 hr in the absence of hormones. It is possible that, t.he washing procedures were inadequate to remove all traces of hormones, particularly if cellular penetration had occurred. However, hepatoma cell cultures apparently require hormone continuously in the medium for t,he maintenance of induced enzyme levels (Thompson ef al., 1966), even if one assumes retention of small amounts of hormone either intra- or extracellularly. Therefore, while it cannot be concluded that, our procedures resulted in complete removal of hormone, induced enzyme activity in mammary explants can be maintained for some time with hormone-free mccliun~. ACIiNOWLEDGMENTS This investigation was supported by a Research Grant (HD-02374) and by a Research Career Development -4ward (E. M. R.) (K3-HD-20.688) from the National Institute of Child Health and Human Development. REFERENCES R. I,.. .4ND MARTIN, R. J. (1968). Effects of hypophysectomy and several hormone replacement therapies upon patterns of nucleic acid and protein synthesis and enzyme levels in lactating rat mammary glands. 1. Dairy Sci.
BALDWIN.
51, 748-753. BALDWIN.
R. L.. ASD
MARTIN,
R.J.
(1969).
Protein
326
ItIVEZU
AND
and nucleic acid synthesis in rat mammary glands during early lactation. Endocrinology 82, 1209-1216. B.~LDWIN, R. L., AND MILLIGAN, L. P. (1966). Enzymatic changes associated with the initiation and maintenance of lactation in the rat. J. Biol. Chem. 241, 2058-2066. B.~RTLEY. J. C., ABRAHAM, S., AND CHAIKOFF, I. L. (1966) _ Activity patterns of several enzymes of liver, adipose tissue, and mammary gland of virgin, pregnant and lactating mice. Proc. Sot. Exp. Biol. Med. 123, 67CM75. BOLTON, C., AND BOLTON, A. E. (1970). Effects of prolactin on pathways of glucose oxidation in explants from rabbit mammary gland. FEBS Lett. 9, 177-179. BURTON, Ii. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. EL-DARWISH, I., AND RIVERA, E. M. (1970). Temporal effects of hormones on DNA synthesis in mouse mammary gland in vitro. J. Exp. Zool. 173, 285-291. EL-DARWISH, I., AND RIVERA, E. M. (1971). Hormonal regulation of RNA synthesis by mouse mammary gland in vitro. J. Exp. Zool in press. EI,IAS, J. J. (1959). Effect of insulin and cortisol on organ cultures of adult mouse mammary gland. Proc. Sot. Eq. Biol. Med. 101, 5OS502. GREEN, M. R., AND TOPPER, Y. J. (1970). Some effects of prolactin, insulin and hydrocortisone on RNA synthesis by mouse mammary gland ira vitro. Biochim. Biophys. Acta 204, 441-448. HEITZMAN, R. J. (1969). The induction by exogrnous hormones of enzymes metabolizing glucose&phosphate in the mammary gland of the pseudopregnant rabbit. J. Dairy Res. 36, 47-51. JONES, E. A., AND FORSITH, I. A. (1969). Increases in the enzyme activity of mouse mammary explants induced by prolact,in. J. Endocrinol. 43, xli-xlii. JUERGENS. W. G., STOCKDALE, F. E., TOPPER, Y. J.. AND ELIAS, J. J. (1965). Hormone-dependent differentiation of mammary gland in vitro. Proc. Nat. Acad. Sci. U. S. 56, 629-634. LEADER, D. P., AND BARRY, J. M. (1969). Increase in activity of glucose&phosphate dehydrogenase in mouse mammary tissue cultured with insulin. Biochem. J. 113, 17.!k182. IBCKWOOD, D. H., TURKINGTON, R. W., AND TOPPER. Y. J. (1966). Hormonedependent development of milk protein synthesis in mammary gland in vitro. Biochim. Biophys. Acta 130, 49iiL501. LOCKWOOD,
Y.
(1967).
D.
H.,
STOCKDALE,
Hormone-dependent
F.
E..
AND
TOPPER,
differentiation
CUMMINS
of mammary gland: sequence of action of hormones in relation to cell cycle. Science 156, 94b946. MAYNE, R., BARRY, J. M., AND RIVERA, E. M. (1966). Stimulation by insulin of the formation of ribonucleic acid and protein by mammary tissue in vitro. Biochem. J. 99, 688-693. MAYNE, R., FORSYTH, I. A., AND BARRY, J. M. (1968). Stimulation by hormones of RNA and protein formation in organ cultures of the mammary glands of pregnant mice. J. Endocrinol. 41, 247-253. MORETTI, R. L., AND DEOME, K. B. (1962). Effect of insulin on glucose uptake by normal and neoplastic mouse mammary tissues in organ culture. J. Nat. Cancer Irlst. 29, 321329. PALMITER, R. D. (1969). Early macromolecular syntheses in cultured mammary t.issue from midpregnant mice. E,Ldocrinology 85, 747-761. RIVERA. E. M. (1964). Differential responsiveness to hormones of C3H and A mouse mammary tissues in organ culture. Endocrinology 74, 853864. RIVERA, E. M. (1969). Some observations on the activities of HGH and HPL in mouse mammary organ cultures. In “Lactation, the Initiation of Milk Secretion at Parturition” (M. Reynolds and S. J. Folley, eds.). Univ. of Pennsylvania Press, Philadelphia. RIVERA, E. M., AND CUMMINS, E. P. (1969). Hormonal st,imulation of pentose phosphate enzymes in organ cultures of mouse mammar> gland. Amer. Zool. 9, 578. RIVER.4, E. M., AND CUXMINS, E. P. (1971). Differential actions of insulin on enzyme activities in mammary organ culture. J. Cell Physiol. 77, 175-178. STOCKDALE, F. E., AND TOPPER, Y. J. (1966). The role of DNA synthesis and mitosis in hormonedependent differentiation. Proc. Nat. Aced. Sci. u. 8% 56, 125%1289. THOMPSON. E. B.. TOMKINS. G. M., AND CURRAN, J. F. (1966). Induction of tyrosine cu-ketoglutarate transaminase by steroid hormones in a newly established tissue culture cell line. Proc. Nat. Acad. Sci. U. S. 56, 29&303. TURKINGTON, R. W., AND WARD, 0. T. (1969). Hormonal stimulation of RNA polymerase activity in mammary gland in vitro. Biochim. Biophys. Acta 174, 29-301. VOYTOVICH, A., AND TOPPER. Y. J. (1967). Hormone-dependent differentiation of immature mouse mammary gland in vitro. Science 158, 1326-1327. WOOL. I. G. (1965). Insulin and protein biosynthesis. In “Actions of Hormones on Molecular Processes” (G. Litwack and D. Kritchevskp, eds.), Wiley. New York.
AND
COMPARATIVE
17, 319-326 (1971)
ENDOCRINOLOGY
Hormonal
Induction
of Dehydrogenase
in Mammary E. &I. RIVERA Depwtment
of
Zoology.
Michigan
Gland AND
State
Enzymes
in Vitro
E. P. CCMMINS University,
Received December
East
Lansing,
Michigan
.@8%
9. 1970
Mammary explants from midpregnant mice undergo functional differentiation when cultivated in vitro with insulin, prolactin, and corticosterone. The activities of two enzymes of the pentose phosphate pathway for glucose metabolism, glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, increase by a-fold after cultivation with insulin and prolactin, and this increase can be maintained by the further addition of corticosterone. Maximal induction occurs by 48 hr after an initial lag period. Actinomycin D and cycloheximide prevented enzyme increases when present from the start of culture. Cycloheximide was largely ineffective when added during the late phase (44 hr) of induction, whereas actinomycin D was still 50% inhibitory at this time. This suggests that the essential peptide elongation is virtually completed by 44 hr, despite a continued need for RNA synthesis. Colchicine and cytosine arabinoside, whether added initially or after the explants had been exposed to hormones for 24 hr. had relatively little effect on enzyme induction. Hence, DNA synthesis and cell division do not appear essential for dehydrogenase increases. Fructose can replace glucose in the culture medium. Increased concentrations of glucose in the medium neither replace nor enhance hormonal effects. Hormones must be present in the medium for at least several hours to effect maximal induction at 48 hr. After this time, the continued presence of hormones is not required to maintain enzyme activity over the following 24 hr.
The functional development of the mammary gland is accompanied by marked increasesin the activities of several enzymes (Bartley et al., 1966; Baldwin and Milligan, 1966), among which are glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two enzymes of the pentose phosphate pathway for glucose metabolism. Since these enzymes are associ.ated wit’h the development of a hormone-dependent organ, an analysis of their regulation by hormones should provide a useful approach to the study of hormoneind.Jced differentiation. Studies of t’he mammary gland in viva and in vitro have shown that glucose-6phosphate and 6-phosphogluconate dehyd.rogenases are indeed responsive to hormones. Prolactin and cortisol increase their activit,ies in the mammary glands of hy319
pophysectomized or pscudopregnant animals (Baldwin and Martin, 1968, 1969; Heitzman, 1969). Prolactin and corticosterone also increase dehydrogenase activity in mammary organ cultures from pregnant mice (Rivera, 1969; Rivera and Cummins, 1969; Jones and Forsyth, 1969; Leader and Barry, 1969), although the effects of these hormones in vitro are dependent upon the presence of insulin. This latter observation is in accord with the requirement, for all three of the above-mentioned hormones (insulin, prolactin, and adrenal corticosteroid) for the induct,ion of several other developmental changes in mammary explants (Rivera, 1964; Juergens et al., 1965; Lockwood et al., 1966). Inasmuch as growth and functional act’ivity appear to be temporally regulated by hormones in mammary cultures (Stock-
320
RIVERA
AND
dale and Topper, 1966; Lockwood et al., 1967; El-Darwish and Rivera, 1970)) it was of interest to analyze the time sequence of hormone action vis iL vis the induction of dehydrogenase activity. Information on the combined and individual effects of hormones on enzymatic activity and the duration and nature of these effects would be of obvious value in understanding the hormonal mechanisms underlying mammary differentiation. Accordingly, the present study is concerned with the sequence of effect and the interactions of insulin, prolactin, and corticosterone on the activities of glucose-6-phosphate and 6-phosphogluconate clehydrogenases in organ cultures of the mouse mammary gland. MATERIALS
AND
METHODS
Orgnn cultzires. Mammary explants were derived from nulliparous pregnant (12-14 days) Swiss albino mice and cultivated as organ cultures as previously described (El-Dar\%-ish and Rivera, 1970). Stock solutions of insulin (I), corticosterone and prolactin (PL) were prepared in (B), medium 199 as before (El-Darwish and Rivera, 1970) and were added in various combinations to yield final concentrations of 5 pg/ml of the protein hormones and 1 @g/ml of the adrenal corticosteroid. Penicillin G was added to all media at a concentration of 50 III/ml. The cultures were maintained for 4, 24, 48, and 72 hr. Three to five experiments were run for every hormonal combination for each time interval studied. For each experiment, 100 mammary explants were dissected from each of two mice and randomly distributed among four groups of culture media. One-half of the explants in each group was used for the enzyme assays and the remainder for DNA determinations. The medium was changed at 48 hr in the cultures maintained for 72 hr. Enzyme nssnys. Explants were ground in an all-glass homogenizer with cold 0.15 M KC1 and a 105,OOOg supernatant was prepared by centrifugation for 1 hr. Glucose-6-phosphate dehydrogenase (n-glucose+phosphate:NADP oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-n-gluconate :NADP oxidoreductase, EC 1.1.1.44) activities were determined spectrophotometrically as described before (Rivera, 1969). The reaction mixture contained 1 mM of substrate (either glucose 6-phosphate or 6-phosphogluconate), 0.2 mM of NADP (nicotinamide adenine dinucleotide phosphate) , 10 mM MgCh, and 56 mM of Tris buffer, pH 8.0,
CUMRIINS
in a final volume of 1.0 ml. The reaction at 25% was started by addition of mammary supernatant, and the initial rate of extinction at 340 rnF was determined on triplicate aliquots of the supernatant. Because of the cell proliferation that occurs in mammary cultures (Stockdale and Topper, 19661, enzyme activities were expressed as micromoles of NADPH formed per minute per 10 mg DNA. In the experimentas with inhibitors, enzyme activities were expressed on the basis of tissue weight, since we found that actinomycin D and cycloheximide inhibited DNA synthesis by the mammary explants. DNA assays. Explants were homogenized in cold 10% trichloroacetic acid and the precipitate subjected to lipid extraction with methanol: chloroform (2:1), absolute ethanol, and ether. The precipitate was then incubated with 0.3 N KOH at 37’C for 3 hr, and the resulting digest was acidified with 10% perchloric acid (PCA) and placed at O’C for 1 hr. The acid-insoluble fraction was washed twice with cold 10% PC.4, and DNA was extracted by incubation with 5% PCA at 70°C for 30 min. Extracts were combined from three washes of acid-insoluble fraction and analyzed for deoxyribose by t.he diphenylamine reaction (Burton, 1956) using calf thymus DN.4 standards. Hormones and chemicals. Culture medium 199 was purchased from BBL Division, Bioquest, Maryland. Bovine insulin (24 IU/mg) was a gift from Dr. 0. K. Behrens, Eli Lilly and Company; nonesterified crystalline corticosterone, a gift from the Upjohn Company; ovine prolactin (P-S7. 24.3 IU/mg), a gift from the National Institutes of Health; and actinomycin D was a gift from Merck, Sharp and Dohme. Sodium penicillin G was purchased from Nutritional Biochemicals, and glucose-6-phosphate, 6-phosphogluconate, NADP, Tris, cycloheximide. colchicine, and cytosine arabinoside purrhascd from Sigma Chemicals.
RESULTS
Initial
Enzyme
Activities
of the Explants
Freshly dissected mammary tissues from 12- and 13-day pregnant mice showed similar dehydrogenase activities (Table 1). However, between the 12th and 14 th day of pregnancy, glucose-6-phosphate dehydrogenase activity increased by 63%, 6-phosphogluconate dehydrogenase by 43%, and total dehydrogenase (the sum of the activities of the two enzymes) increased by
HORMONES
AND
MAhlRlARY
TABLE
ENZYhfES
in
321
vifr0
1
INITLIL ENZYME ACTIVITIES OF MAMMARY EBPL.INTS
PMolesNAI>PH/min/lO mg l>NA lkys
of pregnancy 1’2 1:; 14
G-R-PDH
(a)
4..54 .5.54
* 0.51 *
0 71
7.&L
*
0 5:s
6-PGIIH 3.28 3.02 4.70
(b) f 0.56 * 0.21 + 0.26
Tot’al (a + b) 7.83 S.56 1’2.12
*
0.8'2
+ 0.86 + 0 77
activity, which was particularly apparent at 24 hr but which remained near baseline levels thereafter. Prolactin and corticosterone were required for the augmentation and maintenance of this basal, insulin-dependent induct.ion of enzyme activity, although these hormones alone were ineffective in the absence of insulin (Fig. 1). In 24-hr cultures, insulin Injluence of Hormones on Enzyme and prolactin increased total dehydrogenase .4ctizlity of Explanh in V&o by 70% over the initial levels, and by 48 During the first 4 hr of culture total hr this hormone combination was sufficient dehydrogenase activity was maintained at to cause maximal induction of total enzyme initial levels even without hormones (Fig. (161% over the initial control values), The 1). The absence of hormones was not maintenance of this induced lcvcl through ap larent until 24 hr and was reflected by 72 hr required corticosterone in addition a progressive decline in enzyme activity to prolactin, that is, the full combination to below initial values. Insulin alone caused of three hormones. a small increase in total dehydrogenase It is notewort.hy that the responses to 53%. The changes induced in vitra did not rlii’cr in magnitude according to the stage of pregnancy. For comparison of differential hormonal effects, the results were expressed as the change in activity relative to that of the initial explants at time 0 of culture. Explants were taken from 13-day pregnant mice for most experiments.
24
hrr.
48 hrs.
72
ha.
FIG. 1. Changes in total dehvdmgenase activity of mammary explants iu response to different coml)itlniions of inslllin (I), prolactin (PL), and corticosteroneCB).Resultsare ex-pressed aspercentagec,hnllge~II ettcyme activity from that. at. time 0 of culture.
322
RIVERA
AND
prolactin and corticosterone were not immediate. A similar lag of enzyme response was observed in the mammary glands of pseudopregnant rabbits given prolactin and cort~isol (Heitzman, 1969). In t,hese cultures, no enzyme increases could be attributed to either hormone over the first hours in vitro (Fig. I), although t’hey were present from the start of culture in media that contained t,hem. The activity of prolactin was expressed before that of corticosterone, inasmuch as prolactin and insulin caused t’he highest increase in total dehydrogenase activity at 24 hr, whereas corticosterone and insulin had no effect above that obtained with insulin alone (Figs. 1 and 2). Moreover, prolactin but not corticosterone was essential for maximal enzyme induction in 4%hr cultures. The proportion of total dehydrogenase activity due to glucose-6-phosphate dehydrogenase was always greater than that due to 6-phosphogluconate dehydrogenase in the cultured as well as in the fresh ex-
G-6-
CU.\IilIINS
plants (cf. Table 1 and Fig. 2). However, the magnitude of response to hormones was always greater with the slower enzyme, 6-phosphogluconate dehydrogenase. The maximum increase in the activity of the latter enzyme was 248% over initial values, whereas that of glucose-6-phosphate dehydrogenase was 193% over initial values. It had previously been shown that insulin was necessary for continued glucose uptake by mammary explants (Rloretti and DeOme, 1962). Although insulin’s effect on glucose transport can sometimes be duplicated by increasing glucose concentration (see review by Wool, 1965), enzyme activity in mammary cultures could not be increased by raising t’he concentration of glucose to five or ten times that normally used, 1 mg/ml (Table 2). On the contrary, it appeared that higher glucose levels were slightly inhibitory whether hormones were present or absent from the medium. Such an effect could have resulted nonspecifically from increased osmot’ic pressure rather than
PDH
6- PGDH TOTAL
cl
24
I
DH
hrs.
I*B
48
I+Pl
I*B.PL
FIG. 2. Differential effects of hormones activity in the presence of insulin-containing tions involving 3-5 cultures *SE.
I
hrs.
72
I
I+B
on glucose hormone
6-phosphate combinations.
hrs.
I+ B
and 6-phosphogluconate Each bar is the mean
I+PL
I*B.PL
dehydrogenase of 3-5 observa-
333 TABLE EFFECTS
OF GLUCOSIC
.IND
2
FRUCTOSE
ON ENZYME
ACTIVITIW
Relative Hormone :j ddi tion I None None None I + I iI + I + I + I i-
Gliwose
(mg/ml)
Fruct.ose
(mg/ml) -
B + PL B + PL B + PI, PL PL PL
’ Control
activities
of tot,al
dehydrogenase
24 Hr
48 Hr
10Oa
-
6.5
-
-
56 31
-
-
-
-
1OOa 81 82
1OoC 1
-
99
06
5
activities
for each grollp
were
adjusted
increased glucose per se. It is interesting, however, that glucose can be replaced by fructose with similar results even with fructose levels five times the concentration of glucose used normally (Table 2). To determine whether protein and RNA synthesis are required for enzyme induction, and actinomycin D were cycloheximide, tr:&ed for their effects on dehydrogenase acativity. Actinomycin D, 5 pg/ml, inhibited the incorporation of uridine-14C into mammary explant RNA by 96% within 4 hr; cycloheximide, 10 pg/ml, inhibited the incorporation of 14C-labeled amino acid mixt’ure into mammary explant protein by 87% in 4 hr. Explants cultured wit,h insulin 3rd prolact’in were exposed to inhibitor during the early (0 hrj and late 144 hrj periods of enzyme induction, and assays performed at 48 hr. Cycloheximide and actinc.mycin D almost completely inhibited enzyme increases when administered from the start of culture (Table 3). When addition was delayed until 44 hr, cyclolrcximide caused only 13% inhibition, wllercas actinomycin D caused about 50% inhibition of enzyme activity. To explore the relationship of DNA svnthesis and enzyme induction, the effects of colchicinc and cytosine arabinoside were examined. Colchicine, 0.05 Kg/ml, inhibited thymidine-3H incorporation into mammary expIant DNA by 48% in 24 hr ; cytosine nrabinosicle. 5 ,-cg/ml, inhibited incor-
to 100%.
poration by 80% in 24 hr. The inhibitors were administered to the medium at 0 hr and at 24 hr to explants continuously exposed to insulin and prolactin for 48 hr. Inhibitory effects were less pronounced than with RNA and protein inhibitors. Colchicine prevented enzyme induction by 24% when present from the start of culture (Table 3) ; its effect was even less when administered at 24 hr. Cyt’osine arabinoaide had only negligible effects regardless of the time of administrat,ion. To determine whether hormones were continuously required for enzyme induction. explants were exposed to hormones for 0.5 hr, 4 hr, or 48 hr after which they were washed in several changes of hormone-free TABLE >:FFECTS
OF INHIBITORS
3 ON
RTLYME
Inhibitor
Time of addition (hr)
Cycloheximide Cycloheximide Actinomycin D Bctinomycin D Colchicine Colchicine Cytosine arabinoside Cytosine arabinoside
0 44 0 44 0 24 0 24
ACTIYITIBR~
9;) Inhibition of enzyme act,iGity 91 13 93
55 24 13 10
6
a All media contained insulin and prolactin. The results represent the means of duplicate experiments.
324
RIVERA
AND
medium and cultured again without hormones. Transferral to hormone-free medium after 0.5 hour exposure to insulin and prolactin resulted in 89% inhibition of enzyme induction at 48 hr. Delaying transferral to hormone-free medium for 4 hr resulted in 61% inhibition. When explants were cultured with insulin, prolactin, and corticosterone for 48 hr, washed, then cultured for an additional 24 hr without hormones, enzyme activity was essentially the same as that of the controls which had been continuously exposed to hormones for 72 hr.
CUMMINS
may be the formation of new cells which have the capacity to undergo differentiation in vitro in responseto appropriate hormonal stimuli. Insulin also initiates RNA synthesis (Mayne et al., 1966, 1968; Turkington and Ward, 1969; Green and Topper, 1970; El-Darwish and Rivera, 1971)) which may be a prerequisite for enzyme induction in mammary tissue (Leader and Barry, 1969). In addition, insulin’s role may be to maintain a base-level of dehydrogenase activity (Rivera and Cummins, 1971), the amplification of which requires synergistic action with at least prolactin. The interaction of insulin, prolactin, and DISCUSSIOK corticosterone appears to be manifested These results support previous obser- in a specific sequence and is independent vations of a triple hormonal requirement of whether hormones were all present in for mammary differentiation in vitllo. The the medium from the start of culture. For enzymatic increases found here are in example, the highest increases in total deaccord with the results of Bolton and hydrogenase activity did not occur ur.til 48 hr and was achieved with insulin and Bolton (1970)) who reported a preferential stimulation of the oxidation of glucose prolactin, regardless of whether corticostercarbon atom 1 over carbon atom 6 in one was present or absent. However, this response to the triple-hormone combination peak in total enzyme could be maintained in explants of rabbit mammary gland. In for 72 hr only when all three hormones this study, enzyme induct’ion followed a were present. Thus, prolactin manifests its activity before corticosterone and plays a specific time course, comparable to that observed for nucleic acid and protein syn- dual role in the induction and maintenance thesis in mammary explants (Stockdale of enzyme activity. Although cort,icosterone and Topper, 1966; Lockwood et al., 1967; does not appear to be a strict requirement for enzyme induction, its presence in the El-Darwish and Rivera, 1970). The lack of any significant enhancement 72-hr cultures more effectively maintains of dehydrogenase activity during the first induced enzyme activity than do insulin hours in z~iiro indicates a lag or latent and prolactin alone. Comparable effects of corticosterone on nucleic acid levels (Elphase in enzyme induction. Although insulin alone caused a small rise in enzyme Darwish and Rivera, 1970, 1971) indicate activity, this effect remained relatively that the adrenal steroid may be more important for maintaining than for stimuconstant throughout the duration of culture. This finding is in keeping with insulin’s lating at least certain kinds of mammary capacity to maintain the viability of mam- responses in culture. On the other hand, mary explants (Elias, 1959; Rivera, 1964) maximum induction of milk protein synwhich otherwise deteriorate in the absence thesis appears to require the full triad of of insulin and the failure of insulin alone hormones (.Juergens et al., 1965; Lockwood to induce differentiative responses (Rivera, et al., 1966). 1964; Juergens et al., 1965). The increases in enzyme activity reported It has become increasingly apparent that here cannot be accounted for by mere insulin’s role encompassesmore than sus- increases in cell number, inasmuch as taining tissue survival. Since insulin stim- activities were corrected for DNA increases. ulates Dh’A synthesis (Stockdale and Moreover, most of the cellular proliferation Topper, 1966; El-Darmish and Rivera, occurs by 24 hr and requires only insulin 1970), the consequence of this activity (Stockdale and Topper, 1966; El-Darwish
and Rivera, 1970)) whereas peak enzyme induction occurs subsequently and requires prolactin as well as insulin. The observation that increased glucose concentration neither replaced the effect of insulin nor enhanced the combined effects of insulin, prolactin, and corticosterone indicates that the hormonal effects are probably not mediated through glucose transport. This is supported by t,he observation that fructose can replace glucose in thn hormonal induction of enzymes, as well as of milk protein (Voytovich and Topper, 1967). Actinomycin D and cycloheximide largely prevented rises in enzyme activity when present from the start. of culture. This nolild suggest, that both RNA and protein synthesis are involved in enzyme induction. Actinomycin D was still 50% inhibitory during the terminal phase of induction, whereas cycloheximide was only slightly so. It therefore appears that peptide elongation of the protein (or proteins) involved in the increase of enzyme activity occurs, for the most part, prior to 44 hr, whereas the need for RNA synthesis is continuous. Leader and Barry (1969) rcl:orted that actinomycin D failed to inhibit dehydrogenaee increases if added after 3.5 hr of culture and suggested that all essential RKA synthesis is completed by this time. The reasons for our difference in results are not known, but it ehould also he noted that casein synthesis in mammary explants is also sensitive to actinomycin D throughout 48 hr of culture (Stockdale anal Topper, 1966). The observation that colchicine prevented hormone-stimulated casein synthesis (Siockdale and Topper, 1966) led us to examine the relation of DNA synthesis ancl mitosis to enzyme induct’ion. The relatively minor effects of colchicine and cytosine arabinoside on enzyme increases suggests that DNA synthesis and cell division are less important for this funcGonal parameter than for milk protein synthesis. This is further supported by the ohservation that hydroxyurea also fails to prevent dehydrogenace increases in mammary explants (Leader and Barry, 1969).
Alt,hough the concentration of colchicinc used here inhibited DNA synthesis by only 50010, the extent of inhibition of enzyme activity was not proportional-only 24% when colchicine was present from the star: of culture. Cytosine arabinoside, which inhibited DNA synthesis by SO%, was even less inhibitory to enzyme induction. The results obtained by washing explants after prior exposure to hormones indicate that hormones must be present in the medium for at least the first several hours of culture. This may reflect, a critical time interval during which hormones initiate event’s leading to increased enzyme activity. After peak induction at 48 hr, washing and transferral to hormone-free medium had no effect on induced levels over the subsequent 24 hr. Thus, it appears that t.he events set in motion by hormones can be maintained after a critical period for at least 24 hr in the absence of hormones. It is possible that, t.he washing procedures were inadequate to remove all traces of hormones, particularly if cellular penetration had occurred. However, hepatoma cell cultures apparently require hormone continuously in the medium for t,he maintenance of induced enzyme levels (Thompson ef al., 1966), even if one assumes retention of small amounts of hormone either intra- or extracellularly. Therefore, while it cannot be concluded that, our procedures resulted in complete removal of hormone, induced enzyme activity in mammary explants can be maintained for some time with hormone-free mccliun~. ACIiNOWLEDGMENTS This investigation was supported by a Research Grant (HD-02374) and by a Research Career Development -4ward (E. M. R.) (K3-HD-20.688) from the National Institute of Child Health and Human Development. REFERENCES R. I,.. .4ND MARTIN, R. J. (1968). Effects of hypophysectomy and several hormone replacement therapies upon patterns of nucleic acid and protein synthesis and enzyme levels in lactating rat mammary glands. 1. Dairy Sci.
BALDWIN.
51, 748-753. BALDWIN.
R. L.. ASD
MARTIN,
R.J.
(1969).
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326
ItIVEZU
AND
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